Study of genetic polymorphism of grain pea cultivars

. Pea ( Pisum sativum L., 1753) is an essential pulse crop of global importance, which is a source of complete high-quality protein. Breeding new pea cultivars with improved economic valuable traits and high adaptive abilities is important for increasing the production of valuable high-protein pea grain. The key role in breeding is assigned to the initial material. A decrease in the genetic diversity of the initial material of cultivated plants, including pea, has become the main problem of modern breeding. In this regard, the issue of studying and preserving the genetic polymorphism of pea is relevant. Molecular genetic markers of simple sequence repeat (SSR) are widely used for this purpose. Here we aim to (1) to assess morphobiological and economical valuable traits and (2) analyze the molecular genetic diversity of the original pea material. The object of the research work was 20 grain pea cultivars. The yield structure elements of these cultivars were evaluated, and their molecular genetic polymorphism was analyzed using SSR markers AA5, AA355, AB28, AB71, and AD147. Early-ripening and highly productive cultivars were identified; they can be recommended for use as the initial material for pea breeding. As a result of the SSR analysis, 26 polymorphic alleles were identified, on average 5.2 alleles per locus. All studied cultivars were distinguished by a unique combination of alleles at five microsatellite loci, which allowed them to be identified. Based on the data of molecular genetic analysis, a cluster analysis was carried out, and a dendrogram was constructed. On the dendrogram, the studied cultivars were grouped according to the features of their morphological and economically valuable traits.

Agro-climatic conditions of the Republic of Bashkortostan contribute to the production of pea grain with good saleable quality.In this area, the pea is considered a traditional crop, and the history of its cultivation began in the first half of the 19 th century.In 1970-1980, the pea cultivation area in the republic was maximum and amounted to 290-300 thousand hectares [7].In recent years, the acreage of pea has been decreased to 55-60 thousand hectares.
Pea is used as food for humans and feed for farm animals.Pea grain has high protein content and a significantly higher balance of amino acid composition than cereals.Vegetable protein occupies an important place in the nutrition of the population.Enriching the diet of farm animals with pea can significantly reduce the feed consumption of feed for the production of livestock products and make its cost cheaper [6; 25].
In addition, the pea is used as a green manure crop.Due to symbiosis with nitrogenfixing bacteria, the pea can accumulate up to 100 kg of nitrogen in the soil during the growing season.Thus, pea has a noticeable positive effect on the productivity of other crops when it is introduced into the crop rotation [13; 20; 22].
Breeding of new, highly productive, adaptive, technological pea cultivars plays an important role in increasing the production of pea grain.In this case, the key role belongs to the initial material for pea breeding [8].For many years, the selection of parental pairs by the method of contrasting traits, which is based on genetic remoteness, has been used in the Bashkir Research Institute of Agriculture [14].The use of modern molecular genetics methods makes it possible to reduce the labor intensity of breeding work, shorten the analysis time, and supplement the results of the field evaluation of the genetic diversity of cultivars used as source material for pea breeding.
Some researchers have shown that one of the most applicable DNA markers for detecting genetic polymorphism are microsatellites [5; 24].These markers are simple sequence repeats (SSRs) consisting of monomers with a length of one to six base pairs.SSR markers are characterized by a uniform distribution in plant genomes, and their localization is not limited to non-coding regions [18; 26].Many of them are found in the coding sequences of genes, which allows the development of the so-called gene SSR markers based on available expressed sequence tags (ESTs) [24].
The study of molecular genetic polymorphism of different types of cultivated plants using microsatellite markers is actively performed in Russia and abroad [1; 2; 3; 16].Many microsatellite loci were also found in the pea genome, and the microsatellite genomic library (Agrogene®, France) was created.However, the progress in the development of effective microsatellite markers and marker-assisted breeding is significantly slowed down due to the large size (4.45GB) of the pea genome [28].Allele distribution of microsatellite loci in the genotypes of some pea cultivars remains almost unexplored, which limits the practical use of SSR markers.A set of microsatellites for the genetic identification of grain pea cultivars has not yet been determined.In this regard, the genotyping of pea cultivars using SSR markers is relevant.

Materials and methods
The study aimed to assess the breeding value and genetic diversity of grain pea cultivars.The following tasks were set: (1) to study the phenology of pea cultivars; (2) to evaluate pea samples by productivity elements; (3) to study the genetic polymorphism of pea cultivars; and (4) to conduct a cluster analysis based on genotyping of pea samples.
The study was conducted in 2020-2021.Field experiments were performed in the Laboratory of selection and seed-growing of pulse crops of the Bashkir Research Institute of Agriculture; molecular genetic polymorphism was analyzed in the Laboratory of plant genomics of the Institute of Biochemistry and Genetics.
Twenty grain pea cultivars, obtained from the Vavilov Institute's genebank of plant genetic resources or developed at regional breeding centers, were selected as materials in the current study.The soil of the experimental site is represented by carbonate chernozem with a humus content of 8.1%-8.4%, total nitrogen -0.4%, exchangeable potassium -104 mg/kg, and mobile phosphorus -107 mg/kg.The acidity of the soil is neutral (pH = 7.0).Sowing seeds in the collection nursery was performed with a selection seeder SKS-6-10.A preceding crop in our experiments was winter rye.Agrotechnics was the one generally accepted for the zone.The plant nutrition area was 20 × 5 cm.The experimental plot area was 3 m 2 .
Total DNA from completely dried pea leaves was extracted by cetyltrimethylammonium bromide (CTAB) [21].Moreover, for each plant line, the sample was five plants (n = 5).The quality of the isolated total DNA was determined by electrophoresis in 1% agarose gel.For the SSR analysis, five primer pairs were used to amplify loci AA5, AA355, AB28, AB71, and AD147 (Table 1) from the genomic library of microsatellites (Agrogene®, France), which were previously used for the analysis of pea [17].Source: [17].
PCR was carried out in a total volume of 30 μl.The reaction mixture for the SSR analysis contained the following components: 1 unit Taq polymerase (Evrogen, Russia), 3 μl of a 10-fold buffer for Taq polymerase, 2.5 mM MgCl2, 0.25 mM each dNTP, 90 pM each primer from a pair, and 0.2-0.5 μg of total DNA.The following amplification protocols were used: initial denaturation at 94°C for 3 min, followed by 35 cycles with each at 94°C for 40 sec, at the annealing temperature for each primer (ranging from 50°C to 55°C) for 40 sec, at 72°C for 40 sec, and final extension at 72ºС for 5 min.
The SSR amplification products were resolved by polyacrylamide gel electrophoresis.The acrylamide concentration of the gel was 10 %.Electrophoresis was performed at room temperature at 400 V for 4-6 hours.
Phenological observations, field surveys, and evaluation of breeding material were carried out according to the Methodology of state variety testing of agricultural crops [11].The information content of the studied SSR markers was estimated by the polymorphism information content (PIC) value calculated using the following equation (1): where: Pi -the frequency of the ith allele at locus j.
Statistical analysis was conducted in accordance with the methods of field experience  [9] in Microsoft Office Excel 2010.Cluster analysis was carried out using StatSoft® STATISTICA 13.3 program.

Results
The duration of the growing season is the most important biological and economically valuable trait that determines the distribution area of the cultivar.During the growing season, phenological observations for pea plants were carried out.Dates of germination, flowering, and ripening were marked.In the studied pea cultivars, the duration of the growing season ranged from 67.3±2.2 to 75.2±3.3days (Table 2).We have identified earlyripening cultivars Flagman 7, Multik, Flagman 12, Tyumenets, Orel 11, Ufa 75, and Velvet.
The number of pods per plant depends on the number of productive nodes and pods per productive node.In our experiments, the cultivars Vatan, Gotik, Vakhshskiy 1, Ukaz, and Ufa 75 were distinguished by the maximum number of pods per plant ranging from 6.4±0.4 to 7.3±0.5 pcs (Table 2).
The trait "number of seeds per pod" is a rather variable trait, which is largely influenced by environmental factors.In the studied grain pea cultivars, the number of seeds per pod varied from 3.2±0.1 to 6.3±0.3 pcs (Table 2).The highest values of this trait were observed in the cultivars Vakhshskiy 1, Aksayskiy Usatyi 55, Melkosemyannyi 2, Omskiy 18, and Flagman 12.
The trait "number of seeds per plant" is the most important indicator for evaluating the productivity of cultivars; it is determined by the number of productive nodes, pods per productive node, and seeds per pod.In our study, the cultivars Vakhshskiy 1, Vatan, Gotik, Flagman 7, and Melkosemyannyi 2 were characterized by the highest number of seeds per plant ranging from 25.5±1.5 to 42.3±2.5 pcs (Table 2).The weight of 1,000 seeds depends on cultivar features and environmental factors.This trait is an indicator of the seed size.In the studied grain pea cultivars, the values of the weight of 1,000 seeds varied from 104.3±9.3 to 290.3±15.6 g (Table 2).The cultivars Velvet, Flagman 12, Vatan, and Ukaz were characterized by the largest seed size.
The weight of seeds per plant (seed productivity) is determined by the ratio of many components.Their importance in yield formation is not the same in different conditions of pea growing.Ultimately, seed productivity is influenced by the number of seeds per plant and the weight of 1,000 seeds.In our experiments, the cultivars Vatan, Gotik, Flagman 7, Ukaz, Ufa 75, and Omskiy 18 were distinguished by the heaviest weight of seeds per plant varying from 6.02±0.47 to 8.41±0.60 pcs (Table 2).
According to the results of our research, we have identified grain pea cultivars with a complex of economically valuable traits: Flagman 7, Flagman 12, Gotic, Omskiy 18, Ukaz, Ufa 75, Vatan.These and other cultivars from the collection nursery were used as parental pairs in hybridization.
As a result of molecular genetic analysis of 20 grain pea cultivars using five microsatellite markers, 26 alleles were identified, on average 5.2 alleles per locus.All analyzed cultivars were distinguished by a unique combination of alleles at five microsatellite loci.All markers delivered good electrophoretic profiles and helped amplify a number of alleles per locus varying from three (AB28) to seven (AA355).Fig. 1 shows the electrophoregram obtained by amplifying the SSR marker AB71.According to the number of detected polymorphic SSR amplicons for the studied pea cultivars, the following series was obtained: AA355 > AB71 > AA5 ≥ AD147 > AB28.The polymorphism information content value was calculated for each microsatellite marker.Depending on the locus, the PIC varied from 0.40 (AB28) to 0.78 (AA355, AB71) with an average of 0.70.
The resulting molecular genetic analysis data were clustered using the nearest neighbor method, and the dendrogram showing the distribution of cultivar genotypes according to their genetic relationship was constructed (Fig. 2).In order to interpret the dendrogram, we used the data obtained during the evaluation of the studied grain pea cultivars according to economically valuable traits.It can be seen that early ripening and late ripening cultivars were grouped into two large clusters.Within the first cluster, which includes cultivars with the duration of the growing season ranging from 67.3±2.2 to 71.0±2.5 days, four groups may be distinguished.One of them includes pea cultivars Flagman 7, Omskiy 18, Ukaz, and Ufa 75 characterized by semi-large and large seeds and high seed productivity.The cultivars Flagman 12, Orel 11, Tyumenets, and Velvet with semi-large seeds were combined in another group.Small-seeded pea cultivars Vakhshskiy 1 and Melkosemyannyi 2, as well as Multik and Shrek, were grouped separately.
The second large cluster, including cultivars with the duration of the growing season varying from 67.3±2.2 to 71.2±2.5 days, was divided into three groups.One group is formed by grain pea cultivars Gotik and Vatan distinguished by a large number of pods and seeds per plant and high weight of seeds per plant.The other two groups include pea cultivars with medium (Aksayskiy Usatyi 55, Batrak, and Tomas) and low (Faraon, Fregat, and Kaban) seed productivity.

Discussion
The study of the initial pea material in various natural and climatic zones is important for breeding cultivars adapted to the conditions of the particular region.In modern breeding programs, research on early ripening, increasing productivity, and improving the quality of agricultural products is a priority [27].The literature data analysis shows that in the conditions of different regions, it is recommended to use different pea cultivars as donors of a number of economically valuable traits [19; 25; 27].We have revealed cultivars that are promising for use in grain pea breeding in the Southern Urals.
The results of the SSR analysis obtained in this study are consistent with the data of other authors on the high efficiency of microsatellite markers for cultivar identification [5; 16; 17; 24; 26; 28].It is considered that the larger the PIC value for a given locus, the more informative this locus as a marker.The following classification of PIC values is accepted: (1) for PIC > 0.5, the locus is highly informative; (2) at 0.5 > PIC > 0.25, it is reasonably informative; and (3) at PIC < 0.25, it is slightly informative [4].Thus, in our study, the SSR markers AA5 (PIC = 0.75), AD147 (PIC = 0.77), AB71 (PIC = 0.78), and AA355 (PIC = 0.78) were highly informative, and AB28 (PIC = 0.40) was reasonably informative.
Clustering of the genotypes of grain pea cultivars on the dendrogram (Fig. 2) demonstrates their distribution into groups in accordance with their phenology and productivity indicators, which may specify the linkage of microsatellite markers with the genes of economically valuable traits, and is consistent with the results obtained by us in previous studies [14; 15].

Conclusion
As a result of our experiments, early ripening grain pea cultivars with a large number of seeds per pod, pods and seeds per plant, high weight of 1,000 seeds, and seeds per plant were identified.They can be used in pea selection as sources of economically valuable traits.The constructed dendrogram (Fig. 2) indicates the genetic diversity and shows the features of the studied cultivars clustering in accordance with the assessment of their economically valuable traits.This dendrogram provides additional information for the efficient selection of parental pairs in hybridization.

Fig. 2 .
Fig. 2. A dendrogram showing the genetic relationships among the pea grain cultivars based on the data of the SSR analysis (bootstrap support values greater than 50 are given).Source: Compiled by the authors.

Table 1 .
SSR markers used for molecular genetic analysis of grain pea cultivars.

Table 2 .
Indicators of economically valuable traits of grain pea cultivars.
Source: Compiled by the authors.