Genetic Divergence in French Bean Accessions

The French bean (Phaseolus vulgaris L.) is one of the most important annual diploid leguminous vegetables in India. It is rich source of essential amino acids like lysine in contrast to animal protein. It is also rich in carbohydrates, dietary fibre minerals, antioxidants like polyphenols and vitamins (Broughton et al., 2003). Due to its high nutritional value, importance was given to enhance its yield and conservation by adopting better management practices and thorough understanding of genetic variation among different genotypes of French bean. Genetic diversity has the evolutionary significance for the survival and adaptation of species in different agro-climatic conditions. If there is not enough genetic diversity among genotypes, it is practically not possible to increase the yield and other desirable characters of the crop. A complete understanding of the genetic diversity and population structure of the French bean is essential for its conservation and management.
       
Hence, it is important to know the extent of existing genetic variations in the plant material. Many studies have been devoted to assess genetic diversity within and among cultivated and wild French bean genotypes based on phenotypic traits, isozymes (Belletti et al., 1996) and seed protein analysis (Lioi et al., 2005). Moreover, during the last few decades, the use of different molecular markers was used to study the genetic diversity of plants. Random Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) have been used for genetic characterization in many crop species including common bean (Duran et al., 2005; Tiwari et al., 2005; Kumar et al., 2008; Hanai et al., 2007; Kumar et al., 2014).
       
Because of the limitation of morphological and biochemical markers, efforts were being directed to use Simple Sequence Repeats (SSRs) to study the genetic diversity of common beans (Hamman et al., 1995; Gopinath et al., 2013; Zargar et al., 2016; Jannat et al., 2022). SSRs were used to study the allelic variation within a locus. However, Inter Simple Sequence Repeat (ISSR) is an alternative marker system for RAPD and it is used to study the allelic variation among multi locus. But few studies were conducted to assess the genetic diversity of French bean by using ISSR markers (Galvan et al., 2003, Vyas et al., 2018, Sharma et al., 2020, Janghel et al., 2021). So, in the present investigation, an attempt was taken to study the genetic variation among French bean genotypes collected from different agro-climatic zones of India by ISSR markers.

Plant materials
 
The plant materials used in this investigation consisted of twenty-three french bean genotypes including released varieties collected from various parts of India. The list of the genotypes along with their source is presented in Table 1.


 
Experimental procedure
 
The experiment was conducted during the year of 2015-16 at department of Agriculture Biotechnology, College of Agriculture, OUAT, Bhubaneswar. The data of two growing seasons over four locations were collected, pooled and statistically analyzed under randomized complete block design by SARS software. Planting was done at a spacing of 30 cm between the rows and 10 cm between plants. The experimental fields were well prepared and all the recommended package of practices were followed in raising the crop. The laboratory experiment was conducted in the Department of Agriculture Biotechnology, OUAT, Bhubaneswar, Odisha. Four to five young and fresh leaves of each genotype were collected from field and wrapped in aluminium foil and brought to the laboratory for DNA extraction.
 
Observations recorded
 
The observations were recorded in both the growing seasons on different morpho-physiological characters in the field as well as in the laboratory after the crop harvest. Five randomly selected plants were tagged and observations were recorded on these plants for different characters in each replication. The data recorded on five plants per treatment were averaged over locations over years.
 
DNA extraction                                                                                                                                                     

Total DNA of a single leaf was isolated according to the protocol established by previous investigators Doyle et al., (1997). The isolated total DNA was air-dried at room temperature and re-suspended in 200 µl of 1 x Tris EDTA buffer and stored at -0.20°C for further use.    
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Polymerase chain reaction (PCR) amplification using ISSR markers
 
A total four nos. of markers (USB-807 [17bp;AG(8)T], USB-810 [17bp;GA(8)T], USB-835 [18bp;AG (8)AC], USB-836 [18bp;AG(8)TC]) were employed for  PCR amplification. The PCR reaction was carried out in a thermal cycler (Peglab, Germany) with 45 cycles (94°C-5 minutes, 94°C-1 minute, 50°C-1 minute, 72°C-2 minute, extension at 72°C for 2 minutes and stored at 4°C). The amplified products were separated by electrophoresis on 2% agarose gel with ethidium bromide and photographed under UV light using transillumination system (GelDoc. System, UNIVTECH, Cambridge).
 
Scoring and data analysis
 
Data collected during the two growing seasons on these traits were pooled and analysis of variance was done as suggested by Panse and Sukhatme (1985). Genetic diversity was studied using D² statistics of Mahalanobis (1928) and populations were grouped into clusters by following the method suggested by Rao (1952). Cluster analysis was done using the Sequential Agglomerative Hierarchical Nested (SAHN) cluster analysis procedure of NTSYS software version 2.1 which uses the unweighted pair group method with arithmetic averages (UPGMA) to obtain genetic relationships and to cluster varieties described by Sneath and Sokal (1973). The results were used to construct a final dendrogram showing 23 accessions.

Yield and yield attributes
 
The data obtained in the study were pooled and analyzed. The mean performance of 23 genotypes in respect of different quantitative characters is presented in Table 2. In respect to 50% flowering, the genotypes exhibited a wide range of variation from HAFB-5 (31.14) to Swarna Lata (55.59) with an overall mean of 43.43. Days to 1st green pod picking varied from FAB var-6 (52.17 days) to HAFB-4 (81.33 days) with an overall mean of 65.46 days. The magnitude of variability of plant height varied from IIHR-4 (35.94 cm) to Swarna Lata (121.76 cm) with an overall mean of 61.22 cm. The number of primary branches per plant was maximum in HAFB-6 (5.26) and minimum in IIHR-4 (2.95) with an average value of 3.90. Number of secondary branches per plant ranged from Almora Local-1(4.39) to HAFB-6 (9.49) with an average of 6.50. The variation in basal internodal length ranged between IIHR-4 (1.22 cm) to HAFB-4 (7.73 cm) with an average value of 2.44 cm.
 

       
The variability in basal internodal diameter ranged from 0.34 cm (HAFB-3) to 0.81cm (Arka Anoop) with an average value of 0.58cm. The magnitude of green pod length ranged from HAFB-3 (10.17 cm) to HAFB-5 (16.89 cm) with an average of 13.93 cm. The extent of variability with respect to green pod width ranged from 0.51 cm (IIHR-4) to 1.02cm (HAFB-6) with average value of 0.75 cm. The magnitude of green pod breadth ranged from 0.87cm (Kashi Param) to 1.15 cm (HAFB-5) with average value of 1.00 cm. The mean value for number of pods per plant was 14.34 with a range of 6.98 (PDR-14) to 25.27 (HAFB-5). The genotype Arka Suvidha exhibited maximum single pod weight (9.33 g) with an average value of 7.56 g. The green pod yield per plant ranged between 44.26 g (IIHR-4) to 158.61 g (HAFB-5) with an average value of 93.82 g.
 
Analysis of variance
 
The analysis of variance for thirteen different quantitative characters was carried out and the results are presented in Table 3. It revealed that there was significant difference among genotypes for all the characters under investigation except basal internodal diameter, green pod width, green pod breadth. The environmental differences were significant for all the character except basal internodal diameter, green pod width, green pod breadth. However, the genotype ´ environment interactions were significant for the traits such as days to 50% flowering, days to 1^st green pod picking, plant height, green pod length, number of pods per plant, single pod weight and green pod yield per plant.
 

 
Genetic diversity
 
Genetic diversity was studied using D² statistics of Mahalanobis (1928) and populations were grouped into clusters by following the method as suggested by Rao (1952). The genotypes were grouped into seven clusters. Among the clusters, maximum number of genotypes (7) were included in cluster II followed by cluster I (7), cluster III (4), cluster IV (2), cluster V (2). Cluster VI and VII were mono-genotypic cluster consisting of one genotype each indicating their independent identity and importance due to various unique characters possessed by them (Table 4). The formation of largest cluster II comprising 7 genotypes might be due to a free flow (or) exchange of breeding material from one place to another.
       

 
The present pattern of grouping of genotypes indicated that the genotypes have grouped or diverged into different clusters irrespective of their geographical origin which means that the genetic constitution of the varieties was more dominant than their geographical origin while forming a cluster (Choubey et al., 2003, Singh, 2006). This kind of genetic diversity was recorded among the genotypes belonging to the same geographic origin might be due to differences in adoption, selection pressure and selection criteria and environmental condition as suggested by Gokulkrishnan et al., (2012). According to Nancee et al., (2013) choice of the parents for hybridization should be decided on the basis of genetic diversity rather than geographic diversity. Inter cluster distance values were greater than intracluster distance value suggesting heterogeneous and homogeneous nature of the strains between and within the clusters respectively (Table 5). The highest inter cluster distance value was observed between cluster V and VII, followed by IV and VI. Based on the above studies, it could be suggested that crosses involving genotypes from divergent clusters (V and VII, IV and VI, IV and VII, III and V) are likely to exhibit high heterotic expression for yield component and wider segregation in filial generations. Similar lines of observations were obtained by Patel et al., (2011).
 

 
Morphological diversity by hierarchical cluster
 
An un-weighted pair group method with arithmetic mean (UPGMA) analysis was made and a dendrogram was constructed using Jaccard’s similarity matrix involving data generated from four ISSR primers on 23 genotypes of French bean (Fig 1). These genotypes were grouped into five clusters as observed in the dendrogram. One genotype, Kashi Param was completely isolated from all other clusters. If Kashi Param is excluded, Swarna Priya and IIHR4 were the most distantly related parental pair as evident by lowest similarity value: 0.32 (Table 6). The genotypic pairs- Swarna Priya and VL Bean-3; Swarna Priya and HAFB-3; Swarna Priya and Contender; Swarna Priya and Arka Anoop; HUR 137 and HAFB-5; Arka Komal and FAB Var-3 also appeared to be fairly distant from all other genotypes. By excluding Kashi Param, at a similarity index value of 0.52, the remaining genotypes were divided into two cluster viz. I and II. The first cluster include 15 cultivars which was further divided into two sub-clusters viz. I-A and I-B. The first sub-cluster (I-A) contained four French bean cultivars (FAB Var-3, HAFB-6, HAFB-5 and FB-53). The second cluster (I-B) having eleven French bean cultivars was further grouped into two sub-clusters (I-B-1 and I-B-2) at a similarity value 0.65. Cluster I-B-1 contained five cultivars (PDR-14, IIHR-4, Vl bean-1, Swarna Lata and Arka Sharat); whereas cluster I-B-2 contained six cultivars (Arka Anoop, Contender, HAPB-4, HAFB-3, Pant Anupam and VL Bean 3). In this sub cluster, cvs. Arka Anoop and Contender clustered at a similarity value 0.88 depicting very close ancestry relationships. In cluster II, cv. Arka Komal cluster at a similarity value of 0.55, appeared to be distant from the other three accessions (Almora Local 1, Arka Suvidha and VL Bean 2). The cluster III was made up of three phenotypically divergent genotypes viz. Swarna Priya, HUR137 and HAFB-4, although Swarna Priya separated itself from the rest of the two genotypes.
 

 

 
Polymorphism of genotypes based on ISSR markers
 
Divergences among genotypes were analyzed by ISSR markers. The average numbers of clear bands generated per polymorphic primer were 7, with a maximum of 8 for primer USB-835 (Table 7). The four di-nucleotide motif primers under the current study resulted in the amplification of a total of 27 bands and among them 18 (66.66%) were found to be polymorphic. The percentage of polymorphic bands (PPB) relative to the total number of amplified bands, ranged from 11.11% (USB-836) to 22.22% (USB-807). Out of four primers used, primers USB-835 and USB-810 provided the clearest banding pattern. Although most of the bands generated with primer USB-836 were monomorphic, some diversity between and within cultivars was observed (Fig 2).
 

 

       
Our study showed that a high degree of genetic diversity existed within French bean germplasm tested, (64.28% bands are polymorphic). In previous survey of Galvan et al., (2001) in French bean, 53% of the ISSR makers generated with tri-nucleotide motif primers were polymorphic. However, our results suggest that ISSR- PCR products generated with di-nucleotide motif primers can be useful markers for analyses of French bean germplasms. Similarity value based on Jaccard’s coefficient clearly demonstrates that overall similarity between genotypes was low, reflecting strong genetic differentiation among the lines tested and our results were in accordance with previous investigators (Alghamdi et al., 2011; Immaculee et al., 2015, Jannat et al., 2022).

The genotypes used in the present studies showed a wide variation in morphological characters like plant height, pods per plant, pod weight, pod length and yield per plant. The genotype HAFB-5 had shown the maximum potential for pods yield per plant due to their high number of pods per plant and high pod length. ISSR analysis revealed high levels of genetic variation, even with the use of a limited set of primers. The present study also shows that ISSR markers can be a useful tool for the assessment of genetic diversity among French bean genotypes for the efficient utilization of germplasms in the breeding programme.

None.