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Evaluation of Finnish apple cultivars (Malus domestica Borkh.)

The purpose of this research was to study apple cultivars obtained from Luke (previously MTT Piikkiö, Finland) in order to have good candidates for breeding within the central zone of Russia. The studies were carried out in accordance with the methods of cultivar study and artificial freezing by modeling damaging factors using a freezing chamber. The assessment of the cultivars by yield, commodity and consumer qualities of fruit, resistance to adverse abiotic factors of the winter period is given. The ‘Vuokko’ cultivar was identified with a yield significantly higher than Antonovka. By modeling winter damage factors, ‘Sandra’, ‘Samo’ and ‘Make’ cultivars were identified with the resistance of the cambium and wood at the level of ‘Antonovka’, when the temperature in the of mid-winter dropped to –40 °C after hardening off, with the ability to keep the frost resistance at –25 °C after thaw and ability to restore the hardy state by lowering the temperature to –30 °C after thaw and re-hardening off. Cultivars ‘Sandra’, ‘Samo’ and ‘Make’ are recommended for creating new hardy cultivars with high-quality fruits for the temperate continental climate of Europe.

Introduction
The effectiveness of horticulture in a temperate continental climate depends largely on abiotic environmental factors (Chen et al. 2014), including early frosts, low critical temperatures, thaws, spring frosts, and a short and unstable growing season. Successful horticulture requires hardy, disease-resistant cultivars with high fruit quality, mainly with long-term consumption (Khanizadeh et al. 2000, Onishchenko andBelous 2019). To ensure that high productivity and high quality of fruits do not depend on the whims of nature, it is necessary to ensure that the cultivar combines all the necessary signs of adaptation at the maximum level (Kozlovskaya 2015). The effectiveness of breeding work is largely determined by the presence and correct selection of cultivars with a high level of valuable traits and transmitting these traits to offspring (Kalinina and Makarenko 2013). Currently, the problems of conservation and rational use of plant genetic collections are of strategic importance for each country and remain one of the most important areas of science (Dzyubenko 2015). The experience of modern breeders shows high efficiency of correct assessment and selection of donors of economically valuable traits (Savelyeva 2016).
The All Russian Research Institute of Fruit Crop Breeding began to collect the genetic collection at the end of the 19th century, when a collection of cuttings and apple trees were brought to Russia from North America, which was later supplemented by Central Russian, Western European, and new domestic and foreign cultivars. The preserved apple gene pool is the main resource for creating new cultivars and improving the assortment, it is constantly updated with new accessions from domestic and foreign scientific institutions, including the Baltic countries and Finland.
Finnish cultivars bred to survive in harsh winter conditions were imported to the Institute in 2008. These cultivars were created on the basis of frost-resistance of 'Huvitus', 'Antonovka', 'Kaneli' and 'Atlas' which were crossed with cultivars with high taste qualities -'Melba', 'Lobo' and 'Yellow Autumn Callville'. Unfavorable northern conditions, i. e. early frosts, long periods of low temperatures and cool summers, create a risk and limit the industrial cultivation of apple trees in South-Western Finland. Severe cold winters, when the air temperature in the southern part of the country falls below -30 °C, and in Lapland below -50 °C, occur on average once every 10 years. Finland had exceptionally harsh winters for perennials in 1928/29, 1955/56, 1986/87, 2005/06 (Kaukovirta and Syri 1985. Climate and high cost of cultivation complicate and limit apple production in Northern Europe (Lehtonen 2015). For cultivation of apple trees in Northern conditions, it is necessary to choose cultivars that combine hardiness and high quality of fruit (Linden 2002, Sedov 2011, Seppä et al. 2015. Orel region being in the central fruit-growing area of Russia is characterized by periodically recurring severe winters with low temperature, spring frosts, a short growing season, and a lack of heat in summer. The average temperature of the winter months is -8 °C, the absolute minimum temperature is -39.4 °C (2011/2012).

Manuscript received April 2020
The growing season is 175-185 days, the period of active vegetation with a temperature above 10 °C is 139-149 days. The Orel region is located in the Central Russian Upland at an altitude of 203 m above the sea level with moderate continental climate. The experimental station is located Central within the Russian Upland (53°00'N, 36°00'E). The long-term average annual air temperature is 4.6 °C, in winter periods -8 °C, in vegetation periods 15.4 °C. In the Orel region, air temperature of -36 °C is possible (Strashnyi 1972). The sum of positive temperatures above 10 °C is 2250 °C, the growing season lasts 175-185 days. The average annual precipitation is 550 mm, and the spring months are often droughty, with dry winds.
Modern market, in order to get high profits, needs cultivars which are both early-and annually fruiting and possess high fruit quality and resistance to diseases and pests. The cultivars that can combine production capabilities with resistance to abiotic factors are particularly valuable. That is why Finnish cultivars that were developed on the basis of local winter-hardy cultivars can be a precious material for further use in breeding when creating adaptive cultivars with high fruit quality. In this regard, the purpose of this work is to study the main production and biological features of Finnish cultivars in the conditions of the Central Russia and to identify winter-hardy cultivars for breeding.

Material and methods
During the research period (2008-2018), a significant warming was observed in the specified fruit growing zones of Russia in comparison with the average annual indicator, the average temperature of the winter months was -6.4 °C with the average long-term temperature -8.0 °C. Only 2 of 10 winters had temperatures slightly below normal (by 0.6-1.8 °C), while the rest of the winters were warmer (up to 1.2-3.7 °C) ( Table 1).
For the summer periods of Orel region, warming and decreasing precipitation are also typical during the growing season. The average temperature of the growing season in 2008-2018 exceeded the long-term average temperature by 1.1 °C and amounted to 16.5 °C with a decrease in precipitation by 72 mm (from 312 to 240 mm). The growing seasons in 2010, 2012 and 2015 were particularly hot and very dry in the Orel region, when on certain days the air temperature rose to 35 °C, at the soil surface it rose to 43 °C with a deficiency of precipitation ( Table 2).
Cuttings of 11 apple cultivars (Jaspi, Jättimelba, Konsta, Make, Petter, Pirja, Samo, Sandra, Talvikaneli, Tobias, Vuokko) obtained from MTT Piikkiö, Finland were grafted in 2008 directly to the orchard branches of the winter-hardy trees 3-4-98 with 3 trees designated for each cultivar. Trees were planted according to the scheme 4 m between rows and 2 m in a row. The observations were made in the period from grafting to 2018 in accordance with the methodology of cultivar study (Sedov et al. 1999, Tyurina et al. 1999 in the field and in the laboratory by modeling damaging factors using the environmental test chamber ESPEC PSL-2 (Japan). Artificial freezing allows screening genotypes of orchard crops for frost resistance at a high level (Ozherelieva and Sedov 2017). The determination of the stability potential under controlled conditions were carried out according to the main components of the frost resistance complex: I component = resistance to early winter frosts (-5 °С, -10 °С, -30 °С); II component = the maximum level of frost resistance when hardening off shoots in January-February in the mode -5 °С, -10 °С, -40 °С; III component = resistance during thaws (-5 °С, -10 °С, +2 °С, -25 °С); IV component = ability to restore resistance when re-hardening off after thaws (-5 °С, -10 °С, +2 °С, -5 °С, -10 °С, -30 °С) Annual shoots (20 shoots of each cultivar) with a standard length (40-50 cm) were taken for freezing at the end of November. Five shoots of each cultivars were studied for four components of frost resistance.
Duration of exposures to temperatures: hardening off -5 °C (5 days) and -10 °C (5 days); then reducing the temperature by 5 °C per hour to the critical temperature. Exposure to freezing -8 hours; thaw +2 °C (3 days). Evaluation of damage to the buds and browning of shoot tissues was performed using a binocular microscope LZOS MBS-2 (Russia) on a scale from 0 to 5 points: 0 -no damage, 5 -death of tissues and buds after growing back.
Damage to plant tissue and organs in the field during winter were evaluated with points awarded 0-5, where 0no signs of freezing; 1 -very weak freezing, i.e. the wood was slightly yellowish, small surface burns of the bark were on the trunk and skeletal branches; 2 -weak freezing: light brown wood, weak surface burns of the bark and drying of annual shoots; 3 -significant freezing: brown wood, significant damage to the bark with deadening of bark to wood, death of individual branches; 4 -very strong freezing: dark brown wood, severe bark burns, most of the crown was frozen; 5 -complete freezing of the tree.
Apple tree damage by scab (Venturia inaequalis [Cooke]) was assessed in the field against the background of natural infection, in favorable years for the development of the disease. To assess the degree of scab damage to fruits and leaves, a quantitative scale was used in points, reflecting the number of affected leaves and fruits, the degree of damage, and the intensity of fungal sporulation: 0 -no visible damage; 1 -very weak isolated spots on leaves, small spots in the form of points on fruits; 2 -up to 10% of the leaves had a few small spots, mediumsized spots were on the fruit, sporulation was slight or moderate; 3 -up to 25% of the leaf surface was damaged, the fruits had small and large scab spots, sporulation was moderate; 4 -severe leaf damage up to 50%, the spots were large, with dark abundant sporulation, numerous large spots with cracks were on the fruits; 5 -very strong infestation: more than 50% leaves had large spots, numerous merging, spots with dark sporulation were on fruits. Based on the examination of 3 trees (25 leaves on each side of the tree), the average score of leaf damage was determined. To account for scab damage during harvesting, a sample of 100 fruits from different trees was taken.
The study program included the following fruit qualities: size, appearance assessment, one-dimensionality, pulp assessment by consistency, taste, aroma, and fruit storage duration according to generally accepted methods of cultivar testing (Sedov et al. 1999).
Grouping of apple cultivars by fruit size (average fruit weight) was carried out according to the following gradation (taking into account the international methodology UPOV Tg/14/5): small = 41-70 g below average = 71-110 g average = 111-150 g above average = 151-200 g large = 201-250 g The appearance and taste of the fruit were evaluated on a 5-point scale and carried out at the time of optimal fruit maturity by a tasting Committee consisting of 7 people. Attractiveness of fruit appearance was evaluated in points on a scale: 5 -fruits are large, with regular shape and beautiful main and cover color; 4 -medium-sized attractive fruits; 3 -fruits of a mediocre type, below average size, not very attractive; 2 -small fruits, unsuitable in color and shape; 1 -fruits are very ugly and very small.
The taste of the fruit was evaluated in points: 5 -excellent dessert taste; 4 -good table taste; 3 -mediocre taste; 2 -not suitable for consumption, 1 -not edible. When determining the average score when summarizing data from a number of tasters, tenths of the score were added to the whole points.
The structure of the fruit pulp can be coarse-grained, fine-grained, dense, crisp, tender, meaty, juicy, medium-juicy or dry. The onset of removable maturity was noted at the time where the fruits reached normal size and the corresponding color, by the ease of separating the fruits and by seed browning. The fruits were stored in a refrigerator at a temperature of 0-5 °C and humidity of 85-90%. The duration of storage of fruits was determined from the moment of harvest to rejection of 10-15% of fruits.
All results were tested by one-way analysis of variance ANOVA (Version 22, SPSS).

Results
During the period of cultivar study, the conditions were generally favorable for good growth and development of trees. During the first 7-8 years, the trees of the studied cultivars successfully suffered a temperature drop to -34.5 °C on the snow surface (without thawing). After the winter of 2014/2015, apple trees were examined in the field. In the autumn 2014 the trees were well tempered with a gradual transition to low temperatures in November-December (in October 2014 average air temperature was 2.8 °C, in November -2.2 °C, and in December -5.1 °C). The average temperature of the winter months was 2.9 °C higher than the average temperature for many years; the minimum temperature did not fall below -24.5 °C. That winter, reversible damage to wood at the level of Melba standard (2.0 points) was revealed in 'Jättimelba', 'Konsta', 'Petteri' and 'Tobias'. Weak damage (1.0-1.1 points) was in 'Jaspi', 'Make', 'Pirja', 'Sandra', 'Samo', 'Talvikaneli' and 'Vuokko' at the level of 'Antonovka'. The bark and cambium were undamaged in the field.
The subsequent winters between 2016 and 2018 were unstable, mild, with frequent thaws and temperature fluctuations. The winters of 2015/16 and 2017/2018 were particularly unfavorable, with a large number of days with thaws. So, in the winter of 2017/2018 after a warmer December (average air temperature 0.6 °C) and January (-4.9 °C) with an average long-term -5.6 °C and -9.7 °C (respectively), a sharp drop in temperature followed in February and March. Unstable conditions with temperature fluctuations in February and March from +2 °C to -26.0 °C temperatures contributed to the reduction of winter hardiness of Finnish apple cultivars.
To find out the reaction of apple cultivars to various factors of the winter period (including thaw) and establish the threshold of stability, the research program included the determination of the resistance of cultivars to climate stresses by modeling damaging factors. Modeling of -40 °C frost after hardening off contributed to bud damage in all cultivars at the level of 'Melba' standard, but stronger than in 'Antonovka'. Damage to the bark of shoots when exposed to this temperature was also stronger than damage to 'Antonovka' (Table 3). At the 'Melba' level, reversible damage to the bark was observed in the 'Make', 'Sandra', and 'Samo' (1.3-2.3 points), the other cultivars significantly exceeded the damage of the bark in 'Melba' and 'Antonovka'. 'Make', 'Samo' and 'Sandra' showed stability of vital tissues of cambium and wood at the level of 'Antonovka' (1-1.5 points). Significant damage to the cambium (3.2 points) and bark (3.3 points) was noted in 'Vuokko'. The damage in the other cultivars was comparable to the level of 'Melba'.
In terms of wood stability, the studied cultivars did not differ significantly from the controlled cultivars. 'Make', 'Samo' and 'Sandra' were identified as the most stable cultivars (Fig. 1), the tissues of which had small reversible damage.
Long-term observations of the condition of fruit trees in the Russian central zone have shown that winter damage to the tissues and buds of apple trees can occur as a result of severe frosts, and in mild winters it could be from frosts after thaws and sharp daily fluctuations in air temperature. Therefore, the stability of a fruit tree to frosts after thaw is essential in winter and early spring. Modeling of -25 °C frost after a three-day thaw of 2 °C showed a different reaction of cultivars to thaw. Vegetative buds and bark of 'Melba' were damaged more than in 'Antonovka', while cambium damage in these cultivars did not differ significantly.
The buds and tissues of 'Pirja', 'Sandra', 'Samo', and 'Vuokko' were resistant to frost after thaw (with damage significantly less than in 'Melba' and at the level of 'Antonovka') (Fig. 2).  For the stability of cambium the studied apple cultivars were at the level of 'Antonovka' and 'Melba', except for the 'Konsta' with damage to cambium significantly higher than in the standard cultivar when exposed to temperatures of -25 °C after a thaw at +2 °C. No damage to wood was observed in this mode of freezing (Table 3).

a) b)
The ability not only to maintain frost resistance during thaws, but also to restore it after frosts is important. 'Melba' did not differ significantly from 'Antonovka' in the ability to restore tissue stability at -30 °C after a three-day thaw at +2 °C and hardening off. At this level (not more than 1.0 point), there was tissue damage in the studied cultivars. The ability to restore the frost resistance of the buds when hardening off after thawing at a simulated frost of -30 °C (frost resistance component IV) was found in all cultivars at the level of 'Melba', while in 'Pirja', 'Sandra' and 'Vuokko' (Fig. 3) it was higher than in 'Melba' and was at the level of 'Antonovka'.

a) b)
Productivity, quality and scab resistance of the cultivars The results of the yield and quality of fruits of the studied cultivars are presented in Table 4.
Productivity and consumer qualities of fruits are largely determined by the genotype of the cultivar, as well as the growing conditions. 'Jaspi', 'Jättimelba' and 'Petteri' bloomed and gave the first fruit in the second year after grafting; the most rapid-growing 'Vuokko' at this age recorded a yield of more than 5 kg per tree. The other cultivars bloomed in the third or fourth year after grafting. The amount of the yield was largely affected by adverse conditions during flowering in some years. So, in mid-May 2017, during apple blossom, the air temperature dropped to -2 °C and on the soil surface to -3.7 °C with a cold strong north and north-east wind. In such extreme conditions, with good flowering, damage to the pistils in the flowers was noted, which negatively affected the yield in most apple cultivars. Under favorable conditions, the yield of 'Tobias', 'Jättimelba', 'Make' and 'Vuokko' was 20-30 kg per tree (25-38 t ha -1 ).
On average, during 2015-2018, the cultivars of Finnish selection showed moderate yields at the level of 'Melba' and 'Antonovka'. 'Vuokko' stood out from this group with an average yield significantly higher than 'Antonovka' and 'Melba' (Table 4).
The date of maturation and the consumption duration of fruit are significantly determined by the origin of the cultivars. Fruits of 'Pirja', 'Vuokko', 'Jättimelba' and 'Samo', in the origin of which 'Melba' and 'Huvitus' have participated, ripened in summer (early-mid-August). In the central zone of fruit-growing in Russia, the earliest fruit maturation was observed in 'Pirja' -in the last 10 days of July. The fruit maturation was observed in the autumn in those cultivars that were derived from the winter 'Lobo' cultivar. A longer period of storage for fruits was observed in 'Konsta' ('Lobo' × 'Antonovka') -until late November -early December.
'Pirja' was identified as an early-summer cultivar with fruit maturation earlier than in other cultivars at the end of July. The fruits of this cultivar are below average size (95 g), nondescript in appearance, with weak ribbing and blurred red stripes, sweet and aromatic.
The 'Vuokko' fruits are medium-sized (120 g), oval-conical, with a red blurred blush, good sweet and sour taste. The fruits of 'Jättimelba' are rounded and conical, with a dark red blush, thin skin. The flesh is white tender and aromatic.
The fruits of 'Samo' are medium-sized (120 g), round-conical, the cover color is dark red blurred, the flesh is dense and fine-grained, with a pleasant sweet and sour taste. The fruits of 'Make' are rounded-conical, slightly ribbed, with a pleasant sweet and sour taste. The fruits of the rapid-fruiting 'Petteri' are one-dimensional, flat-rounded, the skin is thin, with a bluish coating. The flesh is dense, white, and sometimes with red veins. The taste is harmonious, sweet and sour.
Autumn ripening 'Sandra' gives round-conical fruits of medium size, the skin is thin, the cover color is beautifuldark red striped that merge into a thick blurred blush over the entire surface of the fruit. The taste is harmonious sweet and sour. The fruits of 'Tobias' are oval-conical, with a dense skin covered with a solid blurry-striped blush. The flesh is pale cream, sometimes with red streaks, the taste is bland and sweet. The fruits of 'Talvikaneli' are medium-sized, round-conical, with a slight ribbing, with a beautiful red blurred blush, taste with a predominance of sweetness, with a cinnamon flavor. Early winter 'Konsta' produces rounded-conical fruits, with a dark red, dull, blurred blush. The flesh is white, dense, crisp and sour.
The color of all cultivars studied was more pronounced in conditions of sharp fluctuations in air temperature in the daytime and at night during the ripening period. That was observed in 2015, 2016 and 2018, when the difference in day and night temperatures in August was 20-24 °C.
During 2009 to 2018 the studied Finnish cultivars showed insufficient resistance to scab (Venturia inaegualis [Cook]) which causes great harm to apple orchards. The fruits and leaves of 'Konsta', 'Vuokko', 'Pirja' and 'Petteri' were most affected by scab (3.6 points) at the level of 'Melba', but stronger than 'Antonovka'.

Discussion
The Central zone of fruit-growing in Russia is very similar to the conditions in the South-Western part of Finland, where commercial gardening is possible. A short growing season and insufficient summer heat combined with unfavorable winter conditions are the main indicators that limit the cultivation of high-quality apple cultivars in the south-west of Finland and in the central zone of Russia (Kaukoranta et al. 2010, Krasova et al. 2013. According to Seppä et al. 2016 during the growing season the average air temperature for many years in Helsinki is 13.8 °C, while in the Orel region it is 15.4 °C (by 1.6 °C more) the amount of precipitation for this period is 309 and 312 mm, respectively.
In the field during the past winters with frosts to -34°C with good hardening off in the autumn period, the stability of tissues at the level of the winter-hardy cultivar 'Antonovka' was revealed in 'Jaspi', 'Make', 'Pirja', 'Samo', 'Sandra', 'Talvikaneli' and 'Vuokko'. The stability of 'Jättimelba', 'Konsta', 'Petteri' and 'Tobias' did not exceed the level of moderately hardy 'Melba'. The bark and cambium were undamaged in the field.
The study of the reaction of the cultivars to lower temperatures by modeling frost -40 °C showed some differences in the stability of tissues and buds of the studied Finnish cultivars. At the 'Melba' level, reversible damage to the bark was observed in 'Make', 'Sandra' and 'Samo' while the other cultivars significantly exceeded the damage to bark in the standard cultivar.
'Make', 'Samo' and 'Sandra' showed stability of vital tissues, i. e. cambium and wood at the level of 'Antonovka' (the damage was not more than 1-1.5 points). The significant damage to the cambium (3.2 points) and bark (3.3 points) was noted in 'Vuokko'. The damage in the other cultivars was at the level of 'Melba´. In terms of wood stability, the studied cultivars did not differ significantly from the control.
For further use the most stable cultivars 'Make', 'Samo' and 'Sandra' were selected. Their cambium and wood withstood the modeled temperature -40 °C after hardening off with small reversible damage at the level of 'Antonovka'.
In an unstable climate, the ability of the cultivars to keep stability in winter after thaws is important (Krasova 2014).
Frost modeling of -25 °C after a three-day thaw at 2 °C allowed identification of reactions of cultivars to thaw. The previous conclusion that Finnish cultivars did not have the ability to maintain stability during thaws (Linden et al. 1999) was confirmed only partially and largely depended on the duration of the thaw and the severity of the frost. After thaw, buds and bark in 'Melba' and 'Konsta' were more damaged than in 'Antonovka'.
Under the specified freezing conditions in our experiments, 'Pirja', 'Sandra', 'Samo' and 'Vuokko' showed high resistance of buds and tissues to frost after thaw at the level of 'Antonovka' with damage to the buds no more than 1.0 point, to bark no more than 0.5 point, cambium and wood were completely without damage. Thaw with the subsequent impact of -25 °C had the strongest influence on 'Konsta'. Except for this cultivar, all other cultivars showed high resistance of cambium to frosts after thaws. When modeling freezing of -30 °C after a three-day thaw at +2 °C and re-hardening off, the ability to restore frost resistance at the 'Melba' level was determined in most cultivars, except for 'Konsta' and 'Petteri'.
As a result of modeling damage factors of the winter period, the cultivars 'Sandra', 'Samo' and 'Make' in the field showed resistance at the level of 'Antonovka', and they could withstand the temperature of -40 °C at the level of 'Antonovka' when freezing after hardening off with little damage to the cambium to 1.5 points and reversible damage of the bark to 2.3 points ('Samo'). These cultivars showed a high ability to maintain stability in frosts up to -25 °C after a three-day thaw at +2 °C, as well as lowering the temperature to -30 °C after a three-day thaw +2 °C and re-hardening off. 'Vuokko' showed resistance to frost after thaw at the level of 'Antonovka', but it was worse than others when simulating a drop in temperature to -40 °C.
'Samo', according to Linden et al. 1999, with controlled freezing in mid-March, also showed a high stable level of resistance, but it was still slightly below the level of 'Antonovka', as well as in our conditions. The other cultivars showed resistance according to the components of frost hardiness at the level of 'Melba'.
Stability of frost hardiness and the degree of influence of re-hardening off are the main factors for selecting cultivars for growing in the conditions of unstable winters (Tyurina et al. 1999). Rootstocks, location of cultivation and cultivation technology play an important role in the formation of winter hardiness and productivity (Hampson et al. 2004, Thybo et al. 2005, Seppä et al. 2013, Kviklys et al. 2016. Grafting of Finnish apple cultivars into the crown of a hardy skeletal tree allowed to identify the most stable and productive cultivars and preserve all grafted cultivars completely. On average, during the years of the study (2015-2018) 'Vuokko', 'Jättimelba', 'Samo', 'Make', 'Tobias' and 'Konsta' that were grafted into the crown of a hardy skeletal tree, showed yields at the level of 'Melba'. 'Vuokko' stood out from this group, with an average yield of 16.3 kg per tree (20.4 t ha -1 ), that is higher than all other cultivars.
Our observations during the ripening of the studied cultivars mostly coincides with the data of the Finnish authors (Tahvonen 2007). Summer and autumn cultivars ripened a few days earlier in dry and hot years. 'Pirja' was noted as an early-summer cultivar and its fruit ripened earlier than others, at the end of July. The fruits of 'Vuokko', 'Samo' and 'Jättimelba' ripened a little earlier than 'Melba' and were stored for 2-3 weeks. The fruits of 'Make' and 'Petteri' ripened at the 'Melba' level in late August and early September, and were stored until mid-October (4-5 weeks). Fruits harvesting period of 'Sandra', 'Jaspi', 'Tobias', 'Talvikaneli' and 'Konsta' was in mid-September, the fruits of 'Sandra', 'Jaspi', 'Tobias' were stored for 6-7 weeks, and 'Talvikaneli' for 8 weeks. 'Konsta' had the longest period of fruit storage -until the end of December.
According to the fruit size, the studied cultivars were classified as average-sized (101-150 g). Climate change has a significant impact on fruit quality (Suguira et al. 2013). The fruits of 'Tobias' were not one-dimensional by years with fluctuations in fruit weight from 110 to 160 g depending on the conditions of the year; in warm years, the fruits were larger, with a beautiful intense color, more juicy and delicious, with flavor. Lower than average in size were the fruits of 'Pirja', the cultivar of early-summer maturation. Under the conditions of dry and hot July 2012 and 2014, there was a strong shedding of fruits of this cultivar. The fruits of 'Jättimelba' were very similar to 'Melba' in the character and stability of taste, but the cover color was more blurred and the fruits overriped faster. The assessment and stability of the taste and color intensity of fruits was largely determined by the weather conditions of the year. 'Petteri' had a consistently sweet fruit taste, while 'Samo', 'Make' and 'Vuokko' have a good sweet and sour taste. Seppä et al. (2016) reported about a consistent taste of late-ripening fruits under various annual climatic conditions. For ex. ´Konsta´, the winter cultivar, has a constant acidic taste.

Conclusion
In the temperate continental climate of Russia a number of Finnish apple cultivars showed good adaptive potential to adverse environmental conditions both in the field and laboratory. In the field, the Finnish cultivars showed hardiness at the level of Antonovka.
As a result of modeling damaging factors by components of frost resistance in winter, the most winter-hardy cultivars were identified. 'Sandra', 'Samo' and 'Make' survived the critical temperature of -40 °C with minimal damage to the cambium and wood at the level of 'Antonovka'. Damage to the buds and bark in these cultivars was at the level of' Melba'.
All the studied cultivars, except 'Konsta', showed good resistance to frost after thaws at the level of 'Melba' and 'Antonovka'.
'Petteri', 'Jättimelba' and 'Jaspi' began to fructify the next year after grafting in the crown of the skeletal tree. 'Make', 'Tobias' and 'Vuokko' gave high yields during 11 years of growing in the orchard (an average of 12-16 kg per tree).
'Petteri' was selected as a cultivar with fruits of a pleasant sweet taste, dense fine-grained and juicy flesh. According to the fruit appearance, consistency of the flesh and taste, 'Jättimelba', 'Samo' and 'Jaspi' were rated at the 'Melba' level.
As a result of a comprehensive assessment of economic and biological indicators, 'Sandra', 'Make' and 'Samo' are recommended for breeding new adaptive apple cultivars with high taste qualities for a temperate continental climate.


extfieldsofstudy: Biology Medicine

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

text:
Gene polymorphisms influencing yield, composition and technological properties of milk from Czech Simmental and Holstein cows

Objective The aim of the study was to evaluate the influence of polymorphic loci and other factors on milk performance and the technological properties of milk. Methods The analysis was performed on Simmental and Holstein cows in field conditions (n = 748). Milk yield in kg, fat and protein percentage and yield were evaluated. Technological properties were evaluated by milk fermentation ability, renneting, and an alcohol test. Polymorphisms in the acyl-CoA diacylgycerol transferase 1 (DGAT1), leptin (LEP), fatty acid synthase (FASN), stearoyl CoA desaturase 1 (SCD1), casein beta (CSN2), casein kappa (CSN3), and lactoglobulin beta genes were genotyped, and association analysis was performed. Results The DGAT1 AA genotype was associated with higher milk, protein and fat yields (p<0.05). The MM genotype in the LEP gene was associated with a lower protein percentage and the W allele with a higher protein percentage (p<0.05). In cows with the FASN GG genotype, the protein percentage was higher, but the A allele was associated with higher milk, protein and fat yields than the G allele. The TT genotype in SCD1 was associated with the lowest milk, protein and fat yields and with the highest milk protein percentage (p<0.01). The T allele had higher values than the C allele (p<0.05) except for fat percentage. The genotype CSN3 AA was associated with a significantly heightened milk yield; BB was associated with a high protein percentage. The effect of the alleles on the technological properties was not significant. The CSN2 BB genotype was associated with the best alcohol test (p<0.01), and the renneting order was inverse. Milk from cows with the CSN2 A1A1 genotype was best in the milk fermentation ability. CSN3 significantly affected the technological properties. Conclusion The findings revealed the potential of some polymorphic loci for use in dairy cattle breeding and for the management of milk quality. In field research, the pivotal role of farms in milk yield, composition and technological properties was confirmed.

INTRODUCTION
Milk yield and composition substantially impact the economics of dairy farms. For milk manufacturing, in addition to the percentages of fat and protein and the microbial quality, the technological properties of milk are important, as cheese has become a very important product in the dairy industry and enzymatic coagulation of milk is a crucial step in cheese making process [1]. The influence of diet on the cheesemaking properties of milk is often analyzed [2]. Other impacts on technological properties, such as the housing system or the stage of lactation, have also been the focus of studies [3,4].

Animals
All experiments were performed in accordance with relevant guidelines and regulations recommended by the Ministry of Agriculture of the Czech Republic. All animal experiments were under supervision of the Institutional Animal Care and Use Committee of the Faculty of Agriculture of the South Bohemia University, where the experiment was carried out, approval number 22036/2019MZE18134. DNA was extract ed noninvasively from milk samples.
The analysis was performed in cows of the Czech Simmen tal (part of the Simmental group) and Holstein breeds and their crosses; the cows were kept in the Czech Republic. As the crossbreds of Holstein and Simmental with small propor tions of Ayrshire are common in herds in the Czech Republic, they were included into our field research as well. The numbers of purebred and crossbred cows are given in Supplementary  Table S1. Cows were kept by five companies in free housing and fed with maize silage, grass silage, hay and feed concen trates yearround. The ratios differed among companies in terms of the share of constituents and their quality. The cows calved from 2015 through 2017. The 1st lactation was record ed for 748 cows, and the 2nd was also recorded for 660 of those cows. The mean milk yield was 8,036 kg in the 1st lac tation and 8,722 kg in the 2nd lactation. The fat percentages were 4.12 and 4.12, the crude protein percentages were 3.46 and 3.48, the fat yields were 329.8 kg and 358.0 kg, and the crude protein yields were 274.8 kg and 301.3 kg for the first and second lactations, respectively.
The technological properties (milk fermentation ability, renneting measured by classical procedure and by nephe lometry, ethanol test) of milk samples from 242 cows were examined. Of these cows, 81 were sampled once, 86 twice, 53 three times, 16 four times, and 6 five times. The cows were sampled throughout the course of the year.

Genotyping
Milk samples were individually collected, and DNA was iso lated using the DNA/RNA extractor MagCore HF16 Plus (RBC Bioscience, New Taipei, Taiwan). Isolation was per formed according to the manufacturer's instructions using the MagCore DNA Whole Blood Kit and MagCore Genomic DNA Tissue Kit (RBC Bioscience, Taiwan). The quality and quantity of the isolated DNA were verified by electrophoresis and spectrophotometry.
Genotyping of all loci was performed by the polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) method. DGAT1 gene alleles A (alanine) and K (lysine) were genotyped according to the methods of Kuhn et al [14]; LEP gene alleles M and W according to Buchanan et al [15]; FASN gene alleles A and G according to Roy et al [16]; CSN2 gene alleles A and B according to Medrano and Sharrow [17]; and alleles A 1 and A 2 according to Miluchová et al [18]. CSN3 gene alleles A, B, C, and E were analyzed according to the methodology of Barroso et al [19]; LGB alleles A and B according to Strzalkowska et al [20]; and SCD1 gene alleles C and T according to Inostroza et al [21]. The primer sequences are given in Supplementary Table S2.
The resulting genotypes were electrophoretically deter mined, and genotype and allelic frequencies were calculated. To evaluate the HardyWeinberg Equilibrium, the differences between the observed and expected frequencies of the geno types were tested using a χ 2 test with the significance level p<0.05 and p<0.01. Supplementary Table S3 gives the fre quencies of genotypes and alleles of all genotyped Simmental, Holstein and crossbred cows.

Milk performance, composition and analysis of technological qualities
Data on milk performance were collected from the milk re cording breeder´s database. Milk yield in kg, fat and crude protein percentage, and fat and crude protein yield in kg were evaluated. Milk composition (fat and crude protein contents) was determined in breeder milk laboratories of the Czech Moravia Breeders Association using infrared spectroscopy (Foss Electric, Foss A/S, Hilleroed, Denmark; and Bentley Instruments, Chaska, MN, USA) instrumentation. These laboratories are accredited to the ISO standard (CSN EN ISO/ IEC 17025) for official milk performance analysis in the Czech Republic and are working under the ICAR (International Committee for Animal Recording) umbrella (ICAR certificate to Czech Moravian Breeders' Corp, Hradistko; Accredited Milk Laboratory Bustehrad, Czech Republic, for identifica tion of dairy cattle, production recording in dairy cattle, genetic evaluation, milk laboratory operation, linear classification/ scoring, and data processing), regularly taking part in rele vant proficiency testing. Analytical instruments were regularly monthly calibrated according to the reference method results (extraction by the Röse-Gottlieb method for fat content [22] and distillation and titration according to the Kjeldahl method for crude protein content (total nitrogen content ×6.38) [23]). Technological properties were evaluated by a milk fermen tation ability test, renneting was measured subjectively and instrumentally, and an ethanol test was performed.
The milk ethanol stability was determined by milk titra tion (5 mL) with 96% ethanol until the first precipitation flakes of milk protein were visible and is reported as ml of alcohol. This procedure was modified according to Horne [24].
The milk fermentation ability of the yogurt test was carried out according to the Czech milk industry standards. A sam ple of raw milk (50 mL) was heated at 85°C for 5 min and cooled at 43°C±2°C. Subsequently, the sample was inoculated with 2 mL of the thermophilic lactic culture YC18040FLEX (Chr. Hansen, Horshholm, Denmark; Streptococcus thermophilus, Lactobacillus delbrűeckii subsp. lactis, and L. d. subsp. bulgaricus). The inoculated sample was incubated at 43°C for 3.5 hours. The result was expressed as the titration acidity of the yogurt in mL of 0.25 mol×L 1 NaOH×100 mL 1 (or the socalled SoxhletHenkel degree) [25,26].
Rennetability (classical procedure) was determined dur ing the tempering (35°C) of a defined milk volume after the addition of rennet (1% vol.) by measuring the time (rennet coagulation time RCT) until the first flakes of lactoproteins formed (beginning of coagulation). Rennetability was also determined by using nephelometry (turbidimetry measure ment) to assess the milk coagulation time (ML -2 analyzer). This is the use of the optical method (NEF, Nephelo turbi dimetric milk coagulation sensor ML -2) to evaluate the intensity of the socalled diffusely scattered Tyndall light on dispersed particles (coagulating lactoprotein flakes) [26,27].
The milk ethanol stability, milk fermentation ability and milk rennetability are not introduced by an official standard as technological property in world literature references, but they are known according to citations in the scientific litera ture. These procedures were modified according to literature sources cited.

Statistical analysis
Statistical analyses were performed using SAS (SAS 9.3, SAS Institute, Cary, NC, USA). Descriptive statistics for milk yield in kg, protein and fat percentages and protein and fat yield in kg during the first and second lactations are given in Sup plementary Table S4. Descriptive statistics for the indicators of the technological quality of milk, i.e., the milk fermentation ability, rennetability assessed subjectively and instrumentally, and alcohol test are given in Supplementary Table S5. For the descriptive statistics and genotype frequencies in Supplemen tary Table S4, each record was assessed as a separate entry; when two lactations were recorded for the same cow, it was included twice. Similarly, for Supplementary Table S5, when a cow was measured repeatedly, the genotype was included repeatedly as well.
The data set contained repeated measurements per cow. Repeated measurements were obtained for the first and sec ond lactation for milk performance traits. For technological quality, measurements were obtained several times over the course of two consecutive lactations. To analyze the influence of polymorphisms on milk yield and technological quality, the MIXED procedure of the SAS system with repeated mea surements and the least squared mean method were used to compare genotypes. The models were developed as follows.
For milk performance traits, the following mathematical model was used: Y ijk = µ+gen i +lac j +anim k +e ijk www.animbiosci.org 5 Čítek et al (2021) Anim Biosci 34 :2-11 where Y ijk = milk performance trait; μ = population mean; gen i = fixed effect of the genotype (class effect i = 1, 2, 3); lac j = fixed effect of the lactation order (class effect j = 1, 2); anim k = random effect of the animal; and e ijk = random residual effect.
Different mathematical models were used to determine the technological quality of milk: Yogurt ijklmn = µ+gen i +farm j +protein k +casein l +lacs m +anim n +e ijklmn where Yogurt ijklmn = yogurt test values; μ = population mean; gen i = fixed effect of genotype (class effect i = 1, 2, 3); farm j = fixed effect of farm (class effect j = 1, 2, 3, 4, 5); protein k = fixed effect of protein percentage content in milk; casein l = fixed effect of casein content in milk; lacs m = fixed effect of lacta tion stage in days; anim n = random effect of the animal; and e ijklmn = random residual effect.
Rennetability ijklmn = μ+gen i +farm j +protein k +NFS l +season m +anim n +e ijklmn where Rennetability ijklmn = rennetability assessed subjectively or instrumentally; μ = population mean; gen i = fixed effect of genotype (class effect i = 1, 2, 3); farm j = fixed effect of farm (class effect j = 1, 2, 3, 4, 5); protein k = fixed effect of protein percentage content in milk; NFS l = fixed effect of not fat solids content in milk; season m = fixed effect of season (class effect m = 1, 2, 3, 4)*; anim n = random effect of the animal; and e ijklmn = random residual effect. * The fixed effect of season was created as a combination of three months according to natural weather conditions, temperature, pasture quality, etc. A year was divided into four seasons: 1 = December, January, Feb ruary; 2 = March, April, May; 3 = June, July, August; and 4 = September, October, November.
The effect of alleles on milk production traits and the tech nological quality of milk was computed using the following mathematical model: where Y ij = observed trait; μ = population mean; allele i = fixed effect of allele (class effect i = 1,2); anim j = random effect of the animal; and e ij = random residual effect.
For post hoc comparisons, the TukeyKramer test was used [28].

Milk yield and composition
In the DGAT1 gene, the genotype AA and allele A, which codes for alanine, had a higher frequency than the genotype KA and the K allele, which codes for lysine (Supplementary  Table S3); the homozygous genotype KK was not found at all. Other researchers found similarly unbalanced frequencies.
In Israeli Holstein cows, the frequency of the K allele was reported to be 0.09 overall and 0.16 in sires [29]; in another study, the A allele had the highest frequency in dairy breeds, with the exception of Jersey [30]. Similarly, low frequencies of the K allele and KK homozygous genotype were found in Simmentals [31]. The A allele was confirmed repeatedly to be associated with higher milk, fat and protein yields, and its frequency in intensively selected populations increases due to indirect selection [14,32]. In our analysis, cows with the AA genotype outperform the heterozygous ones signifi cantly in milk, protein and fat yields (Table 1). Additionally, when the effects of alleles are evaluated, A is advantageous but not significantly so ( Table 2). This result is generally in agreement with previous findings and confirms our previous finding in German Holsteins regarding the trend of increas ing frequency of the alanine variant [32,33].
Additionally, for the LEP gene, the MM genotype domi nated (Supplementary Table S3). In Holstein cows, a reverse order of genotypes was also published [34]. MM homozy gous cows had a lower protein percentage, and the difference between MM and MW was significant (Table 1). Allele W positively and significantly influenced the protein percentage ( Table 2); differences in other indicators of milk performance were nonsignificant.
For the FASN gene, the protein content was slightly but significantly higher in GG homozygous cows. The A allele had significantly higher milk yield than the G allele, which resulted in significantly higher protein and fat yield. The dif ferences in fat and protein percentages between alleles were negligible and nonsignificant ( Table 2). The frequencies of allele G were markedly higher than those of A, which does not correspond fully with the differences between alleles in terms of performance. However, considering both genotypes and alleles, the performance differences were low, which may explain the differences in frequency.
The TT homozygous genotype in the SCD1 gene was sig nificantly associated with the lowest milk, protein and fat yields and with the highest protein contents ( Table 1). The analysis of allele associations showed superiority of the T allele in all characteristics except fat content. The differences were not high but significant ( Table 2). The differences among geno types hint at intermediate heredity.
For the CSN2 gene, the differences among genotypes were not significant. The B allele had significantly higher milk yield and therefore protein yield than A. In fat yield, the pvalue was near the significance threshold. The differences in con tents were low and nonsignificant (Tables 1, 2). Similarly, for the A 2 and A 1 genotypes, the effect was nonsignificant. Allele A 2 was significantly better in terms of milk, protein and fat yields. The results of Ozdemir et al [35] indicated that none of the CSN2 variants provide an advantage.
Genotype AA in the CSN3 gene was significantly associ LGB, lactoglobulin beta. * Significant at p < 0.05; ** significant at p < 0.01. a,b Different letters between genotypes in the same column represent significant differences at p < 0.05. A,B Different letters between genotypes in the same column represent significant differences at p < 0.01. X Differences between CSN3 genotypes AA and BE in the protein percentage are significant at p < 0.05. Y Differences between CSN3 genotypes EE on the one hand and AB, AE, BE on the other hand in the protein percentage are significant at p < 0.05. LGB, lactoglobulin beta. * Significant at p < 0.05; ** significant at p < 0.01. ated with high milk yield. BB homozygous cows had milk with a significantly higher protein percentage. Although the highest value was associated with the EE genotype, there were only two cows with a total of three lactations with this geno type, making it a minor consideration; similarly, for the BC genotype, there were two cows with four lactations. However, there were 21 cows of the BE genotype with 40 lactations, and they had significantly higher protein percentages comparing with the AA genotype ( Table 1). The lowest protein content was found in AA homozygous cows, and the difference was significant. Thus, the positive influence of the B variant on the protein content was repeatedly shown. This was confirmed when evaluating the effect of alleles, specifically, the following significant effect was observed: E>B and B>A. However, the CSN3 genotypes were not significantly associated with pro tein yield. Additionally, BB and B performed significantly better than AA and A in fat percentage. The differences in fat yield were not significant. Our findings on the prevalence of the AA genotype and its effects on milk yield agree with other results found in Sim mentals [36]. The authors also found the BB genotype to be associated with the highest protein percentage, but the fat percentage and yield were highest in milk from AA cows. In Czech Simmental cows, significant differences were reported among genotypes in daily milk yield, but the differences in protein and fat percentages were nonsignificant [37]. Ozdemir et al [35] conclude their review and metaanalysis by stating that the CSN3 genotypes are ranked BB>AB>AA in terms of protein content and that the B allele could be considered a marker to improve milk protein content. The prevalence of the BB genotype in relation to protein yield was not always obvious. Additionally, for fat content, the BB genotype was better. They report that the associations of genotypes and al leles with milk yields were not significant. These findings are in general agreement with our results.
The AA genotype of the CSN3 gene is usually the most fre quent in both BlackandWhite and Simmental cattle [20,36]. The frequencies of the BB genotype and B allele in our cows, both Holstein and Simmental, were rather low, which is also consistent with the frequencies found by other authors in Czech Simmental [6]. However, one other group of authors also found that, in Czech Simmental, the most frequent gen otype was AB (0.487), and the frequency of the B allele was high (0.418) [37]. The frequency of the E allele (0.030) was the same as in our Simmental group (0.036). Apparently, there is leeway for breeding, and many Czech breeding companies report the genotype of the CSN3 gene for the sires in their catalogues. However, changing genotype frequencies is a long distance run.
For the LGB gene, the AB genotype was significantly as sociated with higher milk yield than in both homozygous genotypes, resulting in higher protein and fat yields. The rank of genotypes may indicate the effect of heterosis. Allele B out performed A in milk, protein and fat yields, but the differences in the contents were not significant (Tables 1, 2). Additionally, other authors found significantly higher milk, protein yield and fat contents in Simmental cattle and higher fat yields (which were nonsignificant) in AB heterozygous cows [36].
The other factors potentially affecting milk performance were evaluated. Farm, breed and lactation order were tested in a general linear mixed model. The milk yield, fat yield and protein percentage were significantly influenced by all the fac tors. Fat content was influenced by farm, and protein yield was influenced by farm and lactation order. Thus, the impor tance of the effect of farms, i.e., specific stable, management, nutrition, veterinary care, milking, etc. was emphasized, even if some polymorphisms showed a significant association with milk performance.

Milk technological characteristics
The testing of milk fermentation ability, renneting and ethanol stability was the final goal of our analysis. The topic is rele vant because genetic background exerts a strong influence on the cheesemaking properties of milk, largely due to ge netic polymorphisms in the major milk protein genes [38]. In our analysis, the effect of alleles was not significant with the exception of CSN2, with the B allele outperforming the A allele (p<0.01) in terms of milk fermentation ability (yo gurt test) ( Table 2). The milk of KA heterozygous cows in the DGAT1 gene significantly exceeded that of homozygous AA cows in milk fermentation ability ( Table 3). The LEP gene had no significant effect, but the differences among geno types in the alcohol test showed rising values in the order MM>MW>WW with significance differences of MM and MW vs WW at p<0.05. The differences among FASN geno types were not significant. For the SCD1 gene, the milk of TT cows was associated with significantly poorer performance in terms of renneting.
Certainly, the effects of polymorphous variants of milk protein genes are in focus. For the CSN2 gene, the BB geno type had the best heat stability of milk as measured by the ethanol test (p<0.01), which is important for ultrahigh tem perature milk production. However, with regard to renneting, the order was reversed. The A 1 A 1 genotype was significantly associated with the best milk fermentation ability and was not significantly associated with renneting. Poulsen et al [39] refers to the negative association of A 2 with coagulation. Ac cording to our results, it is difficult to describe the preferable genotype or allele in the CSN2 gene.
Kappacasein is the gene most often examined, as its influ ence on technological properties has been confirmed repeatedly. In our analysis, the rennetability measured instrumentally was significantly affected (p<0.05). Genotype BC was asso ciated with the best milk, but only four measurements were performed; because of the high standard errors, this associa tion is of limited importance. The BB genotype was associated with significantly better renneting than the AA genotype but not the AB genotype (Table 3). Genotypes with the A and E alleles (AE, AA, BE) were associated with the poorest ren netability. A probable explanation for the differences in milk protein coagulation is the changes in the primary amino acid sequence between the A and B variants of the kappa casein as a protective factor for raw milk casein micelles. The B vari ant of kappa casein differs from the A variant by amino acid substitutions at two positions: 136th, replacing threonine with isoleucine, and 148th, replacing asparagine with ala nine. These changes in the amino acid sequence of the B variant may interact positively with the action of the rennet enzyme that starts cleavage of the casein molecule between the 105th and 106th amino acids of the peptide chain, i.e., not far from the changed amino acids.
For the milk fermentation ability, the pvalue was close to the significance threshold. The ethanol test resistance was the best in milk from the cows with CSN3 BB genotypes. The dif ferences in these scores between the BB genotype on one hand and AA and AB genotypes on the other hand were significant at p<0.01. The difference BB>AE was significant at p<0.05. Overall, the advantage of the CSN3 BB genotype was con firmed, but the B allele was not significantly better than the others (Table 2). Better properties for the BB genotype have also been reported by other authors [39,40], who stated a positive effect of CSN2 B and CSN3 B. They hinted at the ad ditive genetic variation of milk coagulation and the possibility of selective breeding for variants associated with superior milk coagulation.
Lactoglobulin beta is the most important whey protein. In our analysis, the effect of genotype on ethanol number was significant at p<0.05, and BB was associated with a better value than AB at p<0.01.
When evaluating other factors, the farm was found to sig nificantly affect all parameters of technological quality. Protein percentage was associated with the milk fermentation ability and rennetability scores, while fat percentage was associated with ethanol test performance. Nonfat solid content was as sociated with renneting, while somatic cell count was associated with renneting measured subjectively. The content of casein, LGB, lactoglobulin beta. * Significant at p < 0.05; ** significant at p < 0.01. a,b Differences between genotypes with different letters in the same column are significant at p < 0.05. A,B Different letters between genotypes in the same column represent significant differences at p < 0.01. X Differences between CSN3 genotypes AB on the one hand and AA and BE on the other hand in renneting assessed instrumentally are significant at p < 0.05. Y Differences between CSN3 genotypes BE and BC in renneting assessed instrumentally are significant at p < 0.05.


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Appropriate Number of Sex Pheromone Trap for Monitoring Spodoptera exigua Hubner ( Lepidoptera : Noctuidae ) Moths on Shallot Field

Indonesia is one of countries who love flavor taste so much. One of herb that must be in dish is shallot. Besides being used as a flavor, shallot also has high economic and nutrient value. Shallot has big role in life through of people especially to farmers, that’s way most of farmers were attracted to cultivation a shallot. One of the centers of shallots production is Bantul, Yogyakarta. Within a year there are three planting seasons i.e. July to October, November to February, and March to June. Pest problem is one of major constraint for achieving higher production in shallot cultivation. Febrianasari (2014) stated that Spodoptera exigua Hubner (Lepidoptera: Noctuidae) is one of important pest that can be obstacle the farmers from getting the maximum production (20 ton/ha) and good quality. Spodoptera exigua has attacked shallot throughout the year. Larval stage of S. exigua has high feeding activity by eating all a part of leaves. Leaves have attacked by larva of S. exigua seem like transparant and then leaves look like droop and dry up (Kalshoven, 1981). Thus condition can make high losses for production and it will be worst when populations of S. exigua was increasing (Mau & Kessing, 2007; Khasanah et al., 2014). The S. exigua larvae attack shallot during vegetative growth stage and can cause yield loss of about 57–100% (Putrasamedja et al., 2012). According to Nurjanani and Ramlan (2008), variation of demage of S. exigua attack start from 3.89–100%, it depended of management of shallot cultivation. Heavy attack can caused high losses until 100%, if there was no controlling of pest attack and it can caused crop failure and production of shallot should be come down (Haryati & Nurawan, 2009; Khasanah et al., 2014). Commonly, farmers used synthetic insecticides to minimize losses and controlling S. exigua. Farmers were preferring to used synthetic insecticides because they are readily available, highly promoted, inexpensive, easy to apply and quick acting (Islam, 2012). Most of farmer used insecticides as intensively, and the pests are becoming resistant to almost all chemical pesticide as the frequency of spraying is gradually increasing while their efficacy is gradually decreasing. Wagiman et al. (2003) stated that the use of chemical insecticides is feared to leave residues on the shallot bulbs, and if the shallot bulbs were consumed in fresh condition it will have impact on ABSTRACT

INTRODUCTION
Indonesia is one of countries who love flavor taste so much. One of herb that must be in dish is shallot. Besides being used as a flavor, shallot also has high economic and nutrient value. Shallot has big role in life through of people especially to farmers, that's way most of farmers were attracted to cultivation a shallot. One of the centers of shallots production is Bantul, Yogyakarta. Within a year there are three planting seasons i.e. July to October, November to February, and March to June. Pest problem is one of major constraint for achieving higher production in shallot cultivation. Febrianasari (2014) stated that Spodoptera exigua Hubner (Lepidoptera: Noctuidae) is one of important pest that can be obstacle the farmers from getting the maximum production (20 ton/ha) and good quality.
Spodoptera exigua has attacked shallot throughout the year. Larval stage of S. exigua has high feeding activity by eating all a part of leaves. Leaves have attacked by larva of S. exigua seem like transparant and then leaves look like droop and dry up (Kalshoven, 1981). Thus condition can make high losses for production and it will be worst when populations of S. exigua was increasing (Mau & Kessing, 2007;Khasanah et al., 2014). The S. exigua larvae attack shallot during vegetative growth stage and can cause yield loss of about 57-100% (Putrasamedja et al., 2012). According to Nurjanani and Ramlan (2008), variation of demage of S. exigua attack start from 3.89-100%, it depended of management of shallot cultivation. Heavy attack can caused high losses until 100%, if there was no controlling of pest attack and it can caused crop failure and production of shallot should be come down (Haryati & Nurawan, 2009;Khasanah et al., 2014).
Commonly, farmers used synthetic insecticides to minimize losses and controlling S. exigua. Farmers were preferring to used synthetic insecticides because they are readily available, highly promoted, inexpensive, easy to apply and quick acting (Islam, 2012). Most of farmer used insecticides as intensively, and the pests are becoming resistant to almost all chemical pesticide as the frequency of spraying is gradually increasing while their efficacy is gradually decreasing. Wagiman et al. (2003) stated that the use of chemical insecticides is feared to leave residues on the shallot bulbs, and if the shallot bulbs were consumed in fresh condition it will have impact on ABSTRACT consumer health. There are many alternative approaches to control insect pest such as using pheromone technology like sex pheromone traps.
In most insects, pheromones are produced by glandular epidermal cells concentrated in discrete areas beneath the cuticle, but in some species, gland cells are scattered through the epidermis of different parts of the body. For click beetles, the male attractant pheromone is produced by female pheromone glands located at the last abdominal segment (Reddy & Tangtrakulwanich, 2014). According to Wyatt (2010), pheromones are chemical signals that are used to communicate between species of insects. Sex pheromones are specific chemicals released by females into the air to attract specific males for mating. Males follow the sex pheromone upwind to locate and mate with the female (Kong et al., 2014). Pheromone traps, which attract male S. exigua moths, can be an indicator of moth activity in the area. However, trap catch numbers may not necessarily relate directly to numbers of larvae in a field The utilization of sex pheromone to control insect pest is one of advance technology. The sex pheromone is one method to control insect pests for support food security. In other to know the ability of sex pheromone to trap insect the monitoring of insect using sex pheromone is needed. Sex pheromone can be used as a monitoring tool of pest population in the field and for mass capture of male insects. The sex pheromone disturbs the mating activities of insects so they will be fail to mate, as a result the produced eggs will not be fertilizing and hatching. The further impact is that population of S. exigua and damage rates will be reduced. The pheromone traps is environmentally sound due to not toxic. The sex pheromone was more widely used than the other pheromones as sex pheromones are more easily obtained (Martono, 1997). The pheromone traps can be used as monitoring and early detection of S. exigua infestation. A sex pheromone trap contains tetra hexadecenat asetat as active ingredients that are attached to yellow sticky plastic sheet. Number of sex pheromone trap per hectare may determine the effectiveness to trap the S. exigua male moths. This study was aimed to determine the appropriate number of sex pheromone traps for monitoring the S. exigua moths.

MATERIALS AND METHODS
The research was conducted from August until September 2016 in the shallot grown in Sanden, Bantul, Yogyakarta. The sex pheromone trap was obtained from Agritek Tani Indonesia Ltd. The trap is composed of a yellow sticky plastic sheet and the sex pheromone cointaining hexadecenat tetra acetat. The treated area was selected at shallot area with infestation intensity rate of S. exigua more than 20%. Field sampling conducted on shallot age of 3 weeks after planting.
This research is using RCBD experimental design with single factor experiment namely number of trap i.e. 3, 5, 6, 8 units/2,000 m 2 with sex pheromone, and control (3 traps without sex pheromone) with five replications. Experimental plot was a shallot plantation of about 2.000 m 2 or 50×40 m. Each block had a set of treatments that consisted of five treatments including control so that the total trial plot per block was 1 hectare and sum of sample was 125 unit. Inter plots were separated by difference distance depend on number of trap. Distance among 3 traps were 12.5 m, 5 traps were 8.3 m, 6 traps were 7.14 and 8 traps were 6.25 m. Distances among blocks were 200 m and among plots were more than 50 m. Distance among block was separated with other plant, street and houses. According to Kardinan (2003), metil eugenol was attractant compound on mating process (sex pheromone) of fruit flies, the potency of this compounds can attract insect for come around 20-100 m.
The observation parameter was the number of S. exigua moths caught. The observation was conducted at every 5 days since trap installation until the shallot ready to harvest. Sex pheromone trap was effective to catch S. exigua moths if the number of caught moths on treated plot was significantly than control treatment, because control treatment is yellow sticky trap without sex pheromone. The effectiveness of the sex pheromone trap was determined refering to based on the results of analysis of variance (ANOVA) and DMRT ∞ 5.0%. Meanwhile the appropriate number trap was determined according to the significant difference amongst treatment.

RESULTS AND DISCUSSIONS
The result of the number of moths caught for six times from August until September in Sanden, Bantul Regency showed that average cumulative number of S. exigua moths highly significant difference (F <0.0001) amongst treatments including control. Control (yellow sticky trap without sex pheromone) has significant difference with other treatment (3, 5, 6, and 8 trap with sex pheromone). There's no moths were trapped on control treatments, while in others treatments average number of moths were 9 until 36 moths/trap (Figure 1). It was mean sticky yellow trap with sex pheromone more effective to catch moth than sticky yellow trap without sex pheromone. Eight traps can catch until 36 moths higher than three traps of nine moths caught. Differences density of sex pheromone can effect for number of moths trapped.
The noctuid moths are active at night especially at full moon (Sukarsono, 2016;personal communication). At the night, the chemical sense was more functionate to detected synthetic pheromone than visual sense to detected color of trap (Santi & Sumaryo, 2008). There was no moth in control treatment but it was in other treatment, it was mean that at night the noctuid insect may not distinguish the color on trap (sticky yellow trap) but it was attracted on sex pheromone. According to Haryati et al., (2009) Sex pheromone of S. exigua was produced by female moth especially at night to attracted male moth to come n get mate. So can be sure that male moth was trapped in sticky yellow trap.
Average cumulative total number of the moth for these observations was used as a basic for determining the appropriate number of sex pheromone trap per hectare. Trap Installation at several days before planting can be useful for early pest detection. Pheromone with the active ingredient of tetra acetic hexadecenat is specific for male moths. If the trap was installed at beginning of plant, most of male moths were died while stuck on sticky yellow trap and the egg will not be fertilizing and hatching.
The average cumulative number of moth caught in the trap density of 3, 5, 6 and 3 traps without sex pheromone as control units/2000 m 2 were significantly lower than 8 units/2000 m 2 (Figure 1). The appropriate number of trap to monitor the pest infestation was 3 traps units/2.000 m 2 with sex pheromone, it can be appropiate number for monitoring because with a few trap can catch moth S. exigua. Pheromone traps can be useful as a tool for early detection and monitoring of the pest infestation. The egg of S. exigua was discovered when shallots are in 4-7 DAP of age (Sutarya, 1996). If the trap was installed at a week before planting and it caught S. exigua moths, it means that the trap can be used to prevent the pest infestation as early as possible.
In addition the sex pheromone traps are useful for early detection and monitoring of pest infestation and also it has the potential to control the pest by the way of trap and kill. Haryati and Nurawan (2009), stated that the sex pheromono has opportunity to be developed in shallots production centers, particularly in endemic areas of the shallot pest. The most important thing of monitoring is to determine if they are present in area or to determine if enough insect are present to warrant a costly treatment.

CONCLUSION
The sex pheromone trap with the active ingredient of tetra hexadecenat tetra asetat which attached on the yellow sticky sheet was effectively to capture S. exigua moths. The appropriate number of trap to monitor the pest infestation was 3 units/2.000 m 2 .

ACKNOWLEDGEMENT
Thanks and highly appreciation to Agritek Tani Indonesia Ltd. which had facilitated the study, shallot farmers in Sanden District, Bantul Regency who had allowed their shallot field for the study, as well as to all parties for their supports.


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The Potential of SSR Markers to Reveal the Genetic Diversity among Wheat and its Wild Relatives and to Test the Hybridity of F1s

Bread wheat (Triticum aestivum) is one of the big three globally important crops accounting for 20% of the calories consumed by the people and a staple crop of nearly 35% of the global population. The huge bread wheat genome is comprised of 17 Gb (17,000,000,000) base pairs which is about 5 times the human DNA content and about 40 times of rice genome size. However 8090 % of the genome is made up of repetitive sequences. This offers an ample scope for the use of SSRsthe Simple Sequence Repeats as molecular markers studies. Molecular genetics, or the use of molecular techniques for detecting differences in the DNA of individual plants, has got numerous applications in crop improvement.

Introduction
Bread wheat (Triticum aestivum) is one of the big three globally important crops accounting for 20% of the calories consumed by the people and a staple crop of nearly 35% of the global population. The huge bread wheat genome is comprised of 17 Gb (17,000,000,000) base pairs which is about 5 times the human DNA content and about 40 times of rice genome size. However 80-90 % of the genome is made up of repetitive sequences. This offers an ample scope for the use of SSRs-the Simple Sequence Repeats as molecular markers studies. Molecular genetics, or the use of molecular techniques for detecting differences in the DNA of individual plants, has got numerous applications in crop improvement.
The differences are called molecular markers because they are often associated with specific genes and act as "signposts" to those genes. Such markers, when very tightly linked to genes of interest, can be used to select indirectly for the desirable allele, and this The SSR technique was used to reveal the genetic diversity among wheat (Triticum aestivum) and its wild relatives and secondly to test the hybridity of wheat (Triticum aestivum) F 1 s. All the thirteen SSR primers showed 100% polymorphism and except one, all the primers generated unique bands, so these primers can be used for genotype identification. Also four primers showed heterozygous nature of F 1 s by giving two bands, one from each parent at a particular locus, so these markers can be used for screening purpose also. Use of these primers resulted in 180 polymorphic bands out of which 47 were unique.
represents the simplest form of Marker Assisted Selection (MAS). Molecular markers used to probe the level of genetic diversity among different cultivars, within populations, among related species, etc. have many applications like varietal fingerprinting for identification and protection, understanding relationships among the units under study, efficiently managing genetic resources, facilitating introgression of chromosomal segments from alien species and even tagging of specific genes (Hoisington et al., 2002). SSRs involve the use of specifically chosen primers to amplify the repetitive sequences through Polymerase chain reaction. The repetitive DNA of all the species is highly polymorphic in nature. These regions contain genetic loci comprising several hundred alleles, differing from each other with respect to length, sequence or both and they are interspersed in tandem arrays ubiquitously. The term microsatellite was coined by (Litt and Lutty 1989). SSRs are increasingly being used as genetic markers of chromosome segments (Dib et al., 1996), for identification of individuals (Anon, 1996), studying evolution and orthologous and paralogous relatedness (Rubinsztein et al., 1995 andAli et al., 1999) and wildlife conservation (Roca et al., 2001). The present study aimed at the use of SSRs to study the genetic diversity of wheat and its wild relatives at all ploidy levels (diploid, tetraploid and hexaploid) and secondly the codominant marker was also used to to test the hybridity of wheat (T. aestivum) F 1 s.

Materials and Methods
The study was conducted at G.B. Pant University of Agriculture & Technology, Pantnagar, Uttarakhand, India during 2004-08. The seeds of wild relatives of wheat were obtained from Directorate of Wheat Research, Karnal, Haryana, India. The experimental material consisted of 41 genotypes which included 10 wild relatives of wheat, 2 Triticum durum varieties, 15 Triticum aestivum varieties and 14F 1 s among them. The parentage of T. aestivum genotypes is given in Table 1. DNA characterization was done using 13 SSR primers. Primers were provided by Integrated DNA Technologies, Inc. Details of primers are given in Table 2.

Genomic DNA extraction
CTAB procedure was used for the isolation of DNA. CTAB (Cetyl trimethyl ammonium bromide) is a cationic detergent which solubilizes membranes and forms a complex with DNA. After cell disruption and incubation with hot CTAB isolation buffer, proteins were extracted by chloroform: isoamyl alcohol. CTAB-DNA was precipitated with isopropanol. The DNA pellet resulting after centrifugation was washed, dried and redissolved. RNase A treatment was given to remove RNA contamination.

Protocol followed
Two g of fresh wheat seedling leaves were ground to a fine powder using liquid nitrogen and a mortar and pestle. The powder was transferred as fast as possible into 15 ml of pre-warmed (60° C) isolation buffer in an oakridge tube. The oakridge tubes were then incubated in a water bath at 60° C for 30 minutes. It was mixed gently after every 10 minutes. One volume of chloroform: isoamyl alcohol (24: 1) was then added. The tube was capped and shaken gently and thoroughly for 10 minutes by hand, enough to ensure emulsification of the phase. Then it was centrifuged for 10 minutes (5000 rpm, room temperature). The (upper) aqueous phase was extracted once again with fresh chloroform: isoamyl alcohol. The final aqueous phase was transferred to a fresh tube using micropipette with a wide bore microtip (that of 1000 µl capacity). 0.6 volume of chilled isopropanol was added, the tube was capped and mixing was done gently but thoroughly by inverting the tube several times. At this stage, the DNA-CTAB complex precipitated as a whitish network. The solution was placed at -20° C for 30 minutes to overnight. Then it was centrifuged (10 min., 5000 rpm, 4° C). It was then washed with 70% ethanol; the pellet was gently agitated for a few minutes, and collected by centrifugation (10 min., 5000 rpm, 4° C). Residual CTAB was removed by this step. The tubes were inverted and drained on a paper towel for about 1 hour taking care that pellet does not slip down the wall of the tube. It was ensured that it neither contained residual ethanol nor it was too dry. In both cases redissolving might be difficult. An appropriate volume of 1 X TE buffer was added (say 500 µl) and the pellet was allowed to dissolve at 4° C without agitation.

Purification and quantification of genomic DNA
5 l RNase (10 mg/ml) was added to 100 l of dissolved DNA and incubated at 37° C for 1 hour. Equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added and mixed gently by inverting the tubes. The tubes were spun at 10,000 rpm for 5 minutes and aqueous layer (i.e. upper layer) was collected and equal amount of chloroform + isoamyl alcohol (24: 1) was added. The tubes were spun at 10,000 rpm for 5 min and the top layer of DNA was removed. To this, sodium acetate (1/10 vol, pH=5.2) and chilled absolute ethanol was added. The contents were mixed and kept at -20°C for 30 min. Finally the pellet was washed with 70 per cent ethanol, dried and dissolved in 100 l TE buffer.
The quantification of genomic DNA was done by taking the absorbance on Genesys UV spectrophotometer. The optical density was measured at 260 and 280 nm. The concentration of the DNA in the sample is related to optical density by the following formula: The ratio of OD 260/280 was an indication of the amount of RNA or protein contamination in the preparation. A value of 1.8 is optimum for the best DNA preparation. A value of the ratio below 1.8 indicates the presence of protein in the preparation and a value above 1.8 indicates that the sample has RNA contamination.

PCR amplification
The reaction mixture consisted of genomic DNA, d NTPs, Taq polymerase, reaction buffer, primers (forward and reverse) and double distilled water. The concentrations and quantity of components is given in Table 3. The PCR thermocycler was programmed according to the Electrophoresis of the amplified PCR products was done in horizontal gel electrophoresis assembly using agarose gel of 2.5 % concentration. Electrophoresis was done at 50 V for 4 hours in 0.5 X TBE buffer. After 75% run of the gel, its image was viewed and its photograph saved in a gel documentation system.

Data analysis
Gels were documented using Gel Doc system (Bio-Rad). Pair-wise similarity and cluster analysis were done on the basis of presence and absence of bands. Computer software (NTSYS) was used to perform the similarity matrix analysis using "UPGMA" with Jaccard"s coefficient of similarity.

Results and Discussion
All the13 SSR primers used in the study were polymorphic. They amplified total 180 bands out of which 47 bands were unique. 12 primers gave unique bands. The size of bands ranged from 100 to 3000 bp. The details of amplification pattern are provided in the Table 5.
All the thirteen SSR primers showed 100% polymorphism and except one, all the primers generated unique bands, so these primers can be used for genotype identification. Also four primers showed heterozygous nature of F1s by giving two bands, one from each parent at a particular locus, so these markers can be used for screening purpose also. Primer Barc 019 amplified maximum number of loci (24) and also gave maximum number of unique bands (10) followed by Barc 062 (7), Barc 142 (6), Barc 028 (5), and Barc 228 (4), whereas Barc 065, Barc 119 and Barc 154, each gave 3 unique bands whereas Barc 145, Barc 159 and Barc 025 gave 2, Barc 124 gave one unique band. So these markers can be used for the identification of genotypes. The wild species T. dicoccum showed highest number of unique bands (18) from 6 primers -Barc 019, Barc 025, Barc 142, Barc 154, Barc 159 and Barc 228 followed by Ae. Squarrosa (5) from 4 primers -Barc 019, B. Other wild species T. sphaerococcum, T. polonicum, T. monococcum also showed unique bands. T. durum variety PDW 289 and Secale cereale accession EC 481695 also showed unique bands. It can be inferred that these wild germplasms harbour drought tolerance characteristics and can be used as donor of drought tolerance trait in wheat breeding programmes.
T. aestivum variety WH 730 showed maximum number of unique bands. Varieties like UP 2565 and PBW 373 also showed unique bands which incates the possibility of developing drought tolerance in these varieties. UP 2425 showed 4 unique bands. The study of R. P. Meena et al., (2015) also suggests that UP 2425 performs better under moisture stress conditions based on several stress indices. The hills variety VL 804 also showed unique band confirming its drought tolerant nature. The cross Job 666 X UP 2565 showed unique band as well as the bands present at a particular locus in the parents were also present in the cross, primer Barc 154. This codominant nature of marker was shown in the cross NP 846 x UP 2425 by 2 primers Barc 028 and Barc 159. Barc 025 also revealed its codominant nature in the cross NIAW 34 x UP 2590 ( Fig. 1-3).

Relationship among wheat genotypes
Based on the estimated genetic similarity matrix using UPGMA method, the primers revealed highest genetic similarity value 0.7895 between VL 804 and its cross PBN 51 x VL 804 indicating the involvement of drought lines, followed by 0.7143 between 2 crosses WH 730 x UP 2425 and Job 666 X UP 2565 indicating the presence of drought tolerant parents WH 730 & Job 666 in the crosses, also the other parents UP 2425 and UP 2565 are the varieties released from the same place i.e. Pantnagar and both are recommended for irrigated late sown conditions. It was followed by the similarity value 0.6857 between HI 385 and its cross HI 385 x UP 2425, followed by 0.6786 between PBW 175 and a cross PBN 51 x UP2554 as PBW 175 is drought tolerant and the parental line PBN 51 of the cross is also drought tolerant. It was followed by the similarity value 0.6765 between UP 2590 and its cross NIAW 34 x UP 2590, followed by 0.6744 between 2 crosses Job 666 X UP 2425 and Job 666 x UP 2565 due to the common parent Job 666 between them.
It was followed by the similarity value 0.6667 between 2 Triticum durum varieties PDW 291 and PDW 289, followed by 0.6563 between UP 2338 and a cross NIAW 34 x PBW 373. It was followed by the similarity value 0.6486 between UP 2554 and its cross WH 730 x UP 2554, followed by 0.6250 in 3 pairs i.e. between UP 2338 and its cross PBN 51 x UP 2338, between 2 crosses PBN 51 x UP 2554 and PBN 51 x UP 2338 and between Halna and PBW 175.
The results indicate that SSRs can be very effectively used for molecular characterization of genotypes. SSRs are codominant markers which show bands in both the parents at different loci as well as both the parental bands in the cross. As we know that drought tolerance is a complex Quantitative trait loci therefore a lot of variations can be observed in the banding pattern of crosses. Further for QTL mapping or Gene tagging purposes the populations like Nearly Isogenic Lines, Doubled Haploids, Recombinant Inbred Lines, F 2 , Back cross should be used.    and Halna, followed by 0.0435 between Triticum dicoccum and UP 2590, followed by 0.0444 between Secale cereale EC 481697 and a cross NIAW 34 x UP 2590, followed by 0.0448 between Triticum dicoccum and a cross PBN 51 x UP 2338, followed by 0.0455 between 2 pairs i.e. between Secale cereale EC 481697 and a cross PBN 51 x UP 2338 and between Triticum teemopheevii and Triticum dicoccum, followed by 0.0462 between 2 pairs i.e. between Triticum dicoccum and UP 2338 and between Triticum dicoccum and a cross PBN 51 x UP 2554, followed by 0.0469 between Triticum dicoccum and PBN 51, followed by 0.0476 between Secale cereale EC 481697 and UP 2338, followed by 0.0484 between Triticum dicoccum and NP 846, followed by 0.0488 between Secale cereale EC 481697 and PBN 51 and so on.
The low similarity matrix values clearly indicate low genetic similarities or high genetic diversity and may be attributed to genomic differences in the study material.

Relationship among wheat and its wild relatives
SSR amplification patterns were analyzed to study the genetic diversity between wheat and its wild relatives. The F 1 s were excluded from this analysis. Based on the estimated genetic similarity the highest genetic similarity value (0.667) was observed between PDW 291 and PDW 289, as both of them are the two varieties of T. durum followed by 0.625 between Halna and PBW 175, followed by 0.620 between PBW 373 and NIAW 34, followed by 0.604 between UP 2554 and PBW 373, followed by 0.578 between T. tauschii and T. sphaerococcum, followed by 0.556 between Secale cereale EC 481697 and Secale cereale EC 48195, followed by 0.550 between T. turgidum and PDW 291 followed by 0.545 between PBW 175 and UP2590, followed by 0.525 between Halna and UP 2554, followed by 0.516 between PBW 175 and UP 2338 followed by 0.514 between 2 pairs i.e. UP2554 and UP 2338; PBW 175 and UP 2554.
The lowest genetic similarity value 0.021 was observed between Secale cereal EC 481697 and T. monococcum, followed by 0.028 between T. dicoccum and T. monococcum, followed by 0.038 between 2 pairs i.e. Secale cereal EC 481695 and UP 2554; Aegilops squarrosa and Triticum dicoccum, followed by 0.039 between 2 pairs i.e. Secale cereal EC 481697 and UP 2425; Secale cereale EC 481695 and T. monococcum.

References
Ali, S., Azfer, M. A., Bashamboo, A., Mathur, P. K. and Malik, P. K. 1999. Characterization of species specific repetitive DNA from a highly endangered wild animal Rhinoceros unicornis and assessment of genetic polymorphism by microsatellite