MORPHOLOGICAL CHARACTERIZATION AND GENETIC DIVERSITY IN LENTIL ( Lens culinaris Medikus ssp . culinaris ) GERMPLASM

Genetic divergence of 110 lentil germplasm with checks was assessed based on morphological traits using multivariate analysis. Mahalanobis generalized distance (D2) analysis was used to group the lentil genotypes. Significant variations among lentil genotypes were observed in respect of days to 1st flowering, days to 50% flowering, days to maturity, plant height, and number of pods per peduncle, number of pods per plant, number of seeds per plant, 100 seed weight and yield per plant. Considering the mean values, the germplasm were grouped into ten clusters. The highest number of genotypes (17) was in cluster X and lowest (5) both in cluster II and IV. Cluster IV had the highest cluster mean for number of pods per plant (297.08), number of seeds per plant (594.16), 100 seed weight (1.44 g) and yield per plant (8.53 g). Among them, the highest inter-cluster distance was obtained between the cluster IV and I (24.61) followed by IV and III (22.33), while the lowest was between IX and II (1.63). The maximum value of inter-cluster distance indicated that genotypes belonging to cluster IV were far diverged from those of cluster I. The first female flower initiation was earlier in BD-3812 (49 days) in cluster I and cluster IV had highest grain yield per plant (8.53). BD-3807 produced significant maximum number of pods per plant (298.40) in cluster IV.


Introduction
Lentil (Lens culinaris Medikus ssp.culinaris) is a major and important food legume in Bangladesh.In Bangladesh, all the indigenous landraces and cultivars are microsperma with red cotyledons.Lentil is the early domesticated among crops.It plays an important role in human, animal and soil health improvement.Nutritionally, lentil is very rich and complementary to any cereal crop including rice.It is versatile source of nutrients for man, animals and soil containing, on an average, 25.1% protein, 59% carbohydrate,0.5% fat,2.1% minerals and sufficient amount of vitamins, viz.vitamin A 16 IU ; thiamine 0.23 mg and vitamin C 2.5 mg per gram lentil (Anonymous, 2003;Frederick et al., 2006).Lentil fixes atmospheric nitrogen association with Rhizobium sp for this its cultivation improves soil nitrogen, carbon and organic matter balance in soil.Lentil ranks second in respect of area and production but consumer's preference its rank first among pulses in Bangladesh condition.Farmers are cultivated with sole crop or mixed crop with mustard in rabi season.Production of lentil has long lagged behind domestic demand in Bangladesh, where it is preferred pulse crop for human consumption.
Therefore, it needs to increase its production with high yielding variety of lentil.Effort should be made to develop such disease resistant varieties.Local germplasm of crop plants is an excellent source of economically useful plant characters (Pecetti et al., 1996).The breeders must have a mean of choosing the accession most likely to posses the trait of interest.Quantitative traits provide an estimate of genetic diversity and various numerical taxonomic techniques have been successfully used to classify and measure the pattern of phenotypic diversity in the relationship of germplasm collections in a variety of crops by many scientists as in lentil (Ahmad et al., 1997;Fratini et al., 2007;Tullu et al., 2008), Pea (Amurrio et al., 1995) and Alfalfa (Smith et al., 1991;Smith et al., 1995;Warburton and Smith, 1993).Worldwide, lentil is grown on a total of 1.8 million hectares, of which 60% is in the South Asian region, which includes the lentil producing countries of Bangladesh, Burma, India, Nepal and Pakistan (Nazir et al., 1994).However, lentil in general, does not respond to high inputs such as fertilizer and irrigation.Genetic diversity has been considered an important factor in any crop improvement program.However, the experiment of lentil was conducted to assess the genetic diversity of lentil genotypes in respect of agro-morphological traits.

Materials and Methods
The investigation was carried out at Regional Plant Genetic Resources Center, RARS, Bangladesh Agricultural Research Institute, Ishurdi, Pabna during the rabi season of 2010-11.The experiment was laid out non-replication.Unit plot size was 4m x 1m and seeds were sown in rows with spacing 40 cm.The seeds were sown on 30 October, 2010.The experiment involves two-hundred and twenty lentil germplasm and two varieties BARI Masur 4 and BARI Masur 6 were used as test genotypes.Fungicide, insecticide and other intercultural operations were done as and when necessary.Ten plants were sampled at random to study inter and intraaccession variation.Quantitative traits were recorded on 10 plants following IBPGR Descriptors (Anonymous, 1985).Morphological parameters of quantitative data were recorded on days to flowering, days to 50% flowering, plant height , number of pods per peduncle, number of pods per plant, number of seeds per pod, number of seeds per plant, 100 seed weight, yield per plant and different qualitative characteristics were recorded.Data were analyzed statistically by non-replication with computer and the means were separated.Genetic diversity was studied following Mahalanobis's generalized distance analysis (D2).Statistical analysis was done using the GENSTST 5 programme.

Qualitative characteristics
Qualitative characteristics of 110 genotypes are presented in Table 1.Variation between and within populations of crop species is useful for analyzing and monitoring germplasm during the maintenance phase and predicting potential genetic gain in a breeding programme (Hayward and Breese, 1993;Moore and Collins, 1983).For qualitative characteristics, a considerable level of variability was observed for growth habit, seedling stem pigmentation leaf pubescence, leaflet size, flower ground colour, seed coat colour, seed coat pattern and seed coat pattern colour, cotyledon colour and lodging susceptibility that could be exploited for developing future breeding material in lentil breeding programme (Table 1).Two classes were observed for stem colour, hairiness, tendril, pod pigmentation (anthocianin), pod indehiscence, beak on the pod and cotyledon colour, whereas for other traits more than one class were observed, especially for seed traits that represents the world classification as reported by Erskine and Witcombe (1984).Muehlbauer and Slinkard (1981) reviewed the genetics of Lens and listed 12 genes, which account for morphological and seed variation in lentil.It was observed critically that seed traits in lentil are difficult to record that need standardization.2010) also reported that eight lentil accessions gave seed weight more than 3.1 g and hence could be utilized for the manipulation of this trait as high seed weight in any grain crop.Variability for these traits in lentil germplasm was also reported by Agrawal et al. (1976), Tiwari and Singh (1980), Malik et al. (1984) and Toklu et al. (2009).Singh and Singh (1993) confirmed the wide range of variation in agronomic characteristics of lentil germplam, except for seeds per pod.

Genetic diversity in lentil germplasm
A considerable amount of genetic variability was observed and therefore diversity analysis was carried out through multivariate analysis.
The clustering pattern and distribution

Non-hierarchical clustering
The covariance matrix gave non-hierarchical clustering among 110 genotypes and grouped them into ten clusters (Table 3).They coincided with the apparent grouping patterns performed by PCA.Cluster VII and X both contained the largest number of genotypes (sixteen), followed by clusters I, IX, III,V,VI, VIII, IV, and II.The genotypes of different geographic origin are accumulated in the same cluster indicating that the genotypes are not sharply diversified.Similar results were obtained by Alam et al. (2006) in Barley, Alam et al. (2011) in Lentil.These clusters lead to the highest cluster mean for maximum characters (Table 4).Among the ten characters, the highest mean values for four characters,viz.number of pods per plant (297.08),number of seeds per plant (594.16), 100 seed weight (1.41 g), and yield per plant (8.53 g) were found in cluster IV.Cluster IV had only five genotypes,viz.BD-3807, BD-3821, BD-3902, BD-3894 and BD-3886.Cluster X with sixteen genotypes was able to lead only for two traits in respect of cluster means of six characters.The highest cluster mean was recorded for 100-seed weight (1.41 g).Cluster VI, X and V was moderate yielding associated with desired characteristics like size and early maturity.

Canonical Variate Analysis
Average inter-cluster D2 values among the ten clusters are presented in Table 5.The highest inter-cluster distance was obtained between the cluster IV and I (24.61) followed by IV and III (22.33), while the lowest was between IX and II (1.63).The maximum value of inter-cluster distance indicated that genotypes belonging to cluster IV were far diverged from those of cluster I. Similarly, the highest inter-cluster values between cluster IV and III indicated that the genotypes between each pair of clusters were more diverged.Sultana et al. (2010) also reported that the inter-cluster distance among the accessions revealed that the cluster V consisting of five accessions was obviously very much different from all the other clusters with a genetic distance ranging from 1.99 (Cluster VIII) to 2.85 (Cluster X).

Principal Coordinate Analysis (PCO)
The results obtained from principal coordinate analysis showed that the highest inter-genotypic distance was between genotypes BD-3894 and BD-3970 (1.964), followed by BD-3849 and BD-3894 (1.868), and the lowest (0.083) between genotypes BD-3840 and BD-3805 as well as between BD-3909 and BD-3964 (0.102) (Table 5).The difference between the highest and lowest inter-genotypic distance indicated that moderate variability among 110 germplasm of grass pea.The highest intra-cluster distance was recorded in cluster II (0.717) containing five genotypes viz.).The lowest intra-cluster distance was in cluster X (0.351) and containing also seventeen genotypes genotypes, viz. .Similar types of research were conducted by Malik et al. (1984) who reported high genetic variance in cultivated lentil.

Contribution of characteristics towards the divergence of genotypes
The values of vector 1 and vector 2 are presented in Table 8.The value of vector 1 obtained from PCA for days to first flowering, days to 50% flowering, days to maturity, number of pods per peduncle and number of seeds per pod suggests it was the major characteristics that contributed to the genetic divergence.In vector 2 also obtained from PCA for days to 1st flowering (0.631), days to 50% flowering (0.622), days to maturity (0.115), Plant height (0.122), number of pods per peduncle(0.038),number of pods per plant (0.270), 100-seed weight (0.269) and yield per plant (0.080) showed their important role toward genetic divergence.Both vector 1 and vector 2 revealed that vector 1 had positive values for days to first flowering, days to 50% flowering, days to maturity, number of pods per peduncle and number of seeds per pod and vector 2 had positive values for days to 1st flowering , days to 50% flowering, days to maturity, Plant height (cm), number of pods per plant, 100 seed weight (g) and yield per plant (g) are indicating highest contribution of these traits towards the divergence among 110 genotypes of lentil.
Negative values in both vectors had lower contribution towards the divergence.Similar results were obtained by Alam et al. (2011) in lentil.

Conclusion
Days to 1 st flowering , days to 50% flowering, days to maturity, number of pods per peduncle, number of pods per plant, number of seeds per pod, 100 seed weight (g) and yield per plant (g) had highest contribution towards divergence among 10 characteristics for 110 lentil germplasm.Based on analysis the germplasm grouped into ten clusters.From morphological study, the highest inter-cluster distance was obtained between the cluster IV and I (24.61) followed by IV and III (22.33).The intra-cluster distance was highest (0.717) in cluster II and lowest (0.315) in cluster X. Considering yield performance, cluster distance and cluster mean, the genotypes BD-3894, BD-3821 and BD-3902 from cluster IV and BD-3804, BD-3902 and BD-3869 from cluster III may be considered better genotypes for recombination breeding due to their larger divergence.

Table 1 .
Method for measuring and classifying of plant descriptors in 110 accessions of lens culinaris qualitative characteristics of lentil germplasm

Table 2 .
Range, mean, SD and CV% of yield and yield contributing characteristics of 110 lentil

Table 3 .
Distribution of 110 genotypes of lentil in 10 clusters ,

Table 4 .
Means for quantitative characteristics for 10 clusters in lentil germplasm

Table 5 .
Average inter and intra (bold) cluster distance (D2) for the 110 lentil germplasm obtained on the basis of the morphological characteristics

Table 6 .
Five highest and lowest inter-genotypic distances among one hundred seven genotypes of grasspea

Table 7 .
Eigen values and percentage of variation for corresponding 10 component characteristics of 110 lentil germplasm

Table 8 .
Relative contributions of the 10 characteristics to the total divergence in Lentil germplasm