The local landraces of peas in Kashmir as well in Pakistan indicated larger diversity while comparison were made on yield and yield related characters. To confirm their diversity on the basis of DNA, known primer was employed using PCR techniques and standard gel electrophoresis methodologies. The banding patterns were compared and photographs were taken for reference. Twenty different primers for pea varietal discrimination were used to find out the molecular diversity among 46 local landraces of peas.
Out of 20 amplification product, 595 were found out of which 325 were monomorphic and 257 were polymorphic. The average number of the scorable bands per primer was 29.75% while average numbers of monomorphic and polymorphic bands were 16.25% and 12.85%, respectively. The high frequency of polymorphism was detected with all selected primers. The percentage of polymorphic bands was 100% with three primers i.e; AC58, AD270 and AA90, while low value of polymorphism was recorded for primer AA205 and AB141 (0%), AA103 (21.4%), AA285 (24.4%), AD147 (26.8%), AA175 (27.3%) and AA67 (30%), respectively. Moderate values of polymorphism were recorded for primer AB72 (505), AA92 (52.6%), D21 (53.6%), AA163.2 (55.8%), AA355 (60.7%), AD148 (66.7%), AD51 (68.2%) and AB53 (73.7%), respectively.
A study on genetic diversity among varieties and hybrid lines of Pea (Pisum sativum L.) as revealed by morphological traits and SSR markers was conducted by [2]. The results showed that the number of total bands and polymorphic bands and the percentage of polymorphism in the five parent varieties were generally higher than their corresponding values in their hybrid lines. A maximum number of 72 bands was scored in Var. Master B and include 43 polymorphic bands including two unique bands scoring 59.97% polymorphism. The other four varieties showed lower percentages of polymorphism; the two parents Lincoln and Little marvel have identical percentage of 51.6%; the variety Sugarless has a percentage of 50.0%. The hybrid lines Lincoln x Little Marvel, Lincoln x Sugarless and Little Marvel x Sugarless showed lower proportion of polymorphism (34.1%, 35.6% and 36.9% respectively). On the other hand, the two hybrid lines (Luxer x Master B and Master B x Little Marvel) showed higher percentage of SSR polymorphism (47.2% and 49. 1%) compared to other hybrid lines. The other 17 hybrids have intermediate percentages of polymorphism ranging between 38.3% in the two hybrid lines Little Marvel xLuxer and Sugarless x Master B to 45.3% in the hybrid line Master B x Lincoln.
Simple sequence repeat markers were developed depending upon Expressed Sequence Tag-SSR and utilized for polymorphism between 23 Pea genotypes to help in advancement as well as improvement of pea linkage maps. In general, the SSR markers were constructed to aid in mapping of white mold disease tolerance quantitative trait loci (QTL). Primer pairs were intended for forty six simple sequence repeats detected in EST contiguous sequences gathered from a 454-pyrosequenced transcriptome of the pea genotypes, ‘LIFTER’. Thirty seven SSR markers augmented PCR amplifications, of which eleven (30%) SSR markers reproduced polymorphism in 23 members, with parents of recombinant inbred lines with 2–4 alleles. The observed and expected heterozygosities were between 0 to 0.43 and 0.31 to 0.83, correspondingly [23].
Molecular markers have been utilized to concentrate on inherited diversity as well as breeding to have enormous potential to enhance up the procedure of rising improved cultivars. The simple sequence repeats (SSR), also recognized as microsatellites, have been used on many collections of peas as a key basis of difference [17] and in amalgamation with morphological dissimilarity. Though more than a few 100’s of SSR markers have been documented [23].
Hirrareical Cluster
The cluster diagram constructed by mean of NTSys.pc revealed two main clusters I and II at distance of about 0.38. Cluster I was comprised of only one genotype; L-46. Cluster II was subdivided into two sub clusters IB and IIB. Sub cluster IB was comprised of only two genotypes (M-86 and M-08) corrrelating each other at same linkage distance. Sub cluster IIB was comprised of two sub sub clusters IIB1 and IIB2. IIB1 was comprised of only one genotype namely M-39 whereas cluster IIB2 was further sub divided into three sub sub sub clusters, IIB2a, IIB2b and IIB2c. IIIB2a was comprised of 6 genotypes namely; L-31 and L-33, L-30 and L-34 were correlating each other at same distance while L-29 and L-32 were outliers for the cluster. IIB2b was consisting of two genotypes, M-22 and M-72. IIB2c was comprised of 34 genotypes. L-2 and L-3, L-6 and L-6 and L-7, L-9 and L-10, L-12 and L-16, l-13 and L-14, L-15 and L-18, L-26 and M-25, L-21 and M-116, M-91 and M-07, L27 and L-28 were correlating to each other at same linkage distance. While L-1, L5, L-8, L-11, L-24, L-19, L-17, L-25, L-23, M-102 and M-83 were outliers in the clusters shwing variation. Similar studies were carried out by [9] who studied the genetic diversity among 28 pea (Pisum sativum L.) genotypes using 32 simple sequence repeat markers. Cluster analysis based on Jaccard’s similarity coefficient using the unweighted pair-group method with arithmetic mean (UPGMA) revealed 2 distinct clusters, I and II, comprising 6 and 22 genotypes, respectively. Cluster II was further differentiated into 2 subclusters, IIA and IIB, with 12 and 10 genotypes, respectively.
[1] assessed the inherited variability in thirty five Pisum genotypes by fifteen polymorphic microsatellites located on various Pisum chromosomes. Microsatellites were originated to be polymorphic, amplifying a total of 41 alleles and were able to distinguish all 35 Pea germplasm. These markers were counted by their PIC, ranging from 0.055 (AA206) to 0.660 (AB72) with a mean of 0.460, in addition to their discerning power (D), which values from 0.057 (AA206) to 0.679 (AB72) with an mean of 0.475. UPGMA cluster analysis alienated the thirty 5 Pisum cultivars into two major clusters and eight sub-clusters. The hereditarily changeable genotypes recognized in this study could be used to obtain parental lines for Pea breeding.
Another collection of scientist, [9] assessed 28 pea cultivars to display inherited multiplicity using 32 SSR. Principal component analysis uncovered results same to those of UPGMA. The 1st, 2nd and 3rd PCs displayed 21.6, 16.1, and 14.0% of the variation, correspondingly; total variation of the first 3 PCs was 51.7%.
Correlation Studies
The correlation matrix was computed by mean of Dice similarity coefficient. Similarity matrix was ranging between 0.18 and 0.98. Maximum similarity was noticed among L-7 and L-6 (0.97) followed by L-14 and L-13 (0.97), L-3 and L-1, L-15 and L-13, L-17 with L-13 and L-15, L-19 with L-12, L-15, L-13 and L-17 (0.94), L-5 and L-3, L-20 and L-18 (0.93), L-10 and L-9 (0.92), L-15 and L-14, L-16 with L-10 and L-14, L-19 and L-14 (0.92), L-4 and L-2, L-9 and L-8, L-12 and L-10, L-19 and L-18, L-6 and L-2 (0.91), L-4 and L-3, L-6 and L-5, L-20 and L-12, L-20 with L-17 and L-19 (0.90), L-13 and L-12, L-16 with L-13 and L-15, L-19 and L-16, L-2 and L-10 (0.89), L-5 and L-2, L-7 and L-2, L-10 and L-7 (0.88), L-15 and L-12, L-17 and L-12 (0.88), respectively.
Moderate values for similarity were found for L-15 and L-2 (0.80), L-16 with L-2 (0.81) and L-9 (0.84), L-19 and L-4 (0.75), L-20 and L-4 (0.76), L-25 and L-24 (0.86), L-26 and L-24 (0.80), L-30 and L-24 (0.48).
Minimum values of similarity coefficient were noticed among L-33 and L-46 (0.18), L-42 and L-33 (0.31), M-86 and L-33 (0.29), L-33 and M-08 (0.33), L-33 and M-79 (0.29), M-79 and L-32 (0.22), M-08 and L-32 (0.33), M-79 and L-31 (0.33), L-29 and L-46 (0.29), L-23 and L-24 (0.22), respectively. The genetic diversity among 28 pea (Pisum sativum L.) genotypes was analyzed using 32 simple sequence repeat markers by [9]. A total of 44 polymorphic bands, with an average of 2.1 bands per primer, were obtained. The polymorphism information content ranged from 0.657 to 0.309 with an average of 0.493. The variation in genetic diversity among these cultivars ranged from 0.11 to 0.73. [18] reported a relatively higher similarity range (0.80–0.94) with RAPD markers compared with that obtained using AFLP markers in pea cultivars (0.85–0.94). [3] Obtained a very wide range of similarity (0.0–1.0) in 148 Pisum genotypes using protein and PCR-based markers. In this study, the estimated genetic diversity (0.05–0.82) among pea accessions based on SSR markers was higher than that reported by [20] (0.0-0.66) and [7] (0.05–0.48). The higher estimated genetic distance could be ascribed to differences between accessions owing to diversification in the pedigree of the genotypes.