GEOGRAPHICAL DISTRIBUTION OF LENTILS IN PAKISTAN AS INDICATED BY DATA ON GENETIC MARKERS

Lentils are one of the oldest domesticated plant species that originated in Mediterranean region. Its cultivated species, Lens culinaris belongs to the genus Lens that is classified taxonomically in the order Rosales, sub-order Rosineae, family Leguminoseae and sub-family Papilionacea with special features of nitrogen fixation. It is used as a meat substitute due to high protein contents and quality, also used in gluten-free, diabetic, low salt, low calorie, low cholesterol and high fiber diets. It is an important winter pulse crop, grown on a total of 1.8 million hectares in the world, of which 60% is in the South Asia. Gene pool assemblage is important for exploitation of genetic diversity, an asset provided by nature and Lentils are rich in it. Highly heritable quantitative genetic markers provide one estimate of diversity that is important to study along with other morphological, biochemical and molecular markers. The present study was conducted to access and evaluate genetic diversity for morphological and agronomic traits along with biochemical (storage protein, isozymes) and molecular (RAPD) profiles. The data were used to investigate geographical distribution of lentils in Pakistan and relationships among various markers were investigated to enhance the breeder's efficiency. Three hundred and seventeen lentil accessions collected from all over the country were evaluated for qualitative and quantitative traits at Plant Genetic Resources Programme, NARC, Islamabad (33.40°N and 73.07°E). Out of these, 144 accessions were analyzed for seed proteins and out of which 108 were homozygous based on SDS-PAGE. Depending upon diverse region (Baluchistan), 32 accessions were studied for isozymes, whereas 19 for RAPD. In the conclusion, 17 accessions were compared for all five parameters to investigate genetic diversity and relationships among various techniques to identify the best one. On the basis of characterization/evaluation 76 accessions were heterogeneous and germplasm collected from Baluchistan exhibited high degree of heterogenoisity that indicated presence of high genetic diversity in this area. Genetic diversity conducted for 14 qualitative traits revealed 8 clusters, whereas 12 clusters were observed on the basis of quantitative characters. Inter-and intra cluster diversity indicated the worth of local landraces that are suggested to isolate from heterogeneous mixtures especially from germplasm collected from NA and Baluchistan. Analyses for geographic pattern revealed that more germ plasm is needed to explore from the areas with greater genetic diversity and stresses that includes NWFP, NA, Sindh and Baluchistan. The best accessions for individual characters were selected and suggested to evaluate further for confirmation of their superiority and can also be exploited for their genetic potential in future breeding programme. The accessions collected from Sindh were short statured, whereas accessions from NWFP were tall. The selected accessions from diverse groups are expected to produce better recombinants. Maximum seed weight was observed in accessions from Baluchistan although overall variance was low for seed weight. It was observed that cluster analysis on the basis of quantitative characters were phenotypically more distinct and exhibited more breeding value. Though cluster analysis grouped together accessions with greater morphological similarity, the cluster did not necessarily include all the accessions from the same or nearby sites. SOS-PAGE revealed that 9.4% Acrylamide gel concentration and 10 ul of sample gave the best resolution and 55 protein bands were observed, 13 were polymorphic. Homogeneous accessions based on UPGMA revealed 5 clusters, 3 accessions in cluster I, three in cluster 11, fifteen in cluster Ill, seventy one in cluster IV and sixteen in cluster V. Based on diversity for SOS-PAGE, selected members from different clusters could be used in future breeding programme to combine seed size and yield potential in one cultivar. Intensive sampling from Punjab resulted in high polymorphism, whereas the lowest polymorphism was observed in accessions from NA and NWFP. As NA and NWFP are geographically diverse but due to less representation of area, the germ plasm did not give actual position for diversity. This is suggested to explore these areas intensively to collect maximum genetic diversity that should truly represent the area. Six isozymes (SKOH, PGI, PGM, AAT, ME, LAP) were studied and only two (AA T and ME) exhibited diversity and hence could be used for exploring the genetic variation in lentil germplasm. Out of 20 primers, investigated on 19 accessions (4-5 samples per accession), all were amplified and 10 were polymorphic that varied to resolve variability among accessions exclusively from Baluchistan. Out of total 182 fragments 28 (15.38%) were polymorphic. The levels of polymorphism varied with different primers among various accessions. The number and size of amplified fragments also varied with different primers. The primers OPA 10, OPA 7, OPA 17 and OPA 6 gave maximum polymorphism with 62.5%, 59.5%, 37.5% and 28.6%, respectively. Hence, these primers could be exploited for finding genetic variation in lentil germplasm. As the number of accessions varied for each technique, therefore 17 accessions common (especially for RAPO) for all the five parameters were used to study relationships among various accessions and techniques implied. In general, a low level of genetic variation was disclosed by SOS-PAGE giving 6 cluster indicating that genetic variation within lentil germplasm collected from Pakistan is narrow, which may necessitate the use of novel techniques for creation of genetic variability in lentil. It is suggested that diverse genotypes for seed proteins may be acquired from abroad, especially from centers of diversity to build a comprehensive genetic resources. Isozymes gave interesting results, 17 accessions from the region of maximum diversity revealed 15 clusters, hence could be used more confidently than total seed proteins for fingerprinting of lentil cultivars, but due to less polymorphism more isozyme systems are needed to be included along with other markers. Grouping on the basis of isozyme could identify even intra-accession variation if a particular isozyme is used with polymorphic nature for a particular locus. This enhances the validity for studying segregating populations for gene mapping through isozymes. Genetic diversity due to seed proteins was significantly associated with genetic diversity due to RAPD markers, qualitative and quantitative traits, whereas it had no association with isozymes. Twenty clusters were observed for RAPD that indicated the worth of this technique to investigate even intra-specific diversity and for fingerprinting of lentil germ plasm. The RAPD markers exhibited significant association with three parameters (SDS-PAGE, isozymes and qualitative trait). All the five parameters facilitated to resolve issues related to genetic diversity in lentil, anyhow total seed proteins and isozyme (six in present case) gave low level of genetic diversity that suggested to incorporate more isozymes for investigation due to its precise fingerprinting and wider use. The RAPD could be enhanced including more primers including more germplasm, as 50% primers in general were polymorphic in nature