MOLECULAR CHARACTERIZATION OF LENTIL GERMPLASM

INTRODUCTION

Lentil (Lens culinaris Medik.) is an important cool-season crop in North Africa, West Asia, the Middle East, the Indian Subcontinent and North America (Erskine 1996). Lentils are an old world legume and were probably one of the first plant species to be domesticated (Bahl et al., 1993). Cultivated lentil (Lens culinaris Medik) is thought to have originated in the Fertile Crescent of the Mediterranean region in Western Asia, dating back to the beginnings of agriculture, from where it spread to the rest of world (Ladizinsky, 1979; Duke, 1981). Lentil is unique because it can be grown in marginal environments in which other crops cannot be cultivated (Cubero, 1981). It is one of the oldest food crops of mankind that researchers have traced back to 7000 - 8000 BC and probably originated from fertile crescent from where it spread to adjacent regions of west Asia and Africa and later to Europe and north America (Hawtin et al., 1980; Anonymous, 2003). It is one of the founding crops of agriculture, domesticated at about the same time as wheat and barley in the Fertile Crescent, from today’s Jordan northward to Turkey and southeast to Iran. A substantial portion of global lentil production is still concentrated in this area. Major lentil producing regions are Asia and north Africa region. The crop has the ability to grow under water stress conditions and survive under high range of drought and cold (Cubero, 1981). Plant descriptors coupled with molecular markers provide a valid evidence of diversity as these are least affected by environmental fluctuations (Ahmad et al., 1997; Jha and Ohri, 1996; Margale et al., 1995).However, the largest producers of lentils are India and Canada. Sixty percent of lentil production is in the South Asian region, including Bangladesh, Burma, India, Nepal and Pakistan.

Lentils are highly nutritious, containing protein, vitamin A, fiber, starch, potassium, B vitamins, and iron. As a protein source, lentil contains no cholesterol and virtually no fat, and very low levels of antinutrients (Ferguson et al., 2000). Its high protein content from 19% to 36%, 55% starch, low levels of toxic and antinutrient factors, and its ability to grow under water-deficit stress conditions, are the main attributes that make this an important crop (Savage, 1988; Bhatty, 1998). Nutritionist rank lentil as an excellent source of diet which is high in protein, a majorsource of complex carbohydrates, high in fibers, rich in vitamins A and B, potassium and iron, low in sodium and fat that regulate growth and development (Anon., 2003). Lentil plants provide a number of functions aside from being sources of human food. Lentil straw is an important fodder for small ruminants in the Middle East and North Africa, and the nitrogen sequestrating plant improves soil fertility and therefore increases sustainability of agricultural production systems. It is an important source of dietary protein (25 percent) in both human and animal diets, second only to soybeans as a source of usable protein (CGIAR). It ranks seventh among grain legumes and is grown on over 3.5 million hectares in over 48 countries with a total production of over 3 million metric tons.  The major lentil producing regions are Asia (58 percent of the area) and the West Asia-North Africa region (37 percent of the acreage of developing countries). Worldwide lentil is grown on a total area of 1.8 million hectares (FAO,2005). Worldwide, lentil is grown on a total of 1.8 million hectares, of which 60% is in theSouth Asian region which includes the lentil producing countries of Bangladesh, Burma, India, Nepal and Pakistan (Nazir et al., 1994).  In Bangladesh and Nepal lentil is the most important pulse crop for human consumption.Lentils are an increasingly popular crop worldwide and global production has been rising steadily for the last number of decades, more than tripling since 1980(Ferguson and Erskine,2001).The crop has developed into a range of varieties adapted to diverse growing areas and cultural preferences, and containing unique nutritional compositions, colors, shapes, and tastes.

Identification of pure-lines from local lentil germplasm

Subdividing variance into its components assists genetic resources  conservation, utilizationand it enables planning for use of appropriate gene pools in crop improvement for specific plant attributes (Bekele, 1984 & 1985). Large scale testing of broad base germplasm needs to be built up by making extensive local collection and obtaining germplasm from abroad to develop a sound breeding program (Jain et al., 1975; Ghafoor et al., 1992). Brown (1978), Laghetti et al., (1998) and Gupta & Sharma (2007) advocated that maximum genetic conservation would be achieved by sampling population from as many environments as possible to widen the genetic base of the cultivated lentil. Classification of germplasm gave rise to some elite lines for specific characters and the accessions for days to flowering (45), days to maturity (7), plant height (12), pods per cluster (17) and seed weight (27) have been selected and suggested for exploitation in breeding program. Short duration is one of the important characters in legumes as described by Bakhsh et al., (1992) and should be utilized for the development of short duration lentil cultivar. It was observed that some of the seaccessions possessed desirable genes for more than one character and hence these could be utilized directly or included in hybrid programme for varietal development. Selected accessions are suggested to be tested under a wide range of agroecological conditions for their potential confirmation and if found better under diversified and/or specific environments, should be exploited in lentil selection/breeding program. Indifferent results for correlation in various character pairs indicated that the germplasm collected from different regions is needed to use independently for selecting superior pure-lines from each set of cluster. Although pods per cluster vs seeds per pod were positively associated in same direction and magnitude. Correlation coefficient measures the degree to which a variable varies together or a measure of the intensity of association. It further confirms the interrelationship of the metric traits, which are essential for designing breeding strategy (Islam et al., 1990 and 2254 TAYYABA SULTANA ET AL., Toetia et al., 1983). Thus, knowledge of interrelationship among these characters is very critical. In general, correlation results revealed that selection within different clusters could be practiced for different traits and suitable parents could be selected for further development. Peyghambary (2003) reported similar correlation between flowering and maturity and negative correlation between seeds per pod and seed weight.  Amurrio et al., (1993) who reported positivecorrelation between days to flowering and days to maturity.  Wild Lens species/subspecies are a potential source for increasing genetic diversity in cultivated lentil (Gupta & Sharma, 2007).

The genus Lens

The genus Lens comprises seven taxa within four species including the cultivated type, Lens culinaris spp. culinaris (Ferguson and Erskine 2001).  Cultivated lentil includes two varietal types: small-seeded microsperma and large-seeded macrosperma.  Wild Lens species are represented by L. culinaris spp. orientalis, L. odemensis, L. nigrican and L. ervoides. All members of Lens are self-pollinating diploids (2n = 2x = 14; Sharma et al. 1995).  The haploid genome size of the cultivated genome is 4063 Mbp (Arumuganathan and Earle 1991).

The genus Lens Miller is part of the family Fabaceae (Leguminosae), subfamily Faboideae, tribe Fabeae, or alternatively in subfamily Papilionaceae, tribe Vicieae. According to the latest classification by Ferguson et al. (2000) the genus comprises seven taxa split into four species:

• Lens culinaris Medikus subsp. culinaris, L. culinaris subsp. orientalis, L. culinaris

subsp. tomentosus, L. culinaris subsp. odemensis

• Lens ervoides (Brign.) Grande

• Lens nigricans (M. Bieb.)Godr.

• Lens lamottei Czefr.

Taxa contained within L. culinaris comprise the primary genepool for lentil. The remaining

species constitute secondary-tertiary genepools. All species are diploid (2n=14), annual, and

self pollinating with a low outcrossing frequency. L. culinaris subsp. orientalis accessions have been found to have resistance to drought, cold, wilt, and Aschochyta blight. L. nigricans can hybridize with L. culinaris, but with low seed set(IPGRI and ICARDA,1985). The species is native to parts of Asia, Africa and the Mediterranean region. The Near and Middle East is the primary centre of diversity for both the domestic L. culinaris and its wild progenitor, but wild relatives in the genus are found from Spain to Tajikistan(Redden et al,2007). The wild progenitor of lentil is identified as the species Lens culinaris subsp. orientalis, which looks like a miniature cultivated lentil and bears ponds that burst open immediately after maturation. Selection by early farmers around 7000 BC led to the cultivated species with non-dehiscent pods and non-dormant seeds, more erect plants and a considerable increase in seed size and variety in color (Ferguson et al., 1996).

Germplasm and molecular marker 

Electrophoresis for various biochemical and molecular markers along with field evaluation adds information to taxonomy and should not be disassociated from morphological, anatomical and cytological observation (de Vries, 1996; Piergiovanni and Taranto, 2003; Sultana et al., 2006; Sarker and Erskine, 2006; Sultana and Ghafoor 2008). Among biochemical techniques, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE), isozymes and randomly amplified polymorphism DNA (RAPD) are widely used due to their validity and simplicity in describing the genetic structure of crop germplasm (Murphy et al., 1990). Seed protein profiles and molecular markers obtained by electrophoresis have been successfully used to study taxonomical and evolutionary relationships of several crop plants (Gepts et al.,1989; Rao et al., 1992; Ghafoor and Arshad, 2008). With the advancement in biological research, DNAbased markers provide powerful and reliable tools for discerning variations within crop germplasm and for studying evolutionary relationships (Virk et al., 1995). For better management of genebank, a precise comprehensive knowledge of agricultural and biochemical data (protein and DNA) is essential. The molecular markers have been used for estimating genetic variation at population level and among closely related species (Nienhuis et al., 1995). No single method is adequate for assessing genetic variation because the different methods sample genetic variation at different levels and differ in their power of genetic resolution as well as in the quality of information content.

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; Toklu et al., 2009). Intra-accession variation for stem colour, tendril and beak on the pod gave indication for the prevalence of landraces. Muehlbauer and Slinkard (1981) reviewed the genetics of Lens and listed 12 genes which account for morphological and seed variation in lentil. Seed proteins have been successfully used to study the variation of seed storage protein based on geographic distribution (Erskine and Muehlbauer, 1991; Piergiovanni and Taranto, 2003; Sultana et al., 2006; Yüzba_io_lu et al., 2008). The RAPD has been found important to resolve various levels of inter-and intra-specific polymorphism, which facilitates assessment of genetic relationships, definition of regional grouping and identification of individual accessions (Skroch and Nienhuis, 1995; Virk et al., 1996; Babayeva et al., 2009). The accessions with diverse pattern for RAPD are suggested for use in further study and to select parents for inheritance or linkage groups (Eujayl et al., 1997, 1998). Grouping germplasm into geographical entries and elucidating affinities among these groups can define gene pools and determine gene flow among populations. Variation 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 (Gutierrez et al., 2001).

According to Perry and McIntosh (1991), differentiation by geographic region of origin is useful in substantiating postulated regions of diversity or gene centres. Rare alleles occurring in only one or two apparently random populations can be considered mutants, migrants or the result of other coincidental events (van Hintum and Elings, 1991). Alleles common in restricted areas occur mostly in high mountainous areas. This could indicate that genetic material has been introduced from the foothills of separately, the North Western Frontier Province to the high mountains of the North Western Frontier Province and Northern Area. Migration of landraces into new regions, followed by some degree of contamination by mixing with other landraces, can be expected in a country like Pakistan, where movement of germplasm from one area to another is not restricted. Areas with a high levels of environmental stress will present mixtures with interesting types of tolerance to environmental stresses but which are homogeneous; these areas require less extensive sampling for the purpose of conserving genetic resourcesperhaps due to exchange of germplasm by breeders or transport of pulses to different markets from where seed of various origins is disseminated throughout the country. According to Smith et al. (1995), linkage clustering and PCA are useful for preservation and utilization of germplasm

 Lentil breeding

There have been significant breeding achievements in lentil since the late 1970s. The genetic

base of the crop has been broadened and tolerance to abiotic (drought and cold) and resistance

to biotic stresses from wild genotypes and traditional varieties have been incorporated into

new high yielding cultivars (FAO,1994). Large collections of lentil are stored in genebanks as genetic resources for further breeding efforts. However, there are gaps in the coverage of the genetic diversity of the crop and some of the collections are endangered or deteriorating.

Overview of Lens collections

This strategy identified 43 214 accessions of Lens held in collections worldwide, gathered

from the questionnaire, meetings, and other data sources.

Analysis of information from the regional conservation strategies and lentil position

From 2005 to 2007 with support from the Global Crop Diversity Trust (Trust), regional conservation strategies for the long-term conservation and availability of plant genetic resources were developed for almost all of regions worldwide. Lentil is considered a high priority crop in the regional conservation strategy for West Asia and North Africa, with first priority assigned to the crop in West Asia, and very high priority in North Africa. This is both the centre of origin and the primary centre of diversity for the crop. Lentil constitutes the fourth highest number of accessions in the region (7355 accessions), held mostly in NGB Iran, NGB-AARI Turkey, GCSAR Syria, NGB Egypt, NARC Pakistan, and INRA Morocco. Also within the primary centre of diversity, the Central Asia and the Caucasus strategy prioritizes the importance of lentil, especially for food security in Azerbaijan and Tajikistan.  The Eastern Africa strategy includes lentil amongst the region’s priority crops, given the 19^th ranking out of the 21 crop groups listed. The crop is important in Ethiopia and Sudan. Ethiopia maintains the only major collection in the region at the Institute of Biodiversity Conservation (IBC). Screenings of this material have identified earliness, high seed yield, high harvest index, high number of seeds per pod and cold tolerance. The conservation strategy of South, South-East and East Asia gives lentil an overall priority of 17th out of 28 crops. The ranking is higher in the South Asian sub region where the crop is ranked as the 14th most important crop and is assigned the highest priority category in Bangladesh, India and Nepal, and second priority in Bhutan and Sri Lanka. Lentil is the most important pulse crop in the sub region, with rich variability in India and in Nepal. Collections of lentil in the region identified as of greatest importance as well as priority for support include NBPGR India and the working collection at IIPR India. 3022 lentil accessions are conserved in the region. The Southern Africa regional strategy does not list lentil as of priority importance to the region, but does assign the crop high priority in terms of importance to specific countries (these not specifically named). Twenty six accessions are listed as conserved in Lesotho. The Americas strategy gives lentil medium priority in the region.

 Microsatellite marker

Analysis of microsatellite DNA loci is the current method of choice for population analyses (e.g., Morgante and Olivieri. 1993, Vendramin et al. 1998).  Microsatellite loci consist of short (2-6 bp) tandemly-repeated nucleotide arrays surrounded by unique flanking sequences (Weber and May 1989).  These loci are distributed throughout the genome in high abundance; it is estimated that the mammalian genome may contain in excess of 100,000 to 300,000 such loci, or one locus every 10-30 kilobase pairs (Li 1997).  Allelic diversities and heterozygosities are typically extremely high; the presence of 10 or more alleles, and heterozygosities in excess of 0.85, are not uncommon.  Microsatellite markers in lentil (about 80) have been developed by ICARDA recently and some of them (30) have already been assigned to linkage groups (Hamwieh et al. 2004, Eujayl et al. 1998). Microsatellite-DNA markers is used to obtain baseline data on allelic diversity of a composite germplasm set of lentil.  These datal then used to determine allelic frequency distributions for each locus within the collection as a whole and within source regions, as well as the geographical population genetic structure displayed by these loci among source regions.  The analysis of genetic diversity will help elucidate population structures that influence the analysis of the associations between markers and phenotypes for important traits. Phenotypic data collected for the population.

RAPD and lentil

Lentil was investigated for diversity based on botanical descriptors, total seed proteins, isozymes and RAPD markers. Diversity explored through various techniques revealed validity irrespective of the sample size or geographic pattern, RAPD being the best choice for investigating both inter and intra accession variation in lentil. Although all the techniques were  able to resolve genetic diversity in lentil, anyhow isozymes and seed proteins gave low level of genetic diversity that suggested to incorporate more isozymes and investigation on specific proteins for diversity in lentil. The RAPD being the best option for inter and intra–accessions variation is needed to extend to more Germplasm and primers for further study along with botanical descriptors (Sultana and Gafoor, 2009). RAPD utilized in genetic characterization of lentil due to its high simplicity. (Murphy et al., 1990). Sultan and Ghafoor (2009) study has shown that the RAPD is very efficient in the production of DNA polymorphism in lentil for studying intra-accession variation. Similarly, Botanical descriptors, total seed proteins, isozymes and RAPD markers were applied to identify landraces from indigenous lentil germplasm exclusively collected from the province of Baluchistan,Pakistan. The Germplasm revealed the prevalence of landraces, especially on the basis of isozymes and RAPD markers. Dversity explored through various techniques revealed validity irrespective of the sample size from a particular district, RAPD being the best choice for investigating both inter and intraaccession variation that is needed to extend to more germplasm study along with botanical descriptors(Sultana and Gafoor, 2009).

Significant variation among the lentil genotypes were observed in respect of days to first flowering, days to maturity, plant height, pod/plant, 100-seed weight and yield. The genotype BLX-02009-06-3 flowered and matured earlier. Among the test entries, BLX- 02009-18-3 and BLX-02009-18-1 were tall. The highest number of pod per plant was obtained in BLX-02009-04-5. The large seed size was found in LR9-130 and LR9-25. The highest yield was found in BLX-02009-04-1 followed by BLX- 02009-04-5 than the two check varieties. Difference between genotypic coefficient of variation and phenotypic coefficient of variation was small for the traits as plant height, days to maturity, days to first flowering and 100-seed weight. Among different traits, grain yield had high variation both at genotypic and phenotypic level due to the differences of genetic materials and also the differences of the environment. Grain yield was found to be positively and significantly correlated with plant height, pods/plant, 100-seed weight at genotypic and phenotypic levels (Alam et al. 2011).

Lentil germplasm collection was chosen to perform molecular analysis based on ISSR markers. This markers proved to be useful for distinguishing among closely related genotypes and for possibly substantiating the genetic peculiarity of some interesting material (Laghetti et al., 2008).

Usually seed gene banks store a large number of accessions per each crop/taxon ex situ. During the characterization process of this material several quantitative and qualitative data are recorded. Usually, a wide variation is recorded at the intra accession level in addition to interaccession one. The management of all this information becomes very diffi cult without eff ective statistical tools able to combine diff erent types of data of this sort. At the Institute of Plant Genetics (IGV), National Research Council (Bari, Italy) this problem has been addressed by testing many statistical approaches (Laghetti et al., 1990; Perrino et al., 1984; Polignano et al., 2001). However they were old methods that studied separately the quantitative and qualitative data and, in addition, did not consider the intra accession variability using an average datum.

Generally, characterization and preliminary evaluation data are based on agronomic traits linked to yield performance, but they oft en give little information on the actual genetic constitution of the examined material. Conversely, molecular markers precisely defi ne the genetic constitution of a sample, but give no information on yield attitude. Th is is particularly true in some crops like lentil (Lens culinaris Medik), in which it is reported by several authors that genetic variation as examined at a molecular level, does not go along with the level of variation assessed at the morpho-productive level. In fact, domestication pressure in lentil has fi xed few Mendelian characters, e.g. absence of dormancy or pod shattering, and few quantitative traits, like seed size (Fuller, 2007). Th ese characters account for a small proportion of the genome that is oft en not associated to molecular markers, most of which are therefore evolutionary neutral (Hammer, 1984; Grandillo et al., 1999). For this reason lentil was selected as a case study and a subset of the lentil collection was analysed also using molecular markers. Th e present contribution reports on the results of this study.

Linking Genotype with Phenotype in Wild Lentil

There are many production constraints, in western Canada, which can cause decreases in productivity and quality of cultivated lentil (Lens culinaris). Wild lentil species can be a source of traits that could help overcome these production and quality constraints. Currently collections of wild lentil species exist, which could potentially be used in future interspecifc crosses. Before this begins, studies will need to be to done to in order to help plant breeders determine the potential utility of these crosses. The use of wild species for gene introgression comes with a cost: other, potentially deleterious, genes can also be introgressed due to linkage drag. Understanding the genetic make-up of wild species genomes relative to that of the cultivated genome can help provide tools to minimize linkage drag and therefore maximize the benefits from the interspecific cross. Having markers associated with traits of interest in the wild germplasm should allow breeders to keep track of the introgression of these beneficial segments of the wild genome while at the same time eliminate the other parts of the wild genome. Past studies have shown that association mapping can help breeders understand and determine complex traits. Association mapping is a method in which natural and wild populations can be assayed using molecular markers, which are then examined, for associations with a phenotypic trait by measuring linkage disequilibrium (Zhu et al., 2008). This allows for the identification and mapping of QTLs, identification of polymorphisms responsible for variation in phenotypes and the identification of the alleles linked to genes possibly responsible for the variation seen within populations (Gupta et al., 2004). Association mapping has shown to have advantages versus linkage analysis (Yu and Buckler, 2006). It can provide greater mapping resolution, researches don’t need to spend time developing mapping populations, and the markers used are not cross-specific therefore more than just two alleles can be assessed (Skot et al., 2005).

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