Genetic Diversity Analysis of Lentil (Lens culinaris Medik) Cultivars Using Inter Simple Sequence Repeats Markers

Molecular markers have emerged as useful tools to assess the genetic diversity across crops. In lentil, molecular markers are limited. The objective of the study was to explore genetic diversity and relatedness Indian and exotic lentil accessions. Genetic diversity was studied in 25 lentil cultivars using 100 ISSR markers. Out of 100 markers, 24 amplified PCR products and total of 156 alleles were identified with a mean of 6.5 alleles per marker. Genetic similarity among the genotypes ranged from 37 to 84%. UPGMA cluster analysis revealed two main clusters, and the second cluster comprised majority of genotypes with the exception of germplasm line Precoz, which did not fall in either cluster. Eleven genotype specific unique bands were also obtained which showed amplification only in particular genotypes. These unique bands can serve as potential diagnostic markers and therefore, may be of immense importance. The polymorphic markers will enhance marker repertoire to study genetic diversity in lentil and also improve understanding about the genetic base lentil cultivars.


Introduction
Lentil (Lens culinaris Medikus), an autogamous diploid (2n = 2x = 14) species with haploid genome size of 4 063 Mbp, is an important cool-season food legume crop of South Asia, North America, West Asia and North Africa and Australia (Hamweih et al., 2009).Globally, it is cultivated for its protein-rich grains in as many as 52 countries on 3.64 million ha area with annual production of 3.60 million ton (FAOSTAT, 2011). However, about 95% of the global production comes from just ten countries, namely Canada, India, Turkey, Nepal, Australia, China, Iran, USA, Syria, and Ethiopia. India accounts for 39% (1.47 million ha) of the global acreage with 0.90 million ton production.
Assessment of genetic diversity in germplasm is prerequisite for any breeding program so that genetic gain is not limited because of narrow genetic base of parental lines (Kumar et al., 2004). Though morphological data and pedigree information have been used in the past to assess genetic diversity among the lentil varieties, these studies could not make much contribution to our knowledge due to limited phenotyping, high genotype X environment interaction, and paucity of accurate record of ancestry.
In earlier studies, molecular markers such as Simple Sequence Repeat (SSR), Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment Length Polymorphism (AFLP) and Random Amplified Polymorphic DNA (RAPD) have been preferred for genetic diversity analysis in lentil (Havey and Muehlbauer, 1989;Abo-Elwafa et al., 1995;Sharma et al., 1995Sharma et al., , 1996Ahmad et al., 1996;Ford et al., 1997;Udupa et al., 1999;Abe et al., 2003;Hamwieh et al., 2005Hamwieh et al., , 2009Reddy et al., 2010) and gene mapping (Eujayl et al., 1998;Tullu et al., 2003;Duran et al., 2004;Kahraman et al., 2004;Hamwieh et al., 2005). SNPs were identified across Palestinian lentil accessions for development of cost-effective and robust genotyping assays (Basheer-Salimia et al., 2015). Kaur et al. (2014) identified SSR and SNP markers from transcriptome and EST for construction of gene-based Molecular Plant Breeding 2016, Vol.7, No.23, 1-9 http://mpb.biopublisher.ca 2 genetic linkage map in lentil. Several reports from our lab have established transferability of SSR markers from one legume genera/species to other (Datta et al., 2010a(Datta et al., , 2010b2012;2013a, 2013b, and observed high level of sequence conservation of microsatellite markers in legumes. ISSR (Inter Simple Sequence Repeat) markers are inexpensive and readily adaptable technique for routine germplasm fingerprinting and evaluation of genetic relationship between accessions or genotypes (Sardana et al., 1998;Dixit et al., 2004, Edossa et al., 2007 and construction of genetic linkage maps (Abo-Elwafa et al., 1995). This technique has been used to assess genetic diversity in germplasm collection (Gilbert et al., 1999;Salimath et al., 1995), to identify cultivars (Prevost et al., 1999). The present investigation was undertaken to assess the diversity and genetic relatedness in Indian and exotic lentil genotypes with the objectives, to study the polymorphism and genetic relationship among and to identify genotype specific markers.

Results and Analysis
A total of 100 ISSR markers were used to test their ability in detection of DNA polymorphism. Only 24 markers produced reproducible amplification in all the selected genotype and therefore only these were considered for polymorphism assay (Table 1). The ISSR profiles generated with markers UBC-888, UBC-891, UBC-886, UBC-815, UBC-889 and UBC-858 respectively showed ( Figure 1).

DNA polymorphism and genetic relationship
ISSR markers analysis of lentil genotypes using Jaccard's (1908) coefficient of genetic similarity are given in Table 1. The similarity coefficients varied from 0.37 to 0.84, the average being 0.69. Genotypes LL147 and L4147 were found to have maximum similarity (84%) which was closely followed by L4147 and VL103 (82.9%). The lowest similarity was found in between T36 and Precoz (37%) which was followed by IPL525 and T36 (40.9%).
A total of 11 unique bands were also obtained which is present only in a particular genotype. A 900 bp band was obtained in genotype DPL 44 with UBC-808 marker. Similarly the marker UBC-815 amplified two unique bands of 1 120 bp and 400 bp in case of Sehore 74-3 and IPL525. Table 2 includes the complete list of such genotype specific bands.

Cluster analysis
The Jaccard's (1908) similarity coefficients based on ISSR profiles were subjected UPGMA analysis and a dendrogram of 25 lentil genotypes was constructed ( Figure 2). It showed two main clusters. Cluster I contains genotypes IPL525, PL234 and PL5 which are tolerant to rust and wilt disease, the similarity between IPL525 and PL 234 is 68.10%. Cluster IIA contains genotypes DPL15, WBL58 and Sehore74-3. The similarity value between WBL 58 and Sehore74-3 is 75.30%. These two genotypes were large seeded and had ash green colour foliage with high pubescence. The cluster II B contains 4 sub-clusters; sub cluster IIB1 contains two small seeded varieties PL406 and L-9-12. The similarity value between these genotypes is 77.7%. In Sub cluster II B2, NDL 1 and LH 84-8 genotypes were grouped together; the reason for this closeness may be due to a common parent L-9-12 in a d b c f e their pedigree. The similarity value as per similarity matrix table is 76.50%. Four genotypes JL-1, LL-147, L-4147 and VL-103 came together in same cluster (sub cluster II B3) and the similarity value of LL-147 with L-4147, L-4147 with Vl-103 and LL-147 with VL-103 are 84%, 82.9% and 77.39% respectively. This cluster showed bootstrap replication of 36 in major cluster and 50 in sub cluster. Sub cluster II B4 comprised of genotypes LH-82-6 & DPL-62 with similarity value 78.90%. The bootstrap value of this cluster is 63.0. Both genotypes are tolerant to rust and wilt diseases also. This exotic cultivar Precoz which is of Argentine origin was sharply isolated from the clusters. This variety is distinct with others morphologically also due to light green foliage, extra large seeded, low pubescence and resistant to rust. The genotypeT-36 is isolated one but it is similar in character to the cluster II. IPL-81, which is derived from K75 and PL639, positioned itself between sub-clusters IIC and IID the first parent was grouped in the adjacent cluster IIC.

Multi scaling analysis with two and three dimensions
The two dimensional plot obtained through multidimensional analysis revealed the spatial separation of genotypes with each other (Figure 3; Figure 4). The Sehore74-3 and JL1, both varieties are very near to each other which Molecular Plant Breeding 2016, Vol.7, No.23, 1-9 http://mpb.biopublisher.ca 5 might be due to their origin and adaptation from same place Jabalpur, in India. Morphologically also they are similar to each other in relation to foliage colour, size and hairiness. The maximum distance was found between genotypeT-36 and Precoz which is also evident by their genetic similarity (37%), which is lowest among all pair of genotypes. The next distant pairwas IPL525 and T36, with genetic similarity of 40.9%. The average similarity was found to be 69.32% among all genotypes based on ISSR marker data.
The genotype LL147 and L4147 were appears closely in three dimensional plot which proves that they are highly similar which is also clear by their high genetic similarity value (84%) followed by L4147 and VL103 (82.9%) (Figure 3; Figure 4). The maximum distance appears between T-36 and Precoz (37.0%) which was followed by IPL525 and T36 (40.9%). The similar kind of result was obtained in two-dimensional analysis also. The average similarity was found to be 69.32% among all the genotypes based on ISSR data, which indicated relatively narrow genetic base of lentil.

Discussion
Genetic variations in crop plants has continued to narrow down due to continuous selection pressure for specific traits i.e. yield, thus rendering them more vulnerable to disease and insect epidemics and jeopardizing the potential for long term sustained genetic improvement.
Therefore, it is extremely important to study the genetic relationship of the existing modern-day genotypes in comparison with their ancestors and related species. This will not only provide information on their genetic distance and phylogenetic relationship but will also indicate a chance of finding new and useful genes. In the present study, genomic diversity was studied in twenty-five lentil genotypes (Indian/Exotic genotypes and collection). Out of 100 ISSR markers tested, 24 produced unambiguous amplicons. Amplified markers produced easily scorable bands ranging from 160 to 3000 bp in length. A total of 156 ISSR fragments were amplified with an average of 6.5 amplicons per marker. The most informative markers were selected on the basis of extent band polymorphism, detected by the individual marker. Also, 11 unique bands were found only in particular genotypes. A 900 bp band was obtained in genotype DPL-44 with marker UBC-808. Similarly the marker UBC-815 amplified two unique bands of 1120 bp and 400 bp in case of Sehore74-3 and IPL525. Efficiency of markers and their utility in terms of polymorphism and quantitative estimation could be expressed in mean heterozygosity and marker index (Choudhury et al., 2007). The average Hav, (Hav) p and MI were found to be 0.579, 0.124 and 0.640, respectively. The minimum (0.16) and maximum (0.88) PIC value were found with markers UBC-861 and UBC-824, while the lowest (0.056) and highest (0.490) heterozygosity were found with markers UBC-815 and UBC-858 respectively. The average genetic distance coefficient value among the all genotypes was 0.31 based on ISSR markers, which indicated a limited degree of genetic variation in the lentil material. The genotype LL147 and L4147 were found to have maximum similarity (84%) which was closely followed by L4147 and VL103 (82.9%). The lowest similarity was found between T-36 and Precoz (37%) which was followed by IPL525 and T36 (40.9%) The average similarity was found to be 69.32 %.
The information on genetic diversity among these lentil cultivars will be helpful to lentil breeders in selection of appropriate hybridizing parents in developing superior cultivars.

Plant materials
The experimental material comprised of 25 lentil genotypes from different lentil growing states of India and other countries ( Table 2).All seed material for this study was obtained from the germplasm unit of Indian Institute of Pulses Research, Kanpur (India).

DNA Isolation and PCR amplification
Seeds of 25 lentil genotypes were germinated under etiolated conditions on paper towel soaked in sterilized water. One-week-old seedlings were ground in preheated CTAB buffer and incubated at 60℃ for 1 h. The aqueous phase containing DNA was separated using chloroform: isoamyl alcohol (24: 1) (Abdelnoor et al., 1995). The DNA was precipitated with chilled iso-propanol and the pellet was dissolved in 100 µl of T 10 E 1 buffer. The RNA was eliminated by adding 0.5 U of RNAse. DNA concentrations were quantified by measuring absorbance using Hoefer® Dyna Quant® 200 DNA fluorometer (Amersham Biosciences, Piscataway, NJ, USA) and stocks were maintained at 25 ng/μl.
One hundred ISSR markers of the UBC series were used to study genetic similarity in 25 lentil genotypes. PCR reaction mixture (20 μl) consisted of 20 ng of template DNA, 1X PCR buffer, dNTPs (Banglore Genei, Bangalore) 2.5 nM each, 10 pM primerand 0.6 U of Taq DNA polymerase (Banglore Genei, Bangalore). The thermal cycling program was carried out in a PTC 200 thermal cycler (MJ Research, Biorad). The PCR program had an initial denaturation step at 94℃ for 2 min, followed by 41 cycles of denaturation at 94℃ for 1 min, primer annealing(depending upon the Tm of respective marker) for 1 min and DNA extension at 72℃ for 3 min. A final extension step was given at 72℃ for 4 min. The amplified DNA fragments were resolved on ethidium bromide stained agarose gel (2%) in 1X TAE buffer at 50 V. A 100 bp DNA ladder (MBI Fermentas) was used as a molecular weight marker for determining the molecular weight of the amplified products.

Scoring and Data analysis
The amplification profiles of each marker in different genotypes were scored and recorded as presence [1] or absence [0] of bands and binary quantitative data matrix was constructed. Unique alleles were defined as thosedetected in only one genotype. The presence and absence of alleles for each marker was recorded for all genotypes and then converted into genetic similarity matrix using Jaccard (1908) similarity coefficient in NTSYS-PC 2.1 software. (Rohlf, 1998). The similarity coefficients were used to construct a dendrogram depicting genetic relationship using unweighted pair group mean average (UPGMA) method (Sneath and Sokal, 1973). The Polymorphism Information Content (PIC) values were calculated following the formula described by Botstein et al. (1980): where, P ij is the frequency of the j th allele for the i th marker, and summed over 'n' alleles.
The arithmetic mean heterozygosity for a marker (Hav) was calculated by Hav = Hn /n [n= number of markers on loci analyzed (Powell et al., 1996). Heterozygosity for a marker (Hn) = 1-pi 2 , where pi is the allele frequency of i th allele (Nei et al., 1979). The average heterozygosity for polymorphic markers was derived by (Hav) p= δHn /np ( np= no. of polymorphic markers or loci). Marker Index was calculated as the product of two functions: DI (Diversity index) and EMR (Effective Multiplex Ratio). DI of a primer is defined as 1-pi 2 , where pi is the frequency of the ith allele (band). EMR of a primer is defined as ''the product of the fraction of polymorphic loci and the number of polymorphic loci for an individual assay'' (Prevost and Wilkinson, 1999).