Introduction

Phaseolus vulgaris L. (Common bean) is an important legume crop having nutritional status. It belongs to the family Fabaceae, subfamily Papilionoideae, tribe Phaseoleae, and subtribe Phaseolinae. Originally, it was domesticated in Central and Southern America [1]. Later on, it was introduced into Africa and other parts of the world by the Spaniards and Portuguese. It is now widely cultivated in the subtropical and temperate regions of the world during the cool, dry season in tropical areas [2, 3]. The species shows wide variation in the growth, habit, pigmentation, pod and seed morphology [4, 5]. In India, common beans were introduced by Portuguese traders around 1700 B.C. [6]. It has evolved over millennia in the wide range of ecological and human environments.

The species is diploid with a chromosome number of 22 (2n = 2x = 22) and an estimated genome size of 650 million base pairs [7]. The species is predominantly self-pollinated with an exceptional 3 % outcrossing and therefore, represents an excellent material to explore and conserve the genetic diversity for human consumption and value addition [8]. It is traditionally a basic food crop in many developing countries serving as a major plant protein source for rural and urban areas [9]. The green immature pods of some varieties are cooked and eaten as vegetable while the seeds of other varieties are harvested at a later growth stage and consumed for their high protein content (22.9 %). The dry seeds are also good source of essential vitamins, minerals, soluble fiber, starch and phytochemicals [10]. The leaf is occasionally used as vegetable and the straw as fodder. The species is also cultivated for green manuring and erosion control globally.

Earlier studies using morphological, physiological and molecular analyses in common bean revealed the existence of two distinct centers of genetic diversity known as the Mesoamerican or small-seeded type developed in Mexico around 7000 years ago and Andean or large-seeded type gene pools developed in Peru around 8000 years ago [3, 11]. By the time Europeans arrived, beans were cultivated throughout the New World, in North America as well as Central and South America. Studies of chloroplast and mitochondrial DNA polymorphisms have also supported this hypothesis and allowed classifying beans from the centers of origin into diverse racial groups and even different domestication events [12–14]. Therefore, it is important to identify and preserve landraces as genetic resources for future generations [11, 15–18].

In India, P. vulgaris is known by the names like ‘Rajmash’ and ‘Frash bean (green bean)’ which grows in certain parts of the country. It is a minor pulse crop confined to Jammu and Kashmir, Himachal Pradesh, Uttarakhand, and eastern states of Uttar Pradesh and Bihar as it requires low temperatures for growth. Indian farmers are also growing it due to its good market price as it is mainly consumed as grain legume. The genetic improvement of P. vulgaris in India has been accomplished primarily by targeting breeding strategies based on consumer preference for seed size, shape and colour. A great diversity for agro-morphological traits like seed size, shape and colour also exists in Phaseolus germplasm of North-Western Himalayan region comprising the parts of Jammu and Kashmir and Himachal Pradesh States. Many common bean varieties have been released in India by different research institutes but still there is a need to select varieties which are genetically diverse.

In the State of Jammu and Kashmir, Phaseolus vulgaris is cultivated all over the Kashmir valley and most parts of Jammu province. Although it is one of the main Kharif (July–October) season crop; some new genotypes have been grown in Rabi (October–March) season too. The Rajouri and Poonch region of the province holds a great diversity in P. vulgaris and a large number of landraces and cultivars are grown in different areas of these two districts of Jammu and Kashmir State of India. There is enormous variation in morphological traits of the seed including size, shape and colour, which prompted the fingerprinting these varieties by using DNA markers. Therefore, the objective of the present study was to assess the magnitude of diversity available in P. vulgaris in Rajouri–Poonch region of J&K State. The information generated thereof can thus be used for two purposes, (1) to conserve the diversity present locally in the crop in this geographical region and (2) to make use of the diversity available in future crop breeding programmes for generating better varieties in the country.

Material and Methods

Material

Seed samples of 28 accessions of Phaseolus vulgaris L. were collected from different sites of Rajouri and Poonch districts of J&K State, in India (Table 1). The selection of seeds was performed on the basis of phenotypic traits. Three to five seeds in duplicate were sown in labeled poly bags (polyethylene bags) containing soil and vermicompost in the ratio of 3:1 for germination, so that leaf material could be collected from them for DNA isolation. These poly bags were then kept in the shade house in Lead Botanical Garden of the Baba Ghulam Shah Badshah University and regularly monitored for seed germination. Before sowing, seeds were kept in normal tap water at least for one day to initiate germination. For DNA isolation young tender leaves were harvested from 2 to 3 seedlings per accession after 10 days when the seedlings were 20–40 cm high and at five leaf stage. Three grams of fresh leaf material of each accession was taken for DNA extraction.

Table 1 Collection sites and number of accessions of Phaseolus vulgaris L. used in the study
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Methods

Morphological Analysis of Seeds and Seedlings

The morphological diversity of seeds of different accessions of Phaseolus vulgaris L. was studied by analyzing the seed coat colour, size, shape, length, breadth, thickness and weight. The average seed length, breadth, thickness and weight of each seed in different accessions were calculated by taking a random sample of 20 seeds from each accession. The size of the seeds was determined on the basis of weight of 100 seeds in grams as small (<25 g), medium (25–40 g), large (40–60 g) or very large (>60 g). This methodology was proposed by Singh et al. [19]. The shape of the seeds was determined following the methodology described by Munoz et al. [20] The morphological diversity of seedlings of different accessions of Phaseolus vulgaris L. was studied by measuring height of seedling above ground, number of leaves per seedling and inter-nodal length.

DNA Extraction

Total genomic DNA from 3 g of fresh young leaf tissue collected from 3 to 4 plants per accession, was extracted following the CTAB method proposed by Doyle and Doyle [21] with minor modifications. The integrity and concentration of the extracted DNA was determined by electrophoresis of these samples in 0.8 % w/v agarose gel and comparing the intensity of the resulting DNA bands with uncut Lambda DNA of the known concentration loaded alongside these samples. After the concentration of each DNA sample was known, it was diluted by adding autoclaved double distilled water, to a concentration of 25 ng/µl for PCR amplification.

Inter Simple Sequence Repeat (ISSR) analysis

ISSR reaction was performed with 50 ng of genomic DNA in 25 µl reaction volume containing 1X Taq buffer, 3 mM MgCl2, 0.2 mM of dNTPs, 0.5 mM primer and 0.5 U of Taq polymerase (all from Banglore GeNei Pvt. Ltd., India). Amplifications were carried out in a Mastercycler (Eppendorf, nexus gradient, Germany) under the following conditions: an initial denaturation at 95 °C for 5 min followed by 35 cycles each with denaturation at 94 °C for 1 min, annealing at 44–58 °C for 1 min depending upon the annealing temperature of the primer used and extension at 72 °C for 2 min and a final extension at 72 °C for 7 min. The amplified products were run on 1.8 % w/v agarose gel under a voltage of 75–80 V and after running for about 2 h, gels were photographed using gel documentation system (Alpha Innotech, USA). Reproducibility of profiles was determined by repeating all ISSR reactions at least two times. Out of 27 screened random ISSR primers 12 were selected for final amplification and diversity analysis on the basis of robustness of amplification, clarity and scorability of the banding patterns.

Statistical Analyses

Analysis of variance (ANOVA) within and between various morphological parameters was statistically tested using SPSS programme.

Amplified bands were scored as either present (1) or absent (0). Only the bands that were consistent across replicates and repeatable on two independent runs were included in the analysis. Population-wise Shannon’s information index (\( I = - \sum {{\text{p}}_{\text{i}} \log_{2} {\text{p}}_{\text{i}} } \), where pi is the frequency of a given amplified fragment) [22] and Nei’s genetic diversity [\( H = 1 - \sum {{\text{p}}_{\text{i}}^{2} } \), where, pi is the frequency of a given amplified fragment (amplicon)] [23] were calculated using POPGENE version 1.32 [24]. The degree of polymorphism was calculated at the population and species level using percentage polymorphic loci (P), Shannon’s information index (I) [25], and Nei’s genetic diversity index (H) [23]. Nei’s [23] genetic diversity statistics were used to measure the total genetic diversity (H sp ) as well as intra- population or within group genetic diversity (H pop ). The genetic differentiation between populations/groups of accessions, also known as coefficient of gene differentiation (G ST ), was calculated as G ST  = 1 − H sp /H pop. The gene flow was estimated by Nm = 0.5 × (1 − G ST )/G ST . Nm < 1 indicates a local differentiation of populations while Nm > 1 is evidence of little differentiation among populations [26]. Genetic distance (GD) estimates among population/groups were calculated using Nei’s [27] unbiased genetic distance coefficient.

Partitioning of ISSR variation in case of Phaseolus vulgaris L. was also estimated by AMOVA (analysis of molecular variance) based on the pair-wise squared Euclidean distances between the molecular haplotypes [28]. F ST [29] based pair-wise distances between groups of accessions were estimated using GenAlEx version 6.5 [30] in case of Phaseolus vulgaris L. The distance matrix was subjected to Neighbour joining cluster analysis by NEIGHBOR programme of PHYLIP version 3.5 [31]. The NJ trees were viewed with Tree View programme [32].

Results and Discussion

Morphological and Genetic Variability

The seeds that were collected from the different sites of Rajouri and Poonch region exhibited enormous amount of morphological diversity. Seed coat colour ranged from dark red, reddish brown, dark brown, light brown, black, purplish and white to mottled. The shape varied from ovoid to elliptic, kidneyed and long almost squared. On the basis of seed size, it appears that Mesoamerican gene pool has predominance in the region (Table 2).

Table 2 Phaseolus vulgaris L. germplasm and its characterization
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The average seed length and breadth varied from 0.99 to 1.70 cm and 0.65 to 0.90 cm respectively. The average seed thickness and weight varied from 1.81 to 2.47 cm and 0.201 to 0.545 g respectively. Analysis of variance (ANOVA) indicated greater variation existing within the groups than between groups of accessions (Table 3).

Table 3 Analysis of variance (ANOVA) for 6 groups of accessions of Phaseolus vulgaris L.
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Seeds of 27 accessions were able to germinate and develop to seedling stage. Variation with respect to height and inter-nodal length was observed in the ten day old seedlings. Number of leaves in all the seedlings of different accessions remained to five after 10 day of growth and development. The height of seedlings varied from 22.5 to 45 cm and their inter-nodal length ranged from 8 to 22.5 cm.

Among 27 accessions from 6 groups, 92 bands were generated of which 5–47 (5.43–51.09 %) were polymorphic within groups of accessions. The number of bands generated per primer was 6–10, in the size range of 250–2500 bp, with an average of 7.66 bands per primer. Out of the 92 bands generated, 51 (55.43 %) were polymorphic and 41 (44.56 %) were monomorphic. Primer ISSR07 showed maximum number of polymorphic bands (5 amplicons). The percentage of polymorphic bands for different primers ranged from 28.57 to 83.33 %. Out of the total 12 selected primers, 7 displayed more than 60 % polymorphism (Table 4).

Table 4 Details of 12 ISSR primers used for the final amplification and level of polymorphism detected
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Analysis of ISSR data revealed that Loran Mandi group of accessions had the lowest frequency of polymorphic bands (5.43 %), while Buddhal group of accessions had the highest polymorphism (51.09 %; Table 5). Nei’s genetic diversity (H) and Shannon’s information indices (I) were lowest for Loran Mandi group of accessions (H = 0.0272; I = 0.0377) and highest for Buddhal group of accessions (H = 0.2228; I = 0.3200; Table 5).

Table 5 Polymorphic percentage, Nei’s genetic diversity (H) and Shannon’s information indices (I) for ISSR in Phaseolus vulgaris L.
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Shannon index based analysis calculated the total species diversity (H sp ) 0.1240 and average diversity within groups of accessions (intra-site) (H pop ) 0.2180 (Table 6). The proportion of diversity among groups of accessions (inter-site) (G ST ) was 0.4311. The latter comprised a 0.5689 proportion of the total diversity i.e., 56.89 % of the total was contributed by within group diversity. AMOVA showed similar results with 75 % variation existing within groups (Table 7).The variation observed among groups of accessions was 25 %.

Table 6 Shannon’s estimates of genetic diversity within and among groups of accessions of Phaseolus vulgaris L.
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Table 7 Analysis of molecular variance for 6 groups of accessions of Phaseolus vulgaris L.
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The F ST distance between groups of accessions ranged from 0.007 (between Thannamandi and Loran Mandi) to 0.730 (between Chandi Marh and Loran Mandi) (Table 8). For confirmation, two accessions from each site were randomly selected and analysed, the results obtained were comparable to that of the total number of accessions. Overall, Loran Mandi (LM) group of accessions had the greatest distance from other groups followed by Bakori (BK) and Chandi Marh (CM). The value of gene flow (Nm) was 0.6598. Neighbour joining tree showed two major clusters, one comprising Loran Mandi (LM) group and the other cluster having rest of the 5 groups of accessions (Figs. 1, 2).

Table 8 F ST distances between 6 groups of accessions of Phaseolus vulgaris L.
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Fig. 1
figure 1

Neighbour joining dendrogram showing relationship among 6 groups of accessions of P. vulgaris L.

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Fig. 2
figure 2

ISSR amplification profile of 27 accessions of Phaseolus vulgaris L. 1–5, Bakori; 6–9, Buddhal; 10, 11, Loran Mandi; 12–15, Thannamandi; 16–24, Manyal Galli; 25–27, Chandi Marh, M, marker (100 bp)

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The importance of Phaseolus vulgaris L. in Indian agriculture, particularly in hilly areas of Jammu and Kashmir State is increasing because of its nutritious food components and good market price. However, the current status of average national yield of the crop is lower than the international average, because full of its genetic potential has not been effectively harnessed in the country. Diversity analysis of the crop is important for two reasons, first to decipher genetic relationships between cultivars including paternity and second its use in breeding programs for the production of improved varieties and efficient management of germplasm.

Morphological variability analysis was performed to establish the unique identity of each accession. Qualitative morphological trait analysis of seeds and seedlings clearly indicate that significant amount of diversity is present in the accessions of Phaseolus vulgaris L. Analysis of variance (ANOVA) clearly indicated that high variation exist within groups than between groups of accessions. Besides, the present study also indicated that all the accessions were different from one another and were also in accordance with the classification and result of Singh et al. [19] and Munoz et al. [20]. These analyses were very useful not only in differentiating them as separate accessions, but to perform further analysis using DNA markers.

It has been observed that significant levels of variation in response to the selection pressure lead to the evolution of varieties in distinct agro-climatic zones [33]. It is, therefore, not surprising to find significant levels of polymorphism within the groups of accessions (intra-site) (56.89 %) and among the groups of accessions (inter-site) (43.11 %) of the species during the present study. This was not unexpected, since the ISSR technique amplifies microsatellite regions that are potentially polymorphic [34]. The success of the present study in identifying polymorphism is due to the use of a number of randomly selected pre-screened highly informative primers.

The values of Nei’s genetic diversity (H) and Shannon information indices (I) for the species are indicative of significant genetic polymorphism existing in the species. Nei’s genetic diversity (H) and Shannon’s information indices (I) were lowest for Loran Mandi (LM) group of accessions (H = 0.0272; I = 0.0377) and highest for Buddhal (BD) group of accessions (H = 0.2228; I = 0.3200). It was observed that although most of this diversity was distributed within groups of accessions (0.5689), a significant amount also exists among groups as indicated by G ST value [23] of 0.4311 (H pop  = 0.2180, H sp  = 0.1240), which also have been reported by some other workers, who studied the species at other geographical regions in the world [35–38].

The pattern and distribution of genetic diversity observed in the species during present study could be attributed to the occurrence of independent domestication events at different locations where breeding system and selection pressure could have played a role in fixing the genetic diversity. Similar kinds of results were observed among numerous wild populations of P. vulgaris in Mexico [39]. The results of the present study also point to the theory of multiple domestication centers [14], and constant germplasm mobilization among producing regions and vice versa.

When results of the present study were analyzed for the extent of molecular variance, it was observed that large amount of genetic diversity (75 %) existed within groups of accessions (intra-site) and (25 %) among groups of accessions (inter-site). Similar kind of results in the species has been recorded by Cattan-Toupance et al. [36], Papa and Gepts [37] and Gill-Langarica et al. [38], indicating that geographic structure is also one of the factors responsible for conditioning genetic differentiation in the species. The present observation also goes in line with these workers, who observed more genetic diversity component within populations. These findings can also be attributed to the seed exchange among farmers and homogenous selection in different environments [37].

High variance component within population has also been observed in chickpea (Cicer arietinum), a member of Fabaceae. AMOVA revealed 73 % variance within populations and 27 % among populations [40]. This species is diploid [41] and strictly self-pollinating [42]. Some reports however, indicate that the level of polymorphism in the species depends on the type of germplasm [43], markers used [44, 45], primers selected [45, 46] and the sampling strategy [46].

Variation presently observed within and among groups of accessions also needs to be exploited for germplasm selection, and broadening the genetic base of the common bean accessions. The amount of genetic diversity encountered in the presently collected germplasm from different sites of Rajouri and Poonch region indicates that there must have been independent domestication events in common bean as all these sites are geographically isolated. The genetic diversity observed in this sample of accessions is a representative sample of the total genetic variability contained in the gene pool of P. vulgaris. Geographically isolated populations accumulate genetic differences as they adapt to different environments as was also observed in the present study.

The present study reveals that F ST distance between groups of accessions ranged from 0.007 (between Thannamandi and Loran Mandi) to 0.730 (between Chandi Marh and Loran Mandi). On the whole, Loran Mandi (LM) group of accessions had the greatest distance from other groups followed by Bakori (BK) and Chandi Marh (CM). Neighbour joining tree also revealed Loran Mandi (LM) group of accessions to be genetically most distinct of all other groups of accessions. Its distinctly different genetic architecture can be ascribed to the kind of climate of the area that this group of accessions inhabit. The other reason might be only a few accessions of Loran Mandi (LM) group taken for analysis. The dendrogram also revealed two major clusters, one comprising Loran Mandi (LM) group and the other cluster having rest of the 5 groups of accessions.

Future Perspective

It is to undertake diversity assessment programme involving all the agroclimatic zones in India where the crop is grown.

Conclusion

On the basis of present study it is concluded that Rajouri–Poonch region is having a rich diversity of P. vulgaris and the gene pool has the predominance of Mesoamerican race. The information generated thereof can thus be used for the conservation of this diversity and future crop improvement programmes in the country.