Crossability and Genetic Diversity Studies in Pea (Pisum sativum)

Crossability of various accessions of pea on the basis of pollen development and fruit set was studied along with genetic diversity by SSR markers. This study was taken up with 37 pea accessions to analyze genetic diversity with the help of 17 SSR primers. From these accessions, 6 random accessions were selected for the crossability studies on the basis of their fruit set. Among the selfing, the maximum pollen fertility was observed in Pant-5 (98.31%) and minimum in VRP-228 (94.11%). Pollen germination after 24 hours in VRP-372 was maximum amongst parents (22.52%) and the least was found in AP-3 (17.73%). In crosses, maximum pollen germination after 24 hours was found in VRP-61 X Pant-5 (17.69 %) and the minimum in VRP-21 X VRP-372 (13.93 %). The longest pollen tubes after 24 hours were observed amongst parents in Pant-5 (895.67μm) and the smallest in VRP-372 (785.89μm). In the crosses the maximum pollen tube growth was in VRP-372 X Pant-228 (1000.44μm) and minimum in VRP-61 X VRP-21 (723.21μm). The maximum fruit set among parents was 95.22% in Pant-5 and the least in VRP-228 (83.22%) and in crosses it was recorded in VRP-61 X VRP-228 (87.50%) and the least in VRP-21 X VRP-228 (76.47%). The data was subjected to correlation studies which showed that there was nonsignificant correlation between pollen germination, pollen tube growth and fruit set. A total of 17 SSR primers were selected to assess genetic diversity of 37 accessions of pea. Based on the PIC value, SSR AA446 and SSR AA473were found to be more informative. The data obtained from SSR primers were subjected to generate Euclidean distance and clustered dendrogram was made [1], which indicated that Morana-2 and PS are more distinct from each other and VRP-262, GS-10, Parthana, VRP-176 and VRP-243 were found to be very less distinct.


Introduction
Pea (Pisum sativum L.) is a leguminous, annual herbaceous plant with a one-year life cycle. Pea is considered a cool season crop with planting taking place from winter to early summer depending on the location. Green pea is eaten cooked as a vegetable and is marketed fresh, canned or frozen. Pea is a self-pollinated, annual and diploid crop having 14 chromosomes (2n=14, n=7). Due to continual self-pollination in pea, undesirable linkage can hamper recombination and hence crop improvement. The yield of pea is low in India as compared to the world average yield owing to the narrow genetic base and limited variability used in the development of improved varieties. The current area and production of pea in India is 475.9 thousand ha and 4651.5 thousand MT, respectively [2]. For the success of hybridization, the interaction between pollen tube and various pre-fertilization processes affect cross ability. For an effective breeding programme, information concerning the extent and nature of genetic diversity within a crop species is essential.
It is particularly useful for characterizing individual accession and cultivar and as a general guide in the selection of the parents for the hybridization. Molecular markers have enormous potential to explore genetic diversity by detecting polymorphisms to improve the efficiency and precision of conventional plant breeding. SSR markers are the most widely applied technique for genetic diversity analyses in crop species [3]. SSR markers are highly reliable because they are reproducible, co-dominant in inheritance and generally highly polymorphic. This paper reports pollen germination and pollen tube behavior in relation to cross ability between various accessions of pea and seed set and genetic diversity using SSR primers.

Materials and Methods
A total of 37 pea (Pisum sativum) genotypes were used in the current investigation. These were planted in the pots with three replications and kept in the Field and the Greenhouse in rabi season, 2016. Complete details of the collected germplasm along with their respective places are given in Table 1. The maximum number of genotypes (19) was collected from Uttar Pradesh, of which 17 genotypes (designated as VRP) were collected from IIVR, Varanasi. The rest of the genotypes (18) were collected from Madhya Pradesh (2 genotypes), Tripura (6 genotypes), Manipur (4 genotypes), Assam (3 genotypes), Uttar Pradesh (2 genotypes) and Telengana (1 genotype). For pollen germination and pollen tube growth observations, the pollinated flowers were gently rinsed in distilled water and pistils were separated from the flowers after which they were kept in a drop of 1N HCl for 10 minutes. They were again rinsed in distilled water and stained in 1 percent aniline blue [4,5]. The time required for staining was 10-20 seconds depending on the thickness of the style and the stage of penetration of the pollen tube in the stigma.
The pistils were then rinsed in distilled water and mounted in pure lactic acid and studied under the microscope. The pollen grains and pollen tubes stained deep blue.

Results and Discussion
Pollen fertility In this study, maximum pollen fertility was observed in Pant-5 (98.31%) whereas the lowest pollen fertility (94.11%) was recorded in VRP-228 (Table 3). However, the average percent pollen fertility did not show too much difference among the accessions. Munhoz [7] performed an experiment related to the fertility of pollen grain in case of Carica papaya. In this experiment, they compared the effectiveness of in vitro germination technique and pollen staining test in papaya variety i.e. Sunrise Solo. In case of in vitro germination technique, germination was higher in the media without essential elements but with a higher concentration of agar (65% compared with 51.5%). In another case, they used five stains namely 2,3,5-triphenyl tetrazolium chloride, Alexander's stain, acetocarmine, iodine-potassium iodide and Sudan IV. They found that the pollen viability estimated with TTC was 67.5% and similar to germination results providing a reliable estimate of in vitro germination. But the four other stains overestimated pollen viability (>90%) (Figure 1).

Pollen germination
In crosses between the parents, the maximum pollen germination was recorded in VRP-61 X Pant-5 (9.68%) and the least was found in VRP-21 X AP-3 (3.71%) after 8 hours of pollination. 446 after 24 hours of pollination. Monika [8] reported that the common pre-fertilization barriers found to occur in many wide crosses are pollen-pistil incompatibility and stated that these barriers are influenced by delayed pollen grain germination and pollen tube growth of one species on the stigmas of another species. Erdogan [9] reported that the effects of sucrose concentration on pollen germination were statistically significant with increase in sucrose concentration resulting in increase of germination and it decreased after that point.

Pollen tube growth
The emergence of pollen tubes on the stigma and the style was    In some crosses pollen tube growth was more than in the selfings Cheung [10] observed that pollen tube penetrates the pollen grains coat through preformed apertures in the exine. They reported that almost all metabolic activity is localized in the tip of the pollen tube. Georgieva [11] reported that the highest germination percentage also correlated with the greatest pollen tube length, regardless of cultural medium. Rangkham [12] reported a positive correlation between pollen germination and fruit set in cowpea accessions. It was like that also between pollen tube growth and fruit set. Similar results were obtained by Debbarama [13] in Capsicum during wide hybridization and also by Kharkongar [14] during wide hybridization in Solanum. Table 4 shows that the maximum fruit set was obtained in selfing as compared to crosses. Among the selfing, the maximum fruit set was obtained in Pant-5 (95.22%) and the least in VRP-228 (83.32%). When the parents were crossed, the maximum fruit set was obtained in VRP-61 X VRP-228 (87.50%) and the least fruit set was obtained in VRP-21 X VRP-228 (76.47%). In general, fruit set was less in crosses. From this study, it was observed that fruit set had a non-significant correlation with pollen fertility, pollen germination and pollen tube growth. Suganiya [15] observed that boron treated plants showed higher percentage (91.4%) of fruit set than untreated control (62.77%). This may be due to role of Boron in maintaining of cell integrity, enhancing respiration rate and metabolic activities. Vez [16] examined the pollinator contribution to the production of cowpea. They found that higher fruit set in the presence of pollinators (83%) than in their absence (77%). They also observed that cross pollination reduced both the number of seeds per pod and fruit set relative to self-pollination.

Correlation studies in crosses of Pisum sativum
Correlation studies on pollen germination showed nonsignificant values for percent viable pollen. However, pollen germination after 8 hours of pollination was highly correlated with pollen germination after 16 hours of pollination and 24 hours of pollination had a highly significant correlation with pollen germination after 8 hours and 16 hours of pollination, respectively (Table 5). Similarly, pollen tube growth recorded non-significant correlation with percent viable pollen. Pollen tube growth after 8 hours of pollination was observed to be negatively correlated with pollen germination after 8 hours, 16 hours and 24 hours of pollination. In the same way, pollen tube growth after 16 hours of pollination was observed to be highly correlated with pollen tube growth after 24 hours of pollination.  Jaitly [17] reported that percent pollen germination was more directly correlated with seed set, and the rate of pollen tube growth and percent pollen tube abnormalities do not seem to be affecting seed set to that extend. And there was also positive correlation with pollen tube growth with fruit set (0.162, 0.123 and 0.030).

SSR analysis
Twenty-one primers were used for genetic diversity analysis Euclidean distance matrix analysis of the data, the genotypes were grouped into two major clusters (Figure 7).   that high level of genetic diversity among the 37 genotypes. These scores are higher than previous pea studies [14]. Kumari [14] analyzed genetic diversity among 28 genotypes using 32 SSR markers. Among them 44 polymorphic bands per primer were obtained ( Figure 10).   Table 1).

Figure 10:
Gel image showing SSR profile obtained by AA473 primers. Lane 1-37 represent the genotypes (Genotypes name given in Table 1).
The variation in genetic diversity among these cultivars ranged from 0.11 to 0.73. In this present study polymorphic information content (PIC) values ranged from 0.23 to 0.50 with an average of 0.33 which is higher in previous study [19]. Smykal [19] surveyed 164 pea accessions using 10 SSR loci. A total of 53 alleles were identified with a minimum three and maximum eight alleles per locus. Twelve rare alleles (22%) with frequencies below 0.05 were found at six SSR loci. Calculated PIC values were high, ranging from 0.697 to 0.964, with an average of 0.89. Pyngrope [25] did genetic diversity study in blackgram with SSR markers and found these markers very useful. Ahmad [26]