Estimation of genetic relatedness among cultivated and wild lens based on morphological and molecular markers  

K Tewari , H K Dikshit , Jyoti Kumari , Neelu Jain , D Singh , Akanksha Singh
Division of Genetics, ICAR-IARI, New Delhi, 110012; Germplasm Evaluation Division, ICAR-NBPGR, New Delhi-110012-India
Author    Correspondence author
Plant Gene and Trait, 2015, Vol. 6, No. 1   doi: 10.5376/pgt.2015.06.0001
Received: 22 Jan., 2015    Accepted: 17 Mar., 2015    Published: 16 Apr., 2015
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Tewari et al., 2015, Estimation of genetic relatedness among cultivated and wild lens based on morphological and molecular markers, Plant Gene and Trait, Vol.6, No.1 1-8 (doi: 10.5376/pgt.2015.06.0001)


Lentil is an important rainfed legume grown in the winter season. Low productivity due to its narrow genetic base is the cause for concern. The objective of this study was to analyze the genetic diversity in sixty cultivated lentil genotypes based on yield and other agronomic traits. The studied genotypes were grouped in three clusters. Hundred seed weight, plant height and number of secondary branches contributed 26.98%, 17.88% and 15.95% respectively towards the genetic diversity. Intercluster distance revealed narrow genetic base of cultivated lentil. The 60 cultivated genotypes alongwith 23 wild types were also characterized using molecular markers. Dendrogram based on Jaccard similarity coefficient and UPGMA analysis revealed two major clusters and one minor cluster. Twenty-one wild accessions of L. orientalis and 1 L. ervoides subspecies grouped separately into cluster 1. Nineteen Indian varieties grouped together in subcluster IIA indicating their narrow genetic base. Subcluster IIB consisted of 41 genotypes including 5 exotic and 36 advanced breeding lines due to use of exotic lines into recent breeding programmes. Therefore it is implied from this study that prebreeding of lentil is very essential using wild spp and exotic collection in hybridization programme, which is required for broadening of the genetic base and introgression of useful traits.

Lens; Lentil; Diversity; Molecular markers; RAPD; SSR; Wild spp

Lentil (Lens culinaris subsp. culinaris) is a self pollinated grain legume of Vicieae tribe and Leguminosae family.This crop originated in Near East and was first domesticated in Fertile Cresent around 7000 B.C. (Zohary, 1972a; 1972b). Lens orientalis is considered to be progenitor of the cultivated lentil. The cultivated lentil is classified as macrospermas or microspermas (Barulina, 1930) which have been considered as subspecies, races or varieties by different researchers and breeders. Lentil is grown mainly in Indian Subcontinent, Middle East, North Africa, Southern Europe, North and South America, Australia and West Asia. Lentil occupied 1.38 million hectare in India with a production of 0.96 million tons during 2008-2009. In India pilosae types of lentil is grown, however during last two decades few macrosperma types were introduced from ICARDA and used in the breeding programmes mainly for transferring the large seed size and earliness in Indian cultivars. In the recent years demand for large seeded varieties has increased from main lentil producing region (Madhya Pradesh and Bundelkhand region of India) due to its export potential and commercial uses such as dalmoth manufacturing.

A narrow diversity in lentil limits the scope for new recombinations. Hybridization of diverse genotypes from different agro-climatic zones of India, and involvement of exotic lines in breeding programmes has helped in broadening the genetic base. Wild species have been found as potent source of donor for several important traits such as biotic (Tullu et al., 2010) and abiotic stresses and quality traits.The highest frequency of resistance was found in accessions of L. ervoides which originated from Syria and Turkey followed by L. nigricans and L. orientalis. These wild accessions represent a useful and untapped source for disease resistance in lentil.
Further, exotic collections from ICARDA have exhibited potential for broadening of the genetic base of indigenous cultivars. Early maturing and bold seeded exotic introduction, Precoz has been successfully used for developing cultivars with short duration, large seed size and rust resistance. The major bottle necks limiting higher yield of this crop are susceptibility to biotic and abiotic stresses, poor harvest index, and lack of genetic variability and non availability of suitable ideotypes for different cropping systems.
The evaluation of genetic diversity promotes the efficient use of genetic variations in the breeding program (Paterson et al. 1991). Conventionally, morphological data and pedigree information were routinely used to assess the genetic diversity howeverthese studies have limitations of less phenotypic markers, high G x E interactions and paucity of accurate records of ancestry. DNA markers provide an opportunity for precise characterization of genotypes and measurement of genetic relationships in better way than other markers (Soller and Beckmann, 1983). DNA-based marker systems have been used, both alone and in tandem with morphological markers to obtain more reliable information on the genetic diversity existing in a number of species. In genus Lens genetic diversity have been assessed by using RFLPs (Havey and Muehlbaur, 1989), RAPD and ISSR (Duran et al., 2004), ISSR (Fikiru et al., 2007) AFLP (Abo-el-wafa et al., 1995; Sharma et al., 1996) and SSRs (Hamweih et al., 2009; Datta et al., 2011). In the present study, genetic diversity was surveyed in lentil germplasm, released varieties and related species employing agronomic traits and molecular markers.
1 Materials and Methods
1.1 Diversity analysis based on morphological traits
Sixty cultivated accessions of lentil (Table 1) were used for multivariate analysis as proposed by Mahalanobis’s D2-statistics, Tochers method of clustering and combined analysis of variance based on agro morphological traits. Material for this study comprised of 60 elite genotypes of lentil (Lens culinaris subsp. culinaris) including 19 released varieties, 5 exotic introductions and 36 advanced breeding lines. Data were recorded on morpho-agronomic traits that include days to flowering, plant height (cm), days to maturity, no. of pods/plant, seeds/pod, primary branches, secondary branches, yield/plant (g) and100 seed weight (g). The experiment was laid out in Randomised Complete Block Design with two replications. Each entry was sown in four rows of 5 m length. The row to row distance was 25 cm and plant to plant distance within row was 5 cm. The standard cultivation practices prescribed for lentil were followed precisely. ANOVA for different characters were tested for genotypic differences by using analysis of variance technique (Panse and Sukhatme 1954). The D2 analysis was performed as per Mahalanobis (1936) and Rao (1952) using SPAR I software.

Table 1 Details of different Lens species used in diversity analysis

1.2 Diversity analysis based on molecular markers
Eighty three genotypes including 23 wild and 60 cultivated lentil (Table 1) were used in molecular diversity analysis using RAPD and microsatellite markers. Total genomic DNA from 5 gm of fresh young leaf tissue, collected from five random plants per accession, was extracted following the cetyl trimethylammonium bromide (CTAB) method as described by Murray and Thompson 1980.The total genomic DNA was diluted to 20 ng/µl for PCR analysis. Twenty nine SSR primers reported by Hamwieh et al. 2005 and 52 RAPD primers procured from Operon Technologies Inc.,USA (kits A, B, C, O, U, V) were analyzed. Eight SSRs and fifteen RAPD primers among them produced repeatable and clear bands and hence used for further analysis (Table 4). The PCR amplicons were resolved by electrophoresis and scored for presence and absence of bands.
1.3 Data analysis
Fragments amplified by primer sets were scored manually in terms of positions of the bands relative to the ladder sequentially from the smallest to the largest-sized bands. Diffused bands or bands revealing ambiguity in scoring were considered as missing data and designated as ‘9’ in comparison with ‘1’ for the presence of a band and ‘0’ for the absence of a band in the data matrix. A binary matrix was then transformed to genetic similarity (GS) matrix using Jaccard’s coefficient (Jaccard, 1908). A dendrogram based on similarity coefficients was prepared by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using statistical software NTSYS-PC 2.02 (Rohlf, 2000). Polymorphism information content (PIC) values for each primer was calculated based on allele frequencies according to Smith et al. (1997). Gene diversity parameters and partitioning of variation between genotypes (GST) (equivalent to FST for biallelic loci) was calculated with Popgene ver 1.32 (Yeh et al, 2000) assuming Hardy-Weinberg equilibrium. Analysis of molecular variance (AMOVA) (Excoffier et al.,2006) was used to estimate variance components among genotypes using Arlequin ver 3.01.
2 Results and discussion
2.1 Diversity analysis based on morpho-agronomic traits
In all breeding programmes knowledge of genetic diversity is an important factor and also an essential prerequisite for hybridization programme to obtain high yielding progenies. Percent contribution of different quantitative traits towards genetic diversity was presented in Table 2 which showed that 100 seed weight, plant height and number of secondary branches contributed 26.98%, 17.88% and 15.95% respectively towards the genetic diversity. Most exotic types and their bold seeded derivatives are macrosperma types having 100 seed weight above 3gms. The other two traits contributing more to genetic diversity were plant height and secondary branches. Exotic types exhibited good plant height and poor biomass whereas indigenous types had better biomass and secondary branches. Sixty lentil genotypes (including released varieties, advanced breeding lines and exotic germplasm lines) were grouped into three clusters (Table 3). Cluster I comprised of L830, L4661, L7434, L7L917, L7929A, L4378, L5120, L4603, L3685, PL1, PL05, LL884, EC382703, PKVL1, Sehore74-3, JL3, 10-3-Y-26, LC74-5-1, Bari Masoor 4, Globe mutant, LL358, 10-2-B-2. Cluster II had L4076, L4147, L4583, L4594, L4596, L7920, L7931, L7933, L4692, L7764, L7830, PL04, PL406, JLS1, P27240, SKL259, Fasciated mutant, LH90-57 whereas L404, L4602, L4605, L4618, L4649, L5125, L7774, L7820, L7903, L7908, DPL58, DPL62, L4582, MC06, Precoz, MC01, L7828, RL1, FLIP96-51were grouped in cluster III. The genotypes belonging to different eco-geographical areas were grouped in the same cluster indicating that there is no association between clustering pattern and eco-geographical distribution of genotypes. Such genetic diversity among the genotypes of common geographic origin could be due to factors like heterogeneity, genetic architecture of the populations, past history of selection, developmental traits and degree of general combining ability.
The cluster means of each of the nine characters was calculated and are presented in the Table 2. Cluster I comprising 23 genotypes had minimum value for all the studied nine traits. Genotypes of this cluster can be used for transferring the earliness. The genotypes from cluster II exhibited highest days to flowering, days to maturity, plant height, number of pods per plant, seeds per pod, primary branches and yield per plant. The genotypes from this cluster can be used for combining these traits. Cluster III exhibited maximum value for 100 seed weight and secondary branches per plant which are the most important yield contributing traits in lentil. The intercluster distance between cluster 1 and cluster 2 was 2.897 and between cluster 1 and cluster 3, it was 2.636. The intercluster distance between cluster II and cluster III was 2.808 (Tewari, 2010). Based on the results it was suggested to cross genotypes from cluster I with genotypes from clusterII for obtaining transgressive segregants. The narrow intercluster distance range is indication of low genetic diversity among the studied genotypes/clusters.

Table 2 Percent contribution of different quantitative traits towards genetic diversity and their cluster means

Table 3 Distribution of studied lentil genotypes in different clusters obtained on the basis of morpho-agronomic traits

2.2 Diversity analysis based on RAPD and SSR markers
Eighty three lentil genotypes produced a total of 92 amplicons by 15 RAPD primers, the percentage polymorphism being57% to 100%. Nine primers OPA01, OPA03, OPA04, OPA10, OPB01, OPC02, OPC18, OPC05 and OPV17 exhibited 100% polymorphism (Tewary, 2010). The PIC value ranged from 0.2 (OPA04) to 0.85 (OPA16) (Table 4). Average polymorphic bands per primer was 5.6 which was higher than described by Abo-Elwafa et al., 1995. Polymorphism generated by RAPD markers was lower in cultivated varieties compared to wild ones as described previously by other workers in lentil (Duran et al., 2004, Abo-Elwafa et al., 1995). Six polymorphic SSRs amplified 18 alleles and polymorphic information content ranged from 0.10 to 0.78 (Table 4). Microsatellite primer SSR11 revealed 2 unique alleles (present in single genotype) (ILWL75 and L4147) and one rare allele (present in <5% frequency). Wild lentil genotypes amplified loci comparable to those of cultivated species indicating their amenability to PCR and microsatellite sets.

Table 4 Polymorphic information content (PIC) and number of scorable bands produced by fifteen RAPD and eight SSRs markers in eighty three Lens genotypes

Variation between different genotypes was analyzed using different estimators-effective number of alleles/loci, percentage of polymorphic loci, expected heterozygosity and shannon’s information index. RAPD marker showed 96.74 % polymorphic loci, 0.0
-0.49 heterozygosity range over all the loci (Tewari, 2010). The overall Gst, a measure of gene differentiation was 0.3 for all the genotypes. Wild lens genotypes seemed to be most diverse based on all the estimators. For microsatellite markers, average number of polymorphic loci among the genotypes was 90 %. Shannon’s information index ranged from 0.0 to 0.69 and average heterozygosity was 0.24. Further analysis of molecular variance (AMOVA) among and within accessions revealed that 36.8 % of the variation resides among the genotypes and 63.1% between the genotypes. The overall Fst value for all the loci was calculated to be 0.306 which was comparable to the Gst values (Table 5). Nei’s unbiased genetic distance revealed more distance between wild types and exotic lines with RAPD markers and between released varieties and exotic lines with SSR markers. However, genetic distance between paired populations by AMOVA for the combined data agreed with just as in RAPD analysis.

Table 5 Gene diversity parameters for RAPD and SSR markers among 83 lentil genotypes

Genetic similarity coefficient values for both the markers ranged from 0.27 (between ILWL147 and RL01) to 0.90 (between L4649 and L4661) for all the genotypes. The critical evaluation of dendrogram revealed
three major clusters (Figure 1). Cluster I comprised 21 wild accessions of Lens orientalis and 1 wild accession of Lens ervoides (ILWL 485). Subcluster II A comprised of 19 released Indian varieties indicating their low genetic base. This might be due to frequent uses of few lines in breeding programme resulting in narrow genetic diversity. Subcluster II B consisted of 41 genotypes including 5 exotic germplasm (EC-782703, P27240, Precoz, Bari masoor 4 and FLIP 96-51) and 36 advanced breeding lines/exotic derivatives. This clustering is due to use of exotic lines in breeding programmes during recent days and hence their similarity with exotic lines. However, Cluster III had only one genotype of orientalis (ILWL 200) exhibiting only 45 % similarity with cluster I and II. The cophenetic correlation between similarity matrix and cluster analysis based on combined data represented a very high goodness of fit (r = 0.9).

Figure 1 Dendrogram obtained by analysis of combined data of RAPD and SSR. G1-83 represents the same serial number of genotypes as given in Table 1

The agro morphological, RAPD and SSR markers have been used by many researchers to assess polymorphism in Lens. Geographically isolated populations accumulate genetic differences with evolution as they adapt to different environment. The clustering of genotypes within group was not similar when RAPD and SSR derived dendrograms were compared. These differences may be attributed to marker sampling error and/or the level of polymorphism detected, importance of the number of loci and their coverage of overall genome. The accessions with most distinct DNA profile are likely to contain the greatest number of novel alleles. It is these accessions that are likely to uncover the largest number of unique and potentially agronomic useful allele. Our advanced breeding lines were exotic derivatives and shared more alleles with exotic varieties compared to past released varieties with narrow base indicating the alternate preference of breeders to select exotic genes.
The present study successfully assessed the level of inter and intra specific diversity and species relationship among different cultivated and wild genotypes of Lens species. Differentiation of wild and released varieties into separate clusters indicated narrow genetic base of released varieties. ICARDA has found good sources of resistance to vascular wilt and ascochyta blight, and cold tolerance in the crossable wild progenitor L. culinaris spp. orientalis (Ferguson, 2000). L. culinaris spp. orientalis is crossable with the cultivated lentil. Therefore for broadening the genetic base, intraspecific hybridization (involving early maturing Mediterranean germplasm lines and indigenous agronomic bases) and interspecific hybridization is suggested.
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