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CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein–Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.

Animal welfare is a crucial aspect of modern animal husbandry. As the global demand for dairy products increases alongside the growth of the population worldwide, animal farming faces new challenges such as increasing food production output without compromising animal welfare and minimizing the environmental footprint 1 . In today's dairy cattle farming the vast majority of cows display a horned phenotype. This, however, poses an increased risk of injury for the animals themselves as well as for farmers, hence a polled phenotype is preferred 2,3 . Naturally polled phenotypes mainly exist in beef breeds such as Angus. It was reported that in some breeds such as Holstein-Friesian (HF) the polled population originates from only two breeding bulls 4 . This demonstrates the necessity to increase the genetic pool of polled individuals within these breeds. Previous studies revealed the genetic background of polledness [5][6][7][8] . Two genetic variants within the polled locus on chromosome 1 are known to cause the polled phenotype. One variant is the Celtic mutation (Polled Celtic, Pc) located within an intergenic region of chromosome 1 of the bovine genome (horned locus). This autosomally inherited structural variant consists of a complex 202 bp insert-deletion (indel) mutation on bovine Chromosome 1 which has been described before (Fig. 1). Another causative mutation is the Polled Friesian (Pf) variant which is the only polled variant present in dairy cattle and consists of an 80 kb duplication accompanied by three single nucleotide polymorphisms (SNPs). More recently, further genetic variants (Polled Mongolian and Polled Guarani) within the same locus were identified which also lead to a polled phenotype 9 . All known variants connected to polledness are collected in the OMIA database (www.omia.org).
The application of modern breeding systems together with advanced knowledge about the bovine genome facilitated breeding for polledness [10][11][12] . In the past, however, the selected high-performance bulls were mostly horned and the breeding towards polledness was not considered relevant by farmers. Therefore, the distribution of the polled trait by classical cross-breeding is complicated by poor breeding and production properties  11,13 . Novel programmable DNA nucleases such as zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system became widely applicable across various species 14 . As the newest member of the family, CRISPR/Cas9 has proven to be highly efficient, cheap in its production and easy in handling 15 . For the application in eukaryotic organisms, an RNA complex was designed to form a single guide RNA (gRNA) which guides the Cas9 nuclease to the respective target sequence (i.e. RNA to DNA binding) 16,17 . The gRNA can be re-programmed to target virtually any genomic locus, however, a protospacer adjacent motif (PAM) sequence at the 3′-end of the gRNA (5′-NGG-3′) is necessary for binding and cleavage of the DNA 18,19 . Other CRISPR/ Cas systems, such as the CRISPR/Cas12a (formerly Cpf1), were designed to overcome this PAM-restriction 20 . CRISPR/Cas12a is a type V class II CRISPR system that only requires a single CRISPR RNA (crRNA) instead of a tracr:crRNA complex like CRISPR/Cas9. Furthermore, a 5′-TTTN-3′ PAM sequence upstream of the gRNA is required for binding and cleavage of the target DNA. This makes CRISPR/Cas12a a suitable alternative for T-rich loci. Another useful feature of CRISPR/Cas12a is its distinct cleavage pattern. CRISPR/Cas9 possesses two cleavage domains (HNH and RuvC) and cutting of the target sequence results in blunt ends, whereas Cas12a possesses only one active cleavage domain in the site of the RuvC domain which cuts one DNA in cis-and the other in trans-position 21 . This leads to the generation of two non-homologous overhangs (i.e. sticky ends) 22 . These sticky ends facilitate the introduction of new DNA sequences, which makes the CRISPR/Cas12a system a promising option for knock-in experiments 23 .
A valuable method for implementing genome editing in cattle is the somatic cell nuclear transfer 24 . It allows the in vitro production of genetically modified somatic cell lines such as fibroblasts which can be characterized prior to being employed as donor cells for the somatic cell nuclear transfer 25,26 . In a previous experiment, the Pc variant was introgressed into dairy cattle using TALEN; the generated offspring showed a polled phenotype 27,28 .
Here, we established the CRISPR/Cas12a system as a novel method to integrate the Polled Celtic variant into the genome of a horned HF breeding bull to produce offspring with a polled phenotype and thereby rendering dehorning unnecessary.

Results
Generation of knock-in cell lines. A gRNA for the CRISPR/Cas12a system was designed to target the wildtype sequence of the horned locus ( Fig. 1). In a first experiment to check the capability of the CRISPR/ Cas12a to target the horned locus, adult fibroblasts derived from a horned Holstein-Friesian bull (total merit index 141) were co-transfected with plasmids expressing CRISPR/Cas12a and gRNA, but without an HDR template carrying the polled Celtic variant. The T7 endonuclease-I assay of lysed cells showed cleavage of heteroduplex DNA resulting in two additional bands, which indicate the on-target efficiency of the transfected nuclease ( Fig. 2A). Additionally, PCR amplicons of the transfected cells were sub-cloned and sequenced. A 1 ± 1 nt indel mutation at the predicted cutting site confirmed the nucleases specificity to introduce an on-target DSB (Fig. 2B).
Subsequently, wildtype fibroblasts from the same bull were co-transfected with CRISPR/Cas12a, gRNA and HDR template for the polled variant. PCR analysis initially showed that only a small proportion of cells carried the desired knock-in. The exact knock-in efficiency could not be assessed directly after transfection since the PCR could not distinguish between the genomic DNA and the HDR template which might have led to false positive results. Using single-cell dilution, we were able to generate a population of knock-in positive cell clones (Pc K.I.) which then served as donor cells for SCNT (Fig. 3A,B). Somatic cell nuclear transfer and embryo transfer. Pc K.I. cell clones were used as donor cells for SCNT. A total of 70 clones were produced of which 66 were successfully fused. Sixty-four embryos showed cleavage on day 5 of in vitro culture of which 18 embryos developed to expanded or hatched blastocysts on day 7 of in vitro cultivation (IVC) (28.1% blastocyst rate). One or two expanded (hatched) blastocysts were transferred into nine synchronized recipients after seven days of IVC (in total 12 cloned embryos, 3 recipients received 2 embryos). The remaining embryos served as a quality control and were maintained in culture until day 8, six showed delayed development. These were not transferred, resulting in a final blastocyst rate of 37.5% (Table 1). Six recipients initially established a pregnancy as determined by ultrasound diagnostic on day 40 of gestation www.nature.com/scientificreports/ (66.7%). Four animals could not maintain pregnancy past the first trimester. One cow was sacrificed on day 90 of gestation in order to analyze the fetus. The remaining pregnancy was carried to term.
Genomic and phenotypic analysis of the fetus. Potential horn buds can be detected at an early stage of development 30 . In the horned WT control, horn buds were detected macroscopically (Fig. 4A). Histological analysis of the frontal skin revealed thickening of the epidermis with 11-13 layers of vacuolated keratinocytes (Fig. 4B). No fetal hair follicles were detected in the dermal layers beneath the horn bud. In the fetus carrying the Pc variant, no horn buds were detected macroscopically (Fig. 4C). The histological examination showed only slight epidermal thickening with two to three layers of vacuolated keratinocytes (Fig. 4D). No hair follicles were detected in the area of a potential horn bud. Taken together, the fetus was phenotypically polled. Fetal liver tissue was used to isolate DNA. PCR analysis showed the successful integration of the Celtic mutation (Fig. 5A), the fibroblast donor served as wildtype control. Sanger sequencing confirmed the bi-allelic integration of the Pc variant (Fig. 5B).    www.nature.com/scientificreports/ Delivery of living offspring. The remaining pregnancy was successfully delivered via caesarian section (Fig. 6).
The calf showed a polled phenotype and the genomic analyses were identical to the previously generated fetus as the Pc variant was detected via PCR and Sanger sequencing (Supplement 5). However, the calf diseased on the day of birth. The calf showed an increased birth weight of 78 kg. Its pathological examination revealed malformations of some internal organs including the liver, heart, diaphragm, lungs and skull, finally resulting in acute cardio-vascular failure.
Vector integration and off-target analysis. Three potential off-target binding sites showed no undesired mutations in the respective loci according to the T7 endonuclease I assay and Sanger sequencing (Supplement 7). During the generation of the knock-in cell line, three plasmids carrying an ampicillin expression cassette were transfected into the bovine fibroblasts. To test for random plasmid backbone integration, PCRs specific to the antibiotic resistance cassette and selected plasmid fragments were conducted (Supplements 2, 3).  www.nature.com/scientificreports/ A faint signal only for the resistance cassette was detected. PCR analysis specific to the HDR template (HP1748-F1 and HP1748-R1) indicates potential integration of another copy of the Pc variant.

Discussion
Polled cattle are easier and safer in handling and the disbudding of calves contradicts animal welfare, therefore genetically polled cattle are preferred in the dairy industry 2,31 . The Polled Celtic (Pc) variant, a 202 bp indel variant within the polled locus, causes a polled phenotype in many beef breeds 5,32 . We hypothesized that the CRISPR/ Cas12a mediated knock-in of the Pc variant into the genome of a polled HF breeding bull causes a polled phenotype in offspring produced via somatic cell nuclear transfer (SCNT) within one generation.
In a similar study, the knock-in of the Pc variant into bovine fibroblasts by transfecting TALEN in form of mRNA together with an HDR template carrying the 202 bp indel mutation resulted in cloned offspring with a polled phenotype 27 . In our project, we employed the CRISPR/Cas12a system as a novel and efficient method for the introduction of the Pc variant into the genome of a high-performance HF breeding bull.
The CRISPR/Cas12a nuclease (formerly CRISPR/Cpf1) is a class II CRISPR/Cas system which requires a 5′-(T)TTN-3′ PAM sequence for targeted DNA binding and cleavage 20 . This feature makes it a favorable option in T-rich target regions, such as the target region in the polled locus. Another benefit of CRISPR/Cas12a is its distinct DNA cleavage pattern. Contrary to Cas9, Cas12a possesses only one cleavage domain (RuvC-like endonuclease domain) which cuts each DNA single-strand at different sites, resulting in non-homologous 4-5 bp overhangs. This facilitates the correct integration of new DNA sequences independently from the cell cycle and the employed repair mechanism. These characteristics of the CRISPR/Cas12a made it the nuclease of choice for the knock-in of the Pc variant. Here, 20 bp long gRNAs were used, however an updated version of the online tool CRISPOR (https ://crisp or.tefor .net) predicted higher on-target efficiencies with gRNAs of 24 bp length. An experiment comparing both gRNA lengths could potentially optimize on-target efficiencies by assessing their respective ability to create indel formation and thereby facilitate the knock-in. However, quantitative knock-in efficiencies could not be assessed directly after transfection since the employed assays could not discriminate between cellular DNA and residual template DNA.
SCNT is a crucial step in the generation of offspring from in vitro edited fibroblasts. Following SCNT, the developmental rates of the reconstructed embryos are significantly lower compared to IVF embryos 33 . We reliably produced cloned gene-edited embryos with blastocyst rates of 28.1% on day 7 of cultivation and 37.5% on day 8 (Table 1). In vitro produced embryos were transferred into nine recipients and two pregnancies could be maintained until day 90 of gestation. Embryonic and early fetal death is more frequent in pregnancies generated with cloned embryos [34][35][36][37] . Several factors are thought to be involved in decreased full-term development rates. Incomplete de-methylation and subsequent aberrant DNA methylation profiles between embryos produced by in vitro fertilization (IVF) and SCNT have been observed and could deteriorate the developmental capacity of the embryos 38 . This impaired epigenetic reprogramming potentially leads to aberrant gene expression, thereby interfering with the full-term development of respective offspring. Another factor that contributes to fetal mortality could be insufficient placentation. In our study, abnormal placentation was detected via sonography in pregnancies which did not develop full term. Placentomes develop from both the inner cell mass (ICM) and the trophectoderm (TE) of the embryo 39 . It was previously revealed that bovine SCNT embryos showed an increased ICM: total cell ratio (i.e. reduced amount of trophectoderm cells) which may lead to insufficient placentation with subsequent deficits of fetal nourishment 40 .
In our project, one pregnancy was terminated on day 90 of gestation for analysis of the fetus. No fetal horn buds were detected macroscopically. Histological examination of the frontal skin also revealed the absence of a thickened epidermis with additional layers of keratinocytes. These findings are in accordance with a previous study in which the horn status of different fetal stages was examined 30 . Taken together, the phenotypic analyses showed a polled phenotype of the fetus cloned from the Pc K.I. cell line. The remaining pregnancy was successfully delivered via caesarian section. The living offspring also showed a polled phenotype; however, it was not viable on the long term due to organ malformation and showed an increased birth weight of 78 kg. This likely represents artefacts of the cloning procedure, which was commonly described as "large offspring" or "abnormal offspring syndrome (type III AOS)" [41][42][43] . However, whole genome sequencing would be necessary to entirely disprove a detrimental effect of the genome editing process itself.
Genomic analyses of the fetus confirmed the integration of the Pc variant. However, the exact pathway of how the Pc variant causes polledness is still unknown. One hypothesis is that this mutation affects the expression of micro RNAs (miRNAs) which are not yet annotated. MiRNAs can interact with mRNA which might lead to translational repression or mRNA deadenylation [44][45][46] . It was also reported that miRNAs are capable of silencing genes or inducing transcription by directly binding to promoter regions of genes [47][48][49] . Therefore, alteration of miRNA expressing regions might significantly affect gene expression patterns. A novel hypothesis is that Pc and Pf are located in boundary regions of topologically associating domains (TADs). TADs are structural subunits of the genome which form loop structures within a chromosome and thereby bringing loci which are usually far apart along the genome into the vicinity of each other, enabling interaction between enhancing regions and promotor regions 50,51 . TADs are separated from boundary regions which consist of hundreds to thousands of non-coding nucleotides. Mutations in boundary regions may lead to partial fusion or a shift of their position. This might induce a functional change of the three-dimensional chromatin structure [52][53][54] . Taken together, these epigenetic frameshifts establish novel enhancer-promotor interactions affecting gene expression in loci which are not located near the causative mutation. To confirm either hypothesis, a refined annotation of the polled locus and its associating regions including respective gene expression is necessary.
Further genomic analyses indicated a potential integration of a second copy of the HDR template (Supplement 4). In-depth sequencing of the entire locus or quantitative PCR approaches such as digital PCR will confirm Scientific RepoRtS | (2020) 10:13570 | https://doi.org/10.1038/s41598-020-70531-y www.nature.com/scientificreports/ or exclude unintended vector integration. In a previous study in which the Pc variant was introduced into the genome of an HF bull via TALEN, a recent publication revealed the unintended integration of the entire HDR template, including another copy of the Pc variant 29,55 . Even though this undesired event did occur, it did not have a negative effect on the generated animals and this unintended plasmid integration could be corrected by classical cross-breeding 56 .
In conclusion, we successfully established the CRISPR/Cas12a system as a novel method to introduce the Pc variant into the genome of a superior Holstein-Friesian bull and could thereby address current issues in today's farm animal housing and breeding. Analysis of the generated fetus showed a polled phenotype. Finally, we successfully delivered a polled calf which also showed the CRISPR/Cas12a mediated knock-in of the Pc variant.

Materials and methods
CRISPR/Cas12a and guide RNA expression. The LbCas12a expressing plasmid SQT1665 (Addgene plasmid #78744) was employed in this study. Three different complementary pairs of DNA oligonucleotides targeting the six base pair deletion of the Celtic mutation (Supplement 1) were annealed and cloned into the BsmBI cloning site of the gRNA expressing plasmid (BPK3082, Addgene plasmid #78742). For subsequent knock-in experiments gRNA "LbCas12a gRNA_4" was used. Cell culture and transfection. Bovine fibroblasts were isolated from an ear notch of a commercially used horned Holstein-Friesian bull (total merit index of 141) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 1-2% penicillin/streptomycin, 1% non-essential amino acids and sodium pyruvate and 10-30% fetal calf serum (FCS) at 37° C and 5% CO 2 . Passaged cells were transfected using the 100 µl kit of the Neon® Transfection System (Invitrogen, Thermo Fisher Scientific) at 1,800 V, one 20 ms pulse. A total of 10 µg plasmid was transfected, respectively. T7 Endonuclease I cleavage assay and Sanger sequencing. In order to asses on-target efficiency, the target sequence was amplified via PCR using P C specific primers (btHP-F1: 5′ GAA GGC GGC ACT ATC TTG ATG GAA 3′; btHP-R2: 5′ GGC AGA GAT GTT GGT CTT GGG TGT 3′) under the following conditions: 95 °C for 2 min followed by 32 cycles of 95 °C for 25 s, 62 °C for 25 s and 72 °C for 60 s. Final elongation was performed at 72 °C for 5 min. The essay was conducted as recommended by the supplier. Somatic cell nuclear transfer and embryo transfer. The somatic cell nuclear transfer and parthenogenetic activation were performed as previously reported 57 . Adult fibroblasts with integrated Pc variant were used as donor cells. Recipient animals were synchronized via repetitive prostaglandin application (alfaCloprost ® forte, alfavet, Germany; 2 ml per animal per injection). One expanded or hatched blastocyst was transferred per recipient, respectively. PCR-based genotyping and phenotypic analysis of fetus. P C specific primers (btHP-F1 and btHP-R2, see above) were used for detection of the knock-in as well as sequencing primers. The first pregnancy was terminated on day 90 for macroscopic and histological analysis of the fetus. Histological slices of the frontal skin were stained with hematoxylin and eosin stain. A horned wildtype control of similar size (i.e. similar day of gestation) was collected from a slaughterhouse.

Off-target analysis and vector integration.
For the induction of a double strand break at the target site a gRNA was employed which does not have likely potential off-target binding sites according to CRISPR RGEN Tools (https ://www.rgeno me.net/cas-offin der/; reference genome: Bos taurus (bosTau7)). A selection of three potential off-target binding sites was analyzed via T7 endonuclease I assay and Sanger sequencing (Supplements 6, 7). Potential vector integration was tested via PCR analysis of plasmid specific fragments from each transfected plasmid (Supplements 2, 3).


extfieldsofstudy: Chemistry Medicine

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Bioactive Compounds and Total Sugar Contents of Different Open-Pollinated Beetroot Genotypes Grown Organically

The growing interest of consumers in healthy organic products has increased the attention to the organic production of beetroot. In this regard, six field experiments were conducted in 2017 and 2018 in three different locations under the specific conditions of organic agriculture, and fifteen beetroot genotypes, including one F1 hybrid as a commercial control and one breeding line, were compared regarding the content of the total dry matter, total soluble sugar, nitrate, betalain, and total phenolic compounds in order to investigate the genetic potential of new and existing open-pollinated genotypes of beetroot regarding the content of their bioactive compounds. The results of this study indicated a significant impact of genotype (p < 0.05) on all measured compounds. Furthermore, results revealed a significant influence of the interactions of location × year (p < 0.05) on the beetroot composition, and, thus, the role of environmental conditions for the formation of tested compounds. The total dry matter content (TDMC) of beetroots varied between 14.12% and 17.50%. The genotype ‘Nochowski’, which possessed the highest total soluble sugar content with 14.67 °Bx (Brix), was among the genotypes with the lowest nitrate content. On the contrary, the cylindrical-shaped genotype ‘Carillon RZ’ (Rijk Zwaan), indicated the lowest sugar content and the highest nitrate concentration. The amount of total phenolic compounds ranged between 352.46 ± 28.24 mg GAE 100 g−1 DW (milligrams of gallic acid equivalents per 100 g of dry weight) and 489.06 ± 28.24 mg GAE 100 g−1 DW for the red-colored genotypes which is correlated with the high antioxidant capacity of the investigated genotypes. Due to the specifics of the required content of bioactive compounds for various products, the selection of suitable genotypes should be aligned with the intended final utilization.

Introduction
Consumption of vegetables due to their various benefiting effects, such as the reduced risk of cardiovascular disease, cancer, and diabetes, is associated with human health promotion [1]. Due to the high amount of biologically active compounds, processing, and product consumption of beetroot (Beta vulgaris subsp. vulgaris L.), a vegetable from the Chenopodiaceae family, has gained attention in recent years [2,3].
Several studies noted beetroot as a vegetable with remarkable health-promoting phytochemicals [2,4]. Beetroot pigmentation can be categorized into two main sources; betalains composing of betanin,

Total Dry Matter Content
In accordance with the findings of this study, the TDMC of the 15 investigated beetroot genotypes varied between 14.12 ± 4.56% and 17.50 ± 4.56%. The results revealed that the genotypes Nochowski, Betina, and Cervena Kulata with 17.50 ± 4.56%, 17.12 ± 4.56%, and 16.41 ± 4.56%, respectively, contained the highest percentage of TDMC (Table 1). On the other hand, the cylindrical-shaped genotype, Carillon RZ with 14.12 ± 4.56%, Nobol with 14.66 ± 4.60%, and Akela RZ with 14.95 ± 4.56% indicated the lowest TDMC. The TDMC of 15 beetroot genotypes grown in Poland within 2009-2012 ranged between 13.5% to 15.3% [19], which were slightly lower than the range found in the current study. With respect to the outcomes of the analysis of variance (ANOVA), a significant impact of genotype (p = 0.0029) and the interactions of year × location (p < 0.0001) have been noted in the TDMC ( Table 1). The assessment of the effect of cultivation method, genotype, and year on the total dry matter of two beetroot genotypes (cv. Czerwona Kula and cv. Opolski) grown in Poland, showed a content of 15 [20]. That not only reveals the differences in the TDMC between the trial year and the genotypes but also significantly higher values in the organic cultivation. The lower TDMC of both cultivars in 2008, was due to the abundant rainfall and cooler days in the growing period compared to the year 2009 [20]. In the current study, the extreme climate condition in Germany (low precipitation level and high temperature) in the second year of the field experiment, resulted in the remarkable impact of the experimental year and location on the TDMC. Concerning the cultivation system, Brandt and Mølgaard (2001) stated that due to mineral nitrogen fertilization in conventional production systems, which increases the yield and the water quantity of the plant cells, often raw materials contain more water which leads to diluted content of the nutrients [21]. Moreover, Hallmann and Rembiałkowska (2012) claimed that the higher TDMC found in sweet bell peppers results from the difference in the metabolism of organic and conventional plants [22]. Nevertheless, Sikora et al. (2008) stated no significant influence of cultivation method on the TDMC of two beetroot cultivars Czerwona Kula and Regulski Cylindryczny [23]. Table 1. Mean values and ANOVA of results of the content of nitrate (mg kg −1 dry weight (DW)), total soluble sugar ( • Bx), total phenolic (mg gallic acid (GAE) 100 g −1 DW), and total dry matter (%) of 15 different genotypes of beetroot grown in three research stations within the trial year 2017 and 2018. Results represent the mean values ± standard error. Means followed by at least one identical letter were not significantly different from each other.

Total Soluble Sugar Content
Due to the importance of carbohydrates as one of the main sources of energy as well as the amount of sugar in the controlled diet of the diabetic patients [24], providing information on the sugar composition and content in vegetables has gained more attention. Furthermore, due to the significant influence of processing practices on different soluble sugars, a need for the information on sugar amount has gained increasing interest for the food processing industry in order to optimize the processing conditions. The sugar contained in beetroot is mainly sucrose with small amounts of glucose and fructose [11].
The total soluble sugar content of the beetroot genotypes in the current work varied between 11.19 ± 0.37 • Bx and 14.67 ± 0.37 • Bx belonged to the genotypes Carillon RZ and Nochowski, respectively, and were statistically different from each other (Table 1). Following Nochowski, the genotypes Cervena Kulata with 13.94 ± 0.37 • Bx and Betina with 13.88 ± 0.37 • Bx possessed the highest total soluble sugar content. The study of Szopińska and Gawęda (2013) stated that the total soluble sugar content of the cylindrical-shaped genotype Regulski Cylinder grown organically in Poland varied between 7.61% and 8.16% within three years [25]. In the current study, higher contents were noted for the cylindrical-shaped genotype, Carillon RZ. Moreover, the amount of the total soluble sugar of 15 beetroot genotypes grown in Poland within 2009-2012 varied between 5.18% to 8.62% [19], which were considerably lower than the range found in the current study.
The amount of sugar content can be impacted by various factors. Statistical analysis of data of the current study determined a significant influence of genotype (p = 0.0028) and interactions of year × location (p < 0.0001) on the content of total soluble sugar (Table 1). Furthermore, Bavec et al. (2010) investigated the impact of different production systems, namely conventional, integrated, organic, and biodynamic, on the sugar content of red beet. Despite the differences in the sugar content of red beet grown under different farming conditions, no statistically significate influence of farming system was determined [11]. Another influencing factor on the sugar content of beetroot is the applied nitrogen in the early stages of growth and nitrogen availability [3]. In the present study, at the trial station De Beersche Hoeve, in the first year, the field was fertilized with compost equivalent to 60 kg N ha −1 and in the second year, compost equivalent to 90 kg N ha −1 was incorporated into the soil. At the Horticulture station Heinze, in both experimental years, no fertilizer was applied to the field and at the research station Kleinhohenheim, in the first year the field was fertilized with Vinasse, equivalent to 8.3 kg N ha −1 , one month after seeding and in the second year, no additional fertilizer was used due to the N fixation by the pre-crop clover grass.
Providing information on the sugar content of each genotype is of great importance for the food industry for developing beetroot products without exceeding the maximum daily consumption and for achieving the best quality after processing. One of the critical products is prepared beetroot juice which contains approximately 62.0-92.0 g/L sugar [2]. Considering the World Health Organization (WHO) recommended daily sugar consumption of 25-50 g for an adult with normal Body Mass Index (BMI), with consumption of 300-600 mL of processed beetroot juice, the limit of daily sugar consumption can be meet [2]. Moreover, for those beetroot products in which intensive thermal treatment should be applied, considering the possibility of the occurrence of the Maillard reaction, the sugar content of the selected genotype should be taken into account.

Nitrate Content
The three predominant sources of human nitrate intake reported as water, cured meat, and vegetables [26], in which the green leafy vegetables like spinach, beets, lettuce, and radishes are the major dietary examples [27]. Heretofore, nitrate amount in the vegetables was discussed as a critical topic. That was due to the point that reduction of nitrate to nitrite leads to methemoglobinemia, which its reaction with secondary amines may sequentially form carcinogenic N-nitrosamines [28]. Latterly, with the reveal of the remarkable physiological importance of nitric oxide, the significant contribution of the dietary nitrate for the formation of the nitric oxide was disclosed [29].
Recently, the potential beneficial effects of dietary nitrate, including reducing the risk of cardiovascular diseases, gastric cancer, and hypertension [26] draw a lot of attention. Several studies noted the possible positive effects of nitrate on muscle efficiency, oxygen transport in the blood, and muscle fatigue of athletes, especially endurance athletes such as professional cyclists or marathon runners [30], therefore, consumption of beetroot as a good source of nitrate has drawn a lot of interest [31,32]. Depending on the final product and purpose of use, in order to have the suitable nitrate content, the choice of the appropriate cultivar has been highly emphasized [33,34].
The nitrate content of the investigated beetroot genotypes in the present study ranged between 4593 ± 1212 mg kg −1 DW and 10,924 ± 1211 mg kg −1 DW. Among all 15 genotypes, the three highest nitrate content were noted for Carillon RZ, Nobol, and Bona with 10,924 ± 1211 mg kg −1 DW, 9447 ± 1210 mg kg −1 DW, and 9364 ± 1221 mg kg −1 DW, respectively, which were statistically non-significantly different from each other (Table 1). On the other hand, the three genotypes, namely, Nochowski, BoRu1, and Monty RZ F1 with 4593 ± 1212 mg kg −1 DW, 4597 ± 1211 mg kg −1 DW, and 5487 ± 1209 mg kg −1 DW, respectively, showed the lowest nitrate content ( Table 1). The reported nitrate values in Table 1 were expressed on a dry weight basis. Most studies express the nitrate content on a fresh weight (FW) basis, wherefore total dry matter content should be taken into account to compare our results to the literature. The findings of the current study were in agreement with the reported mean nitrate value of beetroots from different European countries, which was 1379 mg kg −1 FW [35]. The range of nitrate content of beetroot in Thessaloniki, Greece was between 443-981 mg kg −1 FW [36]. In the study of Rubóczki et al. (2015) the nitrate content in ten different beetroot genotypes grown in Hungry determined by a spectrophotometric method ranged between around 700-850 mg kg −1 FW [37]. Kosson et al. (2011) noted the mean nitrate contents of 1338.5 mg kg −1 FW and 1737.3 mg kg −1 FW for two beetroot genotypes grown in Poland, in 2008 and 2009, respectively [20].
The level of nitrate uptake and accumulation in vegetable tissues can be influenced by three main factors: environmental factors, such as water availability and irradiance, genetic factors, and agricultural factors, such as the application of fertilizers, herbicides, and other nutrients [38]. In accordance with the statistical analysis, the nitrate content was significantly affected by genotype (p = 0.0191) and interactions of year × location (p = 0.0495) ( Table 1). This is in agreement with the findings of Kosson et al. (2011) who noted the significant impact of variety and year on the accumulation of nitrate in two different beetroot genotypes [20]. Furthermore, the effect of cultivation method (organic and conventional) was investigated in the study of Kosson et al. (2011) and 33% lower nitrate accumulation in the beetroot grown organically was reported [20]. In the current work, all beetroots were grown under organic conditions. Moreover, Vasconcellos et al. (2016) assessed the differences in the nitrate content of four beetroot products, including juice, powder, chips, and cooked beetroot. It was revealed that beetroot juice with 12,252.90 mg kg −1 DW had significantly higher nitrate concentration compared to all other products, which indicated the range of 1649.66-2031.20 mg kg −1 DW [5]. Therefore, it can be concluded that the amount of nitrate in beetroot products can be affected by processing. According to the previous studies, the variability in the nitrate content may occur among cultivars of the same species [39,40] genotypes and plant tissues [33,[41][42][43]. In our study, each analyzed sample was a homogenized mixture of three randomly selected beetroot plants of each genotype, which were replicated three times in the field. Consequently, the values demonstrated in Table 1, represent the mean nitrate content of all three replications of each genotype cultivated in three different locations within two years to evaluate the genetic potential in different environmental conditions. Thus, due to the combination of various factors affecting the nitrate content, to have a better evaluation of different beetroot genotypes, a closer look at a larger number of plants per genotype might be appropriate.

Betalain Content
Color is one of the important attributes indicating the aesthetic and quality and consequently the acceptance of food [44]. Recently, due to the point that consumers prefer natural pigments compared to the synthetic ones, focus on the use of carotenoids, anthocyanins, and chlorophylls as examples of plant-based colorants [45] have been increased. Due to the limited edible sources of betalains, these pigments have been less commonly used for food coloring than other natural pigments [46]. Betalains are water-soluble, nitrogenous pigments [46] which are divided into two main structural groups, red-violet betacyanins and yellow-orange betaxanthins [47]. Previous studies reported the high antioxidant capacity of betalains [48]. Furthermore, the other important roles of betalains in human health are their anti-cancer, antilipidemic, and antimicrobial activities [47].
In the present work, two main subgroups of betalain as representatives of the sample pigment content were quantified. Regarding betacyanin content, the studied red-colored genotypes ranged from 5.35 ± 0.49 mg g −1 DW to 7.89 ± 0.72 mg g −1 DW for the genotypes Cervena Kulata and Monty RZ F1, respectively ( Figure 1). The white-and yellow-colored genotypes possessed the lowest betacyanin content with 0.08 ± 0.01 mg g −1 DW and 1.13 ± 0.14 mg g −1 DW, respectively, which were statistically different from each other (Figure 1). Following the F1 hybrid, Monty RZ, two genotypes Nochowski with 7.75 ± 0.70 mg g −1 DW and Ronjana with 7.39 ± 0.67 mg g −1 DW showed the highest betacyanin content. The betaxanthin content of the red-colored genotypes varied between 3.81 ± 0.32 mg g −1 DW and 5.51 ± 0.46 mg g −1 DW. The three highest betaxanthin content belonged to the genotypes Nochowski, Monty RZ F1, and Ronjana with 5.51 ± 0.46 mg g −1 DW, 5.49 ± 0.46 mg g −1 DW, and 5.32 ± 0.45 mg g −1 DW, respectively ( Figure 1). The lowest betaxanthin contents were noted as 0.04 ± 0.01 mg g −1 DW and 0.09 ± 0.01 mg g −1 DW, belonging to the yellow-colored genotype, Burpees Golden, and the white-colored one, Sniezna Kula, respectively ( Figure 1).  (Figure 1). The lowest betaxanthin contents were noted as 0.04 ± 0.01 mg g −1 DW and 0.09 ± 0.01 mg g −1 DW, belonging to the yellow-colored genotype, Burpees Golden, and the white-colored one, Sniezna Kula, respectively (Figure 1).  The results of ANOVA showed a significant effect of genotype (p < 0.0001) and interactions of year × location (p = 0.0277) on the betacyanin content. The effect of genotype (p < 0.0001) and interactions of year × location (p = 0.0069) was noted to be significant on the content of betaxanthin. Means and p-values are available in Table S1 in the supplementary material. The significant influence of genotype on betacyanin and betaxanthin content has been reported by Sawicki et al. (2016) [49]. Felczynski and Elkner (2008) stated the significant impact of weather conditions and consequently year of the experiment on the composition of the betalains in beetroot. Their study investigated the ratio of betanin to vulgaxanthin, which are respectively the main red and yellow pigments in beetroot, in two red beetroot cultivars, Chrobry and Nochowski, within two years and considerable differences have been noted [39], which is in agreement with the outcomes of the present study. Kujala et al. (2000) stated that there is a different betalain distribution among the different parts of red beetroot [50]. The study reported higher betacyanin content in outer parts of red beetroot (cv. Little Ball), increasing in the order flesh, crown, and peel. Slatnar et al. (2015) studied the betacyanin and betaxanthin content in the peel and flesh of three different beetroot genotypes, including two red-colored and one yellow-colored genotype, which were grown in Slovenia, using the HPLC method. Regarding the two red-colored genotypes, the betacyanin content of 20.93 mg g −1 DW and 11.83 mg g −1 DW in the peel and 5.25 mg g −1 DW and 4.13 mg g −1 DW in the flesh of Pablo and Tanus, respectively, was measured [45]. The beetroot samples used in the current work mainly consisted of flesh with a small portion of the peel. Therefore, the slightly higher betacyanin and betaxanthin contents reported in this study ( Figure 2) might be due to the different plant parts. Moreover, the betanin content of four red-colored beetroot genotypes measure by Kujala et al. (2002) ranged between 3.8-7.6 mg g −1 DW in the peel and 2.9-5.2 mg g −1 DW in the flesh. That for the vulgaxanthin content was reported as 1.4-4.3 mg g −1 DW and 1.5-4.0 mg g −1 DW in the peel and flesh, respectively [10]. The results of this study indicated a nearly similar range for the betacyanin content but higher betaxanthin values. Inconsistent to the previous findings, the study of Gasztonyi et al. (2001) presented a significantly lower range of betanin content of five red-colored beetroot genotypes with 0.41-0.50 g kg −1 (equivalent to mg g −1 in the current study) as well as lower range of vulgaxanthin I with 0.32-0.42 g kg −1 [51].
With respect to the betacyanin content of the yellow beetroot genotype, Boldor, Slatnar et al. (2015) stated 0.61 mg g −1 DW in the peel and 0.13 mg g −1 DW in the flesh. Likewise, for the betaxanthin content, the values of 4.71 mg g −1 DW and 0.22 mg g −1 DW in the peel and flesh, respectively, were reported [45]. The findings of our work regarding the yellow-colored genotype, Burpees Golden, indicated higher betacyanin content and remarkably lower betaxanthin value. Furthermore, the outcomes of Lee et al. (2014) demonstrated the betacyanin content of 0.114 mg g −1 FW and betaxanthin content of 0.187 mg g −1 FW for the mixed sample of peel and flesh of the yellow-colored beetroot, Burpees Golden, grown in Texas (the USA) [52].
Providing information on the betalain content of each beetroot genotype, due to the effect on the antioxidant activity as well as the color intensity, is important for the food industry in order to choose the suitable genotype for the final product such as for the use of beetroot in functional processed food or the use as the food colorant.

Total Phenolic Content
Phenolic compounds are plants' secondary metabolites, which affect both sensorial attributes, such as flavor, taste and color, and functional properties, like antioxidant activity, of plant products [53,54]. Furthermore, phenolic compounds also contribute to plants' growth, pigmentation, reproduction [55], and defense mechanisms under different stress conditions. Due to the free radicals scavenging activity, phenolic compounds demonstrate a high antioxidant effect. Antioxidant activity is associated with the prevention of cardiovascular diseases and cancer [56,57].
The total phenolic content ranged from 352.46 ± 28.24 mg GAE 100 g −1 DW to 489.06 ± 28.24 mg GAE 100 g −1 DW for the red-colored genotypes. The F1 hybrid genotype, Monty RZ, showed the highest total phenolic content followed by Cervena Kulata and Nochowski (Table 1). On the other hand, the genotypes Carillon RZ, BoRu1, and Betina with 352.46 ± 28.24 mg GAE 100 g −1 DW, 382.73 ± 28.24 mg GAE 100 g −1 DW, and 393.94 ± 28.24 mg GAE 100 g −1 DW, respectively, indicated the lowest total phenolic content among the studied red-colored genotypes. The yellow-colored genotype, Burpees Golden, and the white-colored genotype, Sniezna Kula, with 172.89 ± 28.78 mg GAE 100 g −1 DW and 216.09 ± 29.87 mg GAE 100 g −1 DW, respectively, demonstrated significantly lower total phenolic content compared to the red genotypes (Table 1). In the study of Rubóczki et al. (2015), the content of total polyphenols in ten different beetroot genotypes grown in Hungary was determined using the Folin-Ciocalteu method and varied between 45.47 mg GAE/100 g and 83.37 mg GAE/100 g [37]. Values were considerably lower than the values measured in our study. Furthermore, Ninfali and Angelino (2013) reported total phenols of 1.77 mg GAE g −1 DW (equivalent to 177 mg GAE 100 g −1 DW) [58], which was lower than the values found in the current study. Kovarovič et al. (2017) investigated the total phenolic compounds of four beetroot varieties in the Czech Republic and noted the range of 368.75 ± 5.14 mg kg −1 (equivalent to 36.87 mg 100 g −1 DW) to 887.75 ± 7.73 mg kg −1 (equivalent to 88.77 mg 100 g −1 DW) belonging to the variety Chioggia (white-colored with red strips) and Cylindra, respectively [59]. Kavalcova' et al. (2015) stated a total polyphenols' range of 820.10 mg kg −1 to 1280.56 mg kg −1 (equivalent to 82.01-128.05 mg 100 g −1 DW) [60]. The investigated genotypes in our study exhibited promising results regarding the amount of total phenolic compounds.
The outcomes of ANOVA indicated a significant impact of genotype (p = 0.0002) and the interactions of year × location (p = 0.0114) ( Table 1). The total phenol content in fresh fruits and vegetables can be affected by different biotic and abiotic factors. Genotype, environmental conditions such as climate and soil, and plant maturity and ontogeny play the most important role in the quantity of phenolic compounds [55]. It was reported that the level of chemical fertilizers, which are used in conventional agricultural practices, can lead to the disturbance in the natural production of phenolic compounds [61]. In this regard, Carrillo et al. (2019) investigated the impact of the production system on the content of total phenols in six different beetroot genotypes. It was reported that the total polyphenol content of the organic beetroots was significantly higher than the conventional ones, although the difference depended on the cultivar [62]. Nevertheless, the study of Straus et al. (2012) demonstrated a higher total phenolic content in beetroots grown under organic condition compared to the conventional ones, however, the difference was not significant [63].
Similar to the betalain content in beetroot, several studies noted a higher total phenolic content in the peel than in the flesh [50,64]. Based on the point that the analyzed samples in the current work mainly consisted of flesh with a small proportion of peel, the differences between the values reported in other studies and the outcomes of this study might be due to the variation in the plant part. Additionally, differences in the measurement methods [65] (e.g., HPLC, capillary zone electrophoresis, and Folin-Ciocalteu), as well as the extraction of the phenolic compounds techniques [66][67][68] in various studies, may lead to a variation in the reported values.

Plant Materials and Sample Preparation
The analyzed beetroot samples were obtained from six field experiments, which were carried out in two years, 2017 and 2018, at three different locations. Locations consisted of two on-farm breeding locations of Kultursaat e.V.: De Beersche Hoeve (Oostelbeers, Netherlands), Horticulture station Heinze (Bingenheim, Hessia, Germany), and at the research station for organic farming Kleinhohenheim (University of Hohenheim, Stuttgart, Baden-Wuerttemberg, Germany). All beetroots were grown organically. In 2017, on each on-farm breeding location 30, and in Kleinhohenheim, 40 genotypes were cultivated in three field replicates. In 2018, 16 genotypes were investigated on each on-farm breeding location while 36 genotypes were assessed in Kleinhohenheim. Data from all genotypes were analyzed together. However, the presented results were limited to the 15 genotypes, which occurred in all location-by-year combinations. The 15 investigated genotypes can be listed as 13 red-colored genotypes, Akela RZ, Betina, Bona, Bordo, BoRu1, Carillon RZ, Cervena Kulata, Detroit 3, Jawor, Monty RZ F1, Nobol, Nochowski, Ronjana, one white genotype, Sniezna Kula, and a yellow genotype, Burpees Golden. Figure 2 illustrates four beetroot genotypes representing different beet shapes and colors, which were investigated in this study. The detailed information on the seeds' origin, experimental design, and the locations can be found in Yasaminshirazi et al. (2020) [69].
Samples were prepared from three randomly selected beetroots per plot, which fulfilled the criteria for marketability (beet diameter between 5 and 13 cm and without any deformation and notable damages). Beetroots were washed directly after the harvest. After cutting out the root tail and the leaves-growth-base, a sectional cut of the beet including the peel were chopped into small pieces and thoroughly mixed to have a homogenous sample. The chopped pieces were collected in a closed plastic flask and immediately were frozen using liquid nitrogen. Afterward, the samples were lyophilized using the Dieter Piatkowski-Forschungsgeraete freeze-dryer (Munich, Germany). The dried samples were ground by a laboratory knife mill (Retsch GM 200, Haan, Germany) until they reached a fine powder texture. The powder was collected and remained in a closed small plastic flask in a cool dark box until the day the chemical analyses were carried out.
Molecules 2020, 25, x FOR PEER REVIEW 9 of 15 one white genotype, Sniezna Kula, and a yellow genotype, Burpees Golden. Figure 2 illustrates four beetroot genotypes representing different beet shapes and colors, which were investigated in this study. The detailed information on the seeds' origin, experimental design, and the locations can be found in Yasaminshirazi et al. (2020) [69]. Samples were prepared from three randomly selected beetroots per plot, which fulfilled the criteria for marketability (beet diameter between 5 and 13 cm and without any deformation and notable damages). Beetroots were washed directly after the harvest. After cutting out the root tail and the leaves-growth-base, a sectional cut of the beet including the peel were chopped into small pieces and thoroughly mixed to have a homogenous sample. The chopped pieces were collected in a closed plastic flask and immediately were frozen using liquid nitrogen. Afterward, the samples were lyophilized using the Dieter Piatkowski-Forschungsgeraete freeze-dryer (Munich, Germany). The dried samples were ground by a laboratory knife mill (Retsch GM 200, Haan, Germany) until they reached a fine powder texture. The powder was collected and remained in a closed small plastic flask in a cool dark box until the day the chemical analyses were carried out.

Total Dry Matter Content
The weight of chopped beetroot samples (i) was measured before and after freeze-drying and the total dry matter content (TDMC) was calculated according to Equation (1):

Total Dry Matter Content
The weight of chopped beetroot samples (i) was measured before and after freeze-drying and the total dry matter content (TDMC) was calculated according to Equation (1): = weight a f ter drying i weight be f ore drying i × 100 (1)

Total Soluble Sugar Content
The determination of total soluble sugar content was carried out using a digital handheld refractometer (Kruess, Germany). One to two drops of freshly pressed beetroot were used to determine the degree of Brix, which is equivalent to the percentage of total soluble sugar content. Each sample was measured in duplicate and a mean value was calculated directly.

Quantitation of Nitrate Content
Approximately 0.5 g of each dried beetroot sample was weighed and added in a 100 mL volumetric flask and was filled up with distilled water until the final volume adjusted to 100 mL and was shaken shortly, manually. The volumetric flask was placed in an ultrasonic water bath at 80 • C for 10 min. After the flask was cooled down, the supernatant was filtered using filter paper. The nitrate content quantitation was conducted by flow injection analysis method (FIA) [27] using FIASTAR 5000 (FOSS Analytical AB, Sweden).

Extraction of Betalains
Approximately 0.04 g of each dried sample was weighed (Precisa 125 A, Dietikon, Switzerland) and added to 30 mL of 50% (v/v) ethanol in a 50 mL falcon tube. The mixture was shaken for two hours with 100 rounds per minute (rpm). Afterward, the obtained extracts were centrifuged (Centrifuge 5810 R, Eppendorf AG, Hamburg, Germany) with 4000 rpm and at 20 • C for 10 min.

Extraction of Total Phenolic Compounds
Approximately 0.5 g of each dried beetroot sample was weighed and collected in a 15 mL falcon tube where 10 mL of methanol was added. The mixture was shaken for 30 min and afterward centrifuged (Centrifuge 5810 R, Eppendorf AG, Hamburg, Germany) at 4000 rpm at 20 • C for 20 min in order to separate the solid phase from the supernatant. These extracts were utilized for the measurement of the total phenolic content.

Total Phenolic Content Quantification
The total phenolic content measurement was carried out following the methodology of Folin-Ciocalteu [71]. Summarily, 0.6 mL of the extracted sample was added to 60 mL of distilled water in a 100 mL volumetric flask. Then, 5 mL of Folin-Ciocalteu reagent was added to the flask. After two to six minutes, 25 mL of sodium carbonate solution 15% was added to the mixture, and the final volume was adjusted to 100 mL with distilled water. After two hours of incubation at room temperature, absorbance at 760 nm was determined by a UV/Visible Spectrophotometer (Ultrospec 3100 Pro, Amersham Bioscience, Buckinghamshire, UK). To draw a calibration curve, six standard solutions consisting of 0.03 g L −1 , 0.12 g L −1 , 0.24 g L −1 , 0.48 g L −1 , 0.9 g L −1 , and 1.5 g L −1 gallic acid in distilled water were used. Lastly, the total phenolic content was stated as mg GAE 100 g −1 DW (milligrams of gallic acid equivalents per 100 g of dry weight).

Statistical Analysis
According to the design of the experiments, the data were analyzed by the linear mixed model used in Yasaminshirazi et al. (2020) [69] on agronomic traits from the same six experiments: where a j and l k are the fixed effects of the j-th year and the k-th location. b ljk , r mjk , and c njk are the random effect of the l-th block, m-th row and n-th column within a year-by-location combination. Note that block effects as well as row and column effects were only fitted if blocks existed and a row-column design was used, respectively. Dummy variables were used to block out these effects for all other experiments but were not mentioned in (2) to simplify the description. τ i is the fixed effect of the i-th genotype. (al) jk is the fixed interaction effect of the j-th year and the k-th location.
(τa) ij , (τl) ik , and (τal) ijk are the random interaction effects between the corresponding main effects. e ijklmn is the error of y ijklmn with year-by-location specific error variance. For the trait total phenolic content, a group-by-location-specific error variance was assumed, where a group refers to beetroots of the same color (red, yellow, white). For betacyanin and betaxanthin, a group-by-year-specific error variance was found to fit well. The Akaike Information Criterion (AIC) was used to select the best error variance structure [72]. The assumptions of normality of residuals and homogeneity of residual variances (in addition to the heterogeneity specified in the model) were checked graphically. For the compounds betacyanin and betaxanthin, a logarithmic transformation of the data was used before analysis. Estimated means were back-transformed afterward for presentation purposes only. These means represent medians on the original scale. Standard errors were back-transformed using the delta method. Depending on the significance of fixed effects, mean comparisons were done for the highest significant interaction term of all factors using Fisher´s Least Significant Difference (LSD) test [73]. Means (or medians in case of back-transformed values) and their (approximate) standard error were presented. A letter display was created using the statistical software SAS version 9.4 (SAS Institute, Cary, North Carolina, United States) macro %mult [73] to present results from multiple comparisons.

Conclusions
Significant differences were found in the content of selected compounds between the assessed open-pollinated beetroot genotypes. This outcome is essential for the selection of genotypes and is of great importance for farmers for having new possibilities for cultivation in organic farming, for the industry for the production and development of beetroot products with desirable characteristics, and for meeting consumers' expectations of health-promoting food products. Furthermore, with the significant impact of the interactions of year × location on all tested compounds, it can be concluded that the abiotic factors influence the biochemical composition of beetroot. Out of the 15 tested genotypes the open-pollinated genotype Nochowski with comparable total phenolic compounds and betacyanin content to F1 hybrid genotype, Monty F1 RZ, indicated a high antioxidant capacity, which makes it a potential genotype for value-added food products. Moreover, this genotype may serve as an option for juice production due to its high sugar and low nitrate content. According to the importance of taste on customers' acceptability, further studies in association with the sensory quality of the genotypes as well as other flavor-relevant components, such as geosmin, are required.


extfieldsofstudy: Biology Medicine

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Intact gastro-intestinal tract removal from pig carcasses in a novel Meat Factory Cell approach

Conventional automated slaughter lines for pigs are organised as disassembly lines with many specialised machines. High costs and capacities make them relevant only for large scale meat production. The ambition with the novel Meat Factory Cell (MFC) concept is to provide the meat industry with a robust and flexible automation platform that is also relevant for smaller scale production. The MFC process deviates radically from conventional processing of pig carcasses after singeing. In MFC, the limbs are removed first. Then the dorsal muscles along the spinal axis from tail to head are removed with the column and rind in one meat cut, followed by removal of the viscera. Finally, the cut ribs and belly are removed. Such approaches to automation in pig abattoirs and cutting plants are highly needed in smaller scale production, and they should produce meat and offal as hygienically as conventional factories. This case study reports the evisceration of 37 pigs in 9 trials performed in 2019. Several approaches were tested with a prototype carcass holding unit. Evisceration could be undertaken without the need to cut through the gastrointestinal tract from tongue to rectum, reducing the probability of accidental faecal contamination of pork carcasses from the gut content. The Meat Factory Cell procedure is an advance towards automated evisceration of pig carcasses which is both simple and hygienic. The traditional separation of internal organs into a pluck set and a set of stomach and bowels was more prone to leakages.

Findings
The Meat Factory Cell (MFC) approach for pig slaughter and primary cutting has recently been suggested and described [1,2]. MFC applies three principal changes to conventional meat production and processing: 1. Work partly organised in cell stations instead of lines. 2. Combine and merge elements of slaughter and meat primal cutting.
3. "Disassemble" the carcass from outside-in, without removal of internal organs before removal of most primary cuts.
Alternative processes should at least be as hygienic as traditional slaughter and cutting. Improved hygiene is expected from the MFC concept as the meaty limbs, neck and loin are removed first. These primary cuts are not subject to faecal contamination from intestinal content. The MFC process results in seven cuts; Four limbs, the saddle including head and tail, the viscera including pluck, stomach and intestines, and a cut comprised of ribs and belly, i.e. the belly-cut. Also, from a meat inspection point of view, detection of lesions and abnormalities should at least be as sensitive as in traditional procedures [2].

Open Access
Acta Veterinaria Scandinavica Avoiding bacterial contamination of carcasses and meat is the most important hygienic challenge in meat industry. Evisceration carries a high probability of carcass contamination due to knife cuts and perforations resulting in leakage of the intestinal content. Good Hygiene Practise for evisceration includes ensuring that the probability of perforating the viscera, alimentary tract, uterus, urinary bladder, and gall bladder is minimised during separation cuts [3]. In addition, and regardless of accidental knife perforations, the two ends of the gastrointestinal tract are potential sources of carcass contamination [3]. In conventional slaughter lines, the pig carcasses are suspended by the hind legs. This allows use "bagging", a technique in which a plastic bag is used to seal the rectum after circum-anal incision [4]. The sealed rectum is then pulled through the transected Os pubis and removed together with the intestines.
The oral cavity, with the tongue and palatine tonsils, is known to harbour a high bacterial load, including zoonotic bacteria [5]. It has been considered optimal to avoid cutting into tonsils while removal of the pluck, and "stunning, bleeding, skinning, evisceration and other dressing must be carried out without undue delay and in a manner that avoids contaminating the meat. In particular, the trachea and oesophagus must remain intact during bleeding" [6]. However, recently an alternative approach leaving the tongue with tonsils, pharynx, larynx and part of trachea and oesophagus intact on the head produced the lowest contamination levels on pig carcasses [7].
According to the Regulation EC No 853/2004, Annex III, Section I, Chapter IV, point 7 c, "measures must be taken to prevent the spillage of digestive tract content during and after evisceration" [6]. It was hypothesised that the MFC approach could possibly allow the alimentary tract to be removed intact [2]. If successful, this approach would significantly improve hygiene and reduce zoonotic risks associated with pork. We are not aware that removal of the entire gastrointestinal tract has been reported before and so, probably for the first time, fulfilling this intention of the EU legislation.
The aim of this study was to test in practice and describe evisceration of gastro-intestinal tract at slaughter of pigs in the MFC approach in a manner feasible for automation. Figure 1 shows the schematic cutting pattern applied in the trials. Five pigs where processed in three iterative trials conducted at a workshop on a farm in Norway from April to June 2019. After three trials (5 pigs), it was concluded how the carcass should be fixed, tools of choice, cutting trajectories and a more detailed procedure. Then, 32 crossbred white pigs, castrates and females, with approximately 110 kg live weight were slaughtered over a 6-week period in an abattoir, in total 37 pigs.
The first five pigs were stunned with a captive bolt pistol and bled indoors at the piggery, transported with tractor to the scalding bath, scalded in a hot water bath (approx. 65 °C), scraped manually and singed with a gas burner.
The pigs were bagged and brought to the MFC after singeing and mounted to a prototype Carcass Holding Unit (CHU) (Tronrud Engineering AS, Hønefoss, Norway) designed for the MFC approach. The system was designed to accept a carcass from the hanging position (e.g. vertically from a rail) and present horizontally for cutting. The carcass could also be rotated 180 degreescutting began with the belly-cut facing upwards and After the pig carcass has been scalded and singed, the primary cutting is undertaken when the carcass is still warm. Then, the forelimbs and hindlimbs are removed and brought out of the cell to a rack. Then the body, (truncus), is turned 180° around its longitudinal axis. In front of the coxa wing, the soft belly wall is punctured and a cut moving cranially is made laterally to the loin (M. longissimus dorsi) (blue line). The saddle (hip bone, loins, neck and head) is then lifted up and the viscera loosened from mesentery ventral to the spine (columna) proceeded to belly-cut facing downwards once all limbs had been removed. The carcass was fixed at the head (snout and eye sockets) as well as at the rear. A serrated clamp fixed Os sacrum after removal of the hind limbs. Vacuum suction cups (n = 5) held the back of the pig to enable initial fixation, stretching during rib sawing, and lifting and removal of the back prior to internal organ removal (Fig. 2).
The slaughter and cutting process was developed iteratively in collaboration with veterinarians, butchers and engineers. Limbs were removed with a knife, and the entire back from hip to neck lifted away as shown in Fig. 2. Then the thoracic and abdominal viscera were exposed, resting on the ribs and sides as shown in Fig. 2. The butcher freed the trachea and oesophagus towards the larynx. With two lateral cuts along the mandibles, avoiding the tonsils, and a transverse cut through the soft palate it was possible to remove the tongue as well as the pharynx with tonsils. While gripping firmly around trachea and oesophagus beneath larynx, the organs where pulled backwards.
Three alternative principal evisceration processes for the pluck were identified: 1. Removal of tongue, larynx, oesophagus, trachea, lungs and heart in one piece. This necessitates a cut through oesophagus close to the diaphragm. 2. Similar to 1, but without cutting the oesophagus.
Instead, the diaphragm was loosened with a knife at the attachment to the thoracic and abdominal wall. The viscera were continuously pulled in the aboral direction, loosening mesenteries and normal adher-ences with a knife until they detached from the caudal end of the belly-cut. The easiest operation was to include the peritoneum and underlying fats (flare fat) in the removed viscera. As a result, the entire gastrointestinal tract and other viscera (tongue, pharynx, tonsils, oesophagus, stomach, liver, gall bladder, mesenteries, flare fat, urinary bladder, genitalia and intestines from duodenum to anus) were removed in one piece. Heart, lungs and spleen followed the set, while the kidneys could either follow or be left on the back to ease meat inspection. The entire set of viscera is shown in Fig. 3. 3. Similar to 2, but a conventional set of plucks was produced by transecting oesophagus at the entrance to the stomach. The resulting pluck consisted of tongue, trachea, lungs, heart, diaphragm and liver. The stomach and intestines then glided off or could be physically torn off including the flare fat.  Figure 2 shows that trachea and oesophagus are being openly exposed. The recently described alternative pluck removal [7] was not explored but would probably further simplify robotic operations within the cell.
Our study reports the methodology and experiences with alternative evisceration methods applied in MFC pilots. Most interestingly, the alternative procedure (2) allowed the butcher to remove the gastrointestinal tract intact in one piece. In fact, the presentation of the carcass made it a relatively easy technique for the butcher. The traditional separation of internal organs into a pluck set and a set of stomach and bowels was more cumbersome with higher probability of ruptures and leakages.
From a veterinary hygienic perspective, we have not so far identified disadvantages with the MFC approach. The biggest challenge is that MFC does not fit with the wording of regulatory paragraphs, e.g. that the viscera should be removed as soon as possible [6]. In MFC, evisceration is almost the last operation, but the whole process is undertaken within 8-12 min in the present demo set-up.
Coincidentally, two pigs presented pathological changes. One had an umbilical hernia filled with greyish debris and turbid fluid. The orifice to the abdominal cavity was practically closed by fat tissues. The flare fat was easily removed, and local condemnation of affected tissue was hygienically undertaken. The second presented chronic fibrous adhesive peritonitis between liver, mesentery and stomach. The individual was also heavily infested with Ascaris suum. Again, the evisceration appeared simpler than if this carcass was presented on a conventional line. The evisceration could also be undertaken hygienically as the whole set could be removed before further examination.
The research group strives to provide the meat industry with a robust, flexible and scalable cognitive robotic platform [8]. The removal of an entire set of internal organs appeared feasible for an automated evisceration procedure because the robot may need only one gripping point (trachea) and one relatively simple operation, compared to delicate identification, gripping and removal of the pluck or specific internal organs.
MFC is more robust against downtime because an automated conventional line stops if a machine in the line is out of order. MFC is more flexible because the processing can adjust to varying sizes of animals without reducing the speed in neighbouring cells, or development and maintenance can be undertaken in one cell without interfering with the others. A factory can operate an optimal number of cells depending on day to day variation. Working from outside in, makes it possible to apply cheaper cognitive elements (sensors, cameras and artificial intelligence), e.g. 3D cameras to control movements and optimise cutting trajectories by artificial intelligence.
Alternatively, conventional automated lines apply many and expensive specialised machines with low flexibility, e.g. Danish Crown's plant in Horsens, Denmark [9]. Another example of an automation solution in cold meat cutting depends on control information obtained from expensive x-ray systems that produce a static picture from one point in time before processing, not able to adapt to the configural changes when a carcass is processed warm [10].
However, from a meat technological perspective there are several pro and cons. The hams, with the present cutting trajectories, are leaving the hip bone attached to the spine and back part, resulting in a cut that, for example, deviates from the Spanish dry-cured ham raw material. On the positive side, meat producers could customise the cooling regimes for specific cuts and improved water binding effects of warm pre-salted trimmings.
A disadvantage was that the set of internal organs needs to be handled afterwards to separate parts and organs to present them for meat inspection. This was not studied as customised equipment needs to be constructed for these operations. It could also be a challenge to rip off the flare fat in an entire piece.
The two pigs with lesions became examples where the evisceration in case of pathology could be performed more hygienically with the MFC approach with reduced probability of accidental contamination of the carcass from ruptures.
In conclusion, Meat Factory Cell evisceration can be undertaken without the need to cut through the gastrointestinal tract. It is a novel procedure reducing the probability of accidental faecal contamination from gut content of pork carcasses, and we also think it will reduce the complexity of the evisceration procedure and therefore be suitable for automated evisceration.


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Physiological features of aggregation of the main formed elements of blood in calves at the beginning of early ontogenesis

The need to intensify milk production urgently dictates the need to continue research on the physiology of cattle of any age. It is customary to attach great importance to blood, consisting of uniform elements and plasma, constantly circulating through the vessels. It provides gas exchange, metabolism and the delivery of hormones and bioregulators in their tissues. The success of hemocirculation strongly determines the completeness of the realization of the genetic growth potential and productivity of the animal and is closely related to the activity of aggregation of blood cells. Purpose: to find out the activity of aggregation of the main formed elements of the blood in newborn calves. The work was carried out on 32 newborn calves of black-motley breed, born of healthy cows after 2–3 pregnancies. The calves were examined on 1–2, 3–4, 5–6, 7–8 and 9–10 days of life. In the work, hematological and statistical research methods are applied. For newborns in calves, a tendency towards increased aggregation activity of red blood cells was revealed. This was combined with a low platelet aggregation, which tended to increase. The low aggregation of neutrophils in these calves also gradually increased. In newborn calves of optimal physiological status, there is a tendency to increase the aggregation of the main formed elements of the blood, which is a response to environmental influences.

Introduction
The need for intensification of milk production urgently dictates the need to continue research on the physiology of cattle of any age [1,2]. It is customary to attach great importance to blood, consisting of uniform elements and plasma [3], which constantly circulates through the vessels. It provides gas exchange, metabolism and the delivery of hormones and bioregulators in their tissue [4]. The success of hemocirculation strongly determines the completeness of the realization of the genetic growth potential and productivity of an animal [5,6] and is closely related to the activity of aggregation of blood cells [7].
Due to the great social significance of the onset of thrombosis, the aggregation of blood cells [8] has been studied in detail in humans [9]. As a result of these works, mechanisms of aggregation processes important for physiology, their age dynamics, and their disturbances in conditions of pathology were elucidated. It has been established that excessive aggregation of red blood cells, platelets, and white blood cells disrupts metabolic processes and weakens the body [10]. It was possible to establish the possibility of influencing the aggregation processes in the blood. In the presence of cardiovascular pathology, it was possible to show the possibility of obtaining a balance between them due to various effects [11,12].
At present, it becomes clear that the aggregation properties of blood cells strongly determine the rheology of blood and, consequently, the optimum microcirculation in animal tissues during ontogenesis [13]. They are very significant for the growth processes and the maximum development of the productive properties of animals [14].
Moreover, despite the great physiological significance of the aggregation of blood cells for life support processes in all productive animals, such studies have not been conducted in cattle. Their level at the beginning of early ontogenesis, in the neonatal phase, has not yet been evaluated.
To close the gap in physiological knowledge, it was necessary to evaluate the aggregation parameters in newborn calves. These data can enrich physiology and practice, since obtaining knowledge of the norm is necessary for judging the severity of aggregation disorders in newborn calves with any diseases and their dynamics in the case of applying options for correcting the state of animals.
The goal of the work is to find out the activity of aggregation of the main formed elements of blood in newborn calves.

Materials and methods
The study was conducted in strict accordance with the ethical principles established by the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes (adopted in Strasbourg on March 18, 1986 and confirmed in Strasbourg on June 15, 2006).
The work was carried out on 32 black-motley calves, which were obtained from healthy cows after 2-3 pregnancies. The animals were taken into the study for 1-2 days of life. They were examined five timeson 1-2, 3-4, 5-6, 7-8 and 9-10 days of life.
In the plasma of calves, thromboxane B2 and 6-ketoprostaglandin F1α levels were determined during enzyme-linked immunosorbent assay using kits manufactured by Enzo Life science (USA).
The activity of erythrocyte aggregation was determined using a light microscope in the Goryaev's chamber by the number of red blood cell aggregates, the number of red blood cells aggregated and not entered into aggregation.
Neutrophil aggregation was evaluated on a photoelectrocolorimeter with inductors: wheat germ lectin 32 μg/ml, concanavalin A 32 μg/ml and phytohemagglutinin 32 μg/ml. Statistical processing of the results was carried out by t-student test.

Results
Initially, the normal number of arachidonic acid metabolites in the blood of calves did not have significant dynamics, being unchanged until the end of the observation (by 9-10 days of life, thromboxane B2 was 104.0±0.09 pc/ml, 6-keto-prostaglandin F1α 74.5±0.13 pc/ml). Note: the reliability of the dynamics of the indicators taken is not received.
During the observation period in calves was found a tendency to increase spontaneous aggregation of erythrocytes, as indicated by the tendency to increase the total number of erythrocytes in the unit (4.7 %), the increase in the quantity of erythrocyte aggregates (2.5 %) and slight downward trend in the number of available red blood cells (2.4 %) (table 1).
All calves during the observation period found a trend to increased platelet aggregation in response to all tested inducers (table 1).
During the phase from the observed neonatal calves revealed a tendency to increase aggregation of neutrophils (table 1) in relation to lectin, concanavalin A and phytohemagglutinin (table 1).

Discussion
Cattle are currently an important source of milk and meat. For this reason, a detailed study of its physiology has been conducted for a long time in order to clarify its biological potential [15]. As a result of systematic research, it was possible to collect a large amount of data on various aspects of the age-related physiology of this species of productive animals. However, the information available cannot be considered complete. There remains a great need for new knowledge and clarification of the collected information on the functioning of the body systems of this species of productive animals at different ages. Closing the gaps in existing physiological knowledge should help in the development of effective approaches to ensure functional optimum in cattle throughout ontogenesis, which will ensure maximum productivity [16].
The normal course of ontogenesis of the organism of productive animals is always ensured by the continuous adaptation of the mechanisms of maintaining homeostasis in it to the action of any environmental factors. Fully based on the genetic program, all biological processes in the ontogenesis of cattle experience age-related dynamics, allowing animals to adapt to living conditions that can, at times, disrupt the functioning of the body.
Of great importance in maintaining homeostasis is blood and its mechanisms of rheology and hemostasis, which largely limit the level of perfusion of animal tissues, and, therefore, the course of many life processes in them. Gradually, there comes an understanding that the ontogenetic dynamics of the rheological parameters of blood, which largely depend on the aggregation of blood cells, should be considered as an important component of the regulation of anabolism in animal tissues [17].
In this regard, research on the physiology of blood in newborn calves, which have begun their independent existence outside the maternal organism, is very important. In the work performed, the observed calves have noted regular changes in the hematological parameters taken into account. The revealed facts were confirmed by the results of earlier studies. It is known that the intensity of thromboxane formation and the level of thromboxane in plasma has a significant effect on the ability to aggregate red blood cells, platelets, and white blood cells.
So, during the neonatal period in calves, a slight tendency was found to increase the formation of thromboxane A2 and enhance the synthesis of prostacyclin [18]. This can explain the changes observed in the observed calves at the beginning of their ontogenesis of the severity of aggregation of their basic blood cells.
During the neonatal period, calves showed a tendency towards an increase in red blood cell aggregation. Obviously, the aggregation of red blood cells in calves is strongly ensured by the optimal electronegativity of their surfaces due to the sufficient number of proteins with a negative charge on it [19].
The effectiveness of control over the formation of reactive oxygen species creates a minimum of oxidative alteration of these proteins in membranes and capable of participating in the aggregation of plasma proteins [20]. Interacting on the surface of red blood cells with receptors, they maintain the optimum activity of adenylate cyclase and phosphodiesterase, maintaining the physiological balance of the amounts of cyclic AMP and Ca 2+ in them [21].
In this regard, there is a reason to believe that the neonatal phase in calves is characterized by the optimum of metabolic and receptor processes in red blood cells. At the same time, a low aggregation of erythrocytes and a high number of free red blood cells recorded in calves at this age provide an optimum perfusion of their internal organs.
The high degree of vascular control over calf erythrocyte aggregation at the beginning of ontogenesis during the course of the observation, obviously, lays the foundation for intensive microcirculation of high supply of animal tissues with oxygen and nutrients.
During the first days of life, calves showed a weak tendency to increase platelet aggregation activity. The calf hemostatic properties of their platelets observed in calves are largely due to the stability of their receptors and the sufficiently functional capabilities of the postreceptor aggregation mechanisms.
In addition, in the blood of calves at the age of the first 10 days, there was a low concentration of von Willebrand factor, which is a cofactor of platelet adhesion with a sufficient number of receptors for it (GPIв) on the surface of these blood cells. This was indicated by a slight tendency to reduce the time of AP in newborn calves in response to ristomycin. The found state of AP dynamics in response to strong and weak aggregation agonists can be explained by physiologically justified fluctuations in the activity of platelet phospholipases A2 and C, which ensure the functioning of the thromboxane and phosphoinositol platelet activation pathways [22].
In addition, the found tendency to accelerate AP in calves of the first 10 days of life was probably a consequence of the tendency to increase the production of thromboxane in platelets and an increase in the density of receptors for it on the platelet surface. The low AP in calves in response to all inducers was associated with the physiologically minimal activity of platelet proteins -carriers of the stimulating signal from receptors into platelets [23,24].
The low aggregation of neutrophils found in calves was associated with the optimum of their aggregation capabilities and the normal composition of glycoprotein receptors on neutrophil membranes that can bind to lectins. The found tendency to an increase in lectin-and concanavalin A-induced neutrophil aggregation in calves was ensured by a tendency to increase the expression of these receptors with an increase in their composition of loci with N-acetyl-D-glucosamine, N-acetyl-neuraminic acid and mannose [25].
The revealed tendency towards an increase in the aggregation of neutrophils with phytohemagglutinin was caused by a tendency to increase bD galactose in their receptors. Also, the low aggregation ability of neutrophils can be associated with their high sensitivity to vascular disaggregation influences, apparently due to the powerful production of prostacyclin and NO in the vessels of newborn animals [17,26].

Conclusion
Now it becomes clear that the aggregation capabilities of blood cells to a large extent determine the rheology of blood, and, therefore, the optimum microcirculation in animal tissues throughout ontogenesis. This is very significant for the growth processes and the maximum development of the productive properties of animals. In newborn calves of optimal physiological status, there is a slight tendency to increase aggregation of red blood cells, platelets and neutrophils. The observed optimum aggregation activity of erythrocytes, platelets and neutrophils in calves during the neonatal phase is extremely necessary to maintain their homeostasis and create conditions for further growth and development. The rapid inclusion of adaptive mechanisms under these conditions maintains the balance of aggregation and disaggregation in the blood of calves at the level required for optimal perfusion of internal organs and metabolism in tissues. The presence of a low severity of the aggregation properties of blood cells is obviously the basis for the successful progress of the adaptation process throughout the body in calves during the newborn and the creation of the basis for their successful transition to the next stage of early ontogenesis.


extfieldsofstudy:
Physics
Environmental Science

text:
Effects of Different Amelioration Materials on Soil Fertility in Abandoned Homestead Reclaimed Soil

Abandoned homestead reclaimed soil has poor structural structure and lack of nutrients. In this study, organic fertilizer (F), curing agent (S) and fly ash (C) were used as amelioration materials to improve nutrient content of abandoned residential land reclamation soil. After 3 years of wheat-corn rotation, soil samples were collected to determine soil organic matter, total nitrogen, available phosphorus and available potassium contents. The results showed that soil organic matter content at the depth of 0-30 cm soil layer in the treatments of F increased significantly due to organic fertilizer input. The content of total nitrogen, available phosphorus and available potassium in the treated soil with improved materials increased significantly compared with the control. It indicated that the addition of amelioration materials and continuous tillage had significant effect on improving soil fertility in the abandoned homestead reclaimed soil.

Introduction
The rational use of land and the effective protection of cultivated land are our basic national policies. In recent years, with the rapid development of the economy and society, especially the rapid advancement of new rural construction, the housing construction phenomenon of rural residents "one house with multiple houses", "abandoning old houses, planning new houses, and occupying cultivated land" is more common and expanding potential [1,2]. This development trend of a large number of occupied farmland, homesteads and idle houses has led to an increasing demand for rural construction land. The contradiction between protecting cultivated land and ensuring development is more prominent, which has caused china to effectively protect cultivated land, achieve food security and stabilize rural society a certain hazard [3]. According to the statistical data of the second land survey of Shaanxi Province in 2009, the construction land area of urban villages in Shaanxi Province is 9.583 million mu, of which rural homesteads account for 73.2%, and the area of abandoned homesteads available for reclamation is nearly 850,000 mu, Accounting for 12.1% of rural homesteads, generally showing a large number, uncoordinated distribution, low land utilization, planning difficulties and increasing trends [4]. Therefore, carrying out the reclamation work of the old abandoned homesteads in rural areas has a very important role in easing the tension between people and land at this stage.
The soil in the homestead reclamation area lost many of its original functions and characteristics due to the large number of bricks and stones, and the physical structure was seriously damaged. Moreover, the soil nutrient status was poor and the output rate was low, which seriously affected the yield and quality of crops. There is still a big gap as the use of farmland soil [5], which must be cultivated to meet the requirements of cultivated soil. Yan Rui et al. [6] research on the farmland reclamation soil in the Chengdu Plain found that under the wheat-jade rotation, "bacterial residue compost return" has a better effect on improving the physical properties of the farmland reclamation soil and improving soil fertility. Hua ying [7] rapidly increased the fertility of reclaimed farmland in rural homesteads in Chongqing by increasing the application of organic fertilizer, ash and bio-organic fertilizer. It can be seen that the addition of organic fertilizer not only improves the fertility of the reclaimed soil at the homestead, but also greatly improves the total amount of microorganisms and enzyme activity.
At present, the main way of fertilizing for homestead reclamation to improve soil quality is organic fertilizer, and there are few studies on other materials. In this study, taking the land remediation project of the Loess Hollow Village as the background, a field plot experiment was used to study the effect of different improved materials (organic fertilizer, curing agent, fly ash) on the fertility improvement of homestead reclamation soil. Looking for the best soil nutrient improvement materials to provide a theoretical basis for the rapid maturation of the soil after the remediation of the hollow village and the improvement of soil fertility.

Overview of the test area
The experiment was set in Chuyuan Village, Ducun Town, Fuping County, Weinan City, Shaanxi Province. The area has a warm temperate continental climate, with an average annual precipitation of 473 mm, July to September precipitation accounts for 49% of the annual precipitation, annual evaporation of 1000 to 1300 mm, frost-free period of 225 d, annual average temperature of 13.4 ℃, and the total annual light energy radiation is 518.6 ~ 535.0 kJ· cm -2 .

Experimental design
The experiment started in June 2015. In order to simulate the land condition of abandoned homesteads for remediation, the soil of 30 cm on the surface of the test plot was stripped, and the raw soil collected from the abandoned homesteads of Yuzihe Village in Chengcheng County was backfilled. The cell size is 2 m×2 m. In this study, organic fertilizer (chicken manure), curing agent, and fly ash were used as improved materials, and their application rates were: 22.5 m 3 · hm -2 . 600 kg· hm -2 . 75 m 3 · hm -2 。Taking the treatment without adding any modified materials as a control, a total of 4 treatments were set, and each treatment was repeated 3 times. Apply the improved materials evenly to the backfill soil, and mix them manually. The planting of winter wheat began in october 2015, and the winter wheat-summer corn rotation planting has been carried out for three years. Among them, the maize variety is Xianyu 335, the seeding amount is 60,000 plants· hm -2 , and the hole seeding; the wheat variety is Xiaoyan 22, the seeding amount is 105 kg· hm -2 , and all treatments are compounded with 10 kg of compound fertilizer before sowing each season· hm -2 .

Data statistics and analysis
The test data was preliminarily sorted out by Excel 2007 and related charts were drawn, and SPSS 19.0 software was used to test the significance of LSD 0.05 difference.

Effect of adding different improved materials on soil organic matter content
Soil organic matter is an important material basis for soil fertility, as a carrier and source of soil nutrients, and has a profound impact on various physical, chemical, biological properties and nutrient accumulation of soil [9]. The effects of adding different improved materials on the organic matter content of the soil profile vary (Fig. 1). Due to the addition of organic fertilizers, the F treatment greatly increased the content of soil organic matter in the 0-30 cm soil layer compared with other treatments, reaching 13.9~19.7 g· kg -1 , but the soil layer below 30 cm was significantly reduced, Only 5.15~7.14 g· kg -1 .

Effect of adding different improved materials on soil total nitrogen content
Before the planting of crops, the reclaimed soil at the homestead was raw soil with a very low total nitrogen content of only 0.21 g· kg -1 . After 3 years of wheat-corn rotation, different levels of soil fertilization treatment in the soil at different levels the content has changed greatly. The total nitrogen content of each treatment showed an "S" type trend of first decreasing, then increasing, and then decreasing (Fig. 2). Compared with the control, adding different improved materials can significantly increase the total nitrogen content of the soil and increase with the depth of the soil layer. There is no significant difference between C, F and S treatments in the 0~15 cm soil layer; the total nitrogen content of each treatment in the 30~45 cm soil layer is C> F> S; The control treatment without adding any improved materials had a total nitrogen content in the 0~60 cm soil layer that was significantly lower than the treatment with the improved materials. In short, adding improved materials can significantly increase the total nitrogen content of the soil.

Effect of adding different improved materials on soil available phosphorus content
The effects of adding different improved materials on the effective phosphorus content of the soil profile vary (Fig. 3). In the 0~15 cm soil layer, F and S treated soil had the highest available phosphorus content, but the difference between them was not significant; C treated soil had lower available phosphorus content, but significantly higher than the control. In the 15~30 cm soil layer, the difference between C, F and S treatment is not significant, and CK treatment is the lowest. In the 30~45 cm soil layer, the available phosphorus content of the soil in F treatment is still the highest and the difference between the remaining treatments is not significant. In the 45~60 cm soil layer, the difference in available phosphorus content between the treatments with the improved materials was not significant, but it was significantly increased compared to the control. In short, the addition of improved materials can significantly increase the effective phosphorus content of the soil, especially the effect of increasing the effective phosphorus content of the soil in the 0~30 cm tillage layer.

Effect of adding different improved materials on the content of soil available potassium
The soil developed with the parent material of loess is generally rich in total potassium, but in the absence of activation, the content of available potassium is often insufficient. Although the raw soil of the reclaimed land belongs to the loess, its available potassium content is relatively low, only about 70 mg· kg -1 , which is a potassium deficiency level. After three years of planting crops, the soil available potassium content changes as shown in Fig. 4. Adding different improved materials has different effects on the available potassium content of the soil profile, and the soil available potassium content of each treatment varies with the depth of the soil layer throughout the soil profile Increase and decrease, and the available potassium content of soil in the 0~30 cm tillage layer is significantly higher than that of the control, and there is no significant difference in the available potassium content between treatments in the soil layer below 30 cm.

Conclusions and discussions
The most important thing in the reclamation of waste homesteads as cultivated land is to improve soil quality. Since there is almost no human cultivation in the homestead, and there is usually a phenomenon of topsoil stripping during the reclamation process, the homestead soil is mostly raw soil, and its fertility is usually lower than that of conventional farming soil. Chen Liping et al. [10] analyzed the reclaimed land in the hilly area of Yizheng City, Jiangsu Province, and found that the content of nutrients such as organic matter, available phosphorus, and available potassium in the reclaimed soil are low, and the soil structure is poor.
Through the improvement and fertilization of the reclamation soil of the homestead, its productivity will be gradually improved. But different fertilization measures have different effects on different regions and different soil types. Ren Shunrong et al. [5] found that the application of high amount of organic fertilizer can increase the organic matter and available nutrients content of the reclamation soil in Tianjin homesteads and improve the physical structure of the reclaimed soil through field experiments. "Improver" and "organic fertilizer + inorganic fertilizer + straw" can be used as the most suitable fertilization method for Tianjin homestead reclamation. Yan Rui et al. [11] studied the influence of organic materials (straw, fungus residue, pig manure) on soil organic carbon and volatile organic carbon on the reclaimed soil of the homestead in the Chengdu Plain. The results showed that the "bacterial residue" under wheat-jade rotation "Compost return to field" treatment has a good effect on improving the soil structure of the homestead reclamation and improving soil fertility. Li Yanlin et al. [12] studied the dynamic changes and correlations of soil enzyme activity and microbial number under the combined application of three types of breeding wastes (straw, pig manure and bacterial residue) and chemical fertilizer, and found that "bacterial residue + chemical fertilizer" was applied which is a more suitable way to cultivate soil on the reclaimed homestead in Chengdu Plain.
The contents of organic matter, total nitrogen, available phosphorus, and available potassium in the soil are important indicators of soil fertility, and are also essential nutrients necessary for the growth of crops [13]. In this study, three different improved materials such as organic fertilizer, curing agent and fly ash were used to improve the reclamation soil of the homestead. By studying the changes of soil organic matter, total nitrogen, available phosphorus, available potassium and other nutrient indicators, it was found that the addition of organic fertilizer after 3 years of "winter wheat-summer corn" rotation, the curing agent and fly ash have a certain effect on the improvement of soil nutrients in the reclamation of the homestead, and compared with the raw soil in the initial stage of reclamation, after 3 years of continuous crop cultivation, The contents of organic matter, total nitrogen, available phosphorus and available potassium have also been significantly increased.