Lens Taxa Research Articles

Phylogeny of Lens Taxa based on morphological and AFLP markers

Phylogeny of Lens Taxa based on morphological and AFLP markers

A New Method of Phenotyping and Utilization of Wild Lens Taxa for Diversification of Primary Genepool

The genus Lens is a member of the Vicieae tribe and comprised four self-pollinating diploid (2n=2x=14) species. Lens culinaris subsp. culinaris Medikus, encompassing two cultivated lentil on the basis of agro-morphological traits, the small seeded (microsperma) and largeseeded (macrosperma) and L. culinaris subsp. orientalis (Boiss.) Ponert, its presumed wild progenitor. The other sub-species and species are L. culinaris subsp. tomentosus (Ladz.), L. culinaris subsp. odomensis Ladz., L. ervoides (Brign.) Grande, L. nigricans (Bieb.) Godr. and L. lamottei Czefr. [1]. Lentil is an important pulse crop of Indian sub-continent, the Middle-East, Southern Europe and Eastern and Northern Africa. A critical appraisal of lentil cultivation and breeding in the past has relied primarily on local landraces of L. culinaris ssp. culinaris. Of late, modern lentil cultivars are derived from only a small number of improved landraces through pure line selection following hybridization between genotypes adapted to specific environmental conditions and seed size. These cultigens have some superiority over traditional varieties in terms of yield potential and disease resistance. Like other pulses, substantial improvement in the yield potential of lentil has not been achieved due to the lack of combination of genes for higher productivity, biotic and abiotic resistance, which are scattered in the cultivated and its wild relatives. Exploitation of wild gene pool for breeding purposes is a common practice in an increasing number of cultivated plants. In many cases, the wild relatives still possess important variation that no longer exists in their cultivated counterparts. Therefore, synthesis of new gene pool following hybridization and introgression from cultivated and wild types would make it possible to recreate some of the lost diversity in respect of productivity and other desirable characters. In this direction, a total of 463 global wild annual Lens taxa were introduced from the Biodiversity and Integrated Gene Management (BIGM) Unit at the International Centre for Agricultural Research in Dry Areas (ICARDA), Aleppo, Syria during 2010-11. These were further multiplied in adequate quantity, characterized and evaluated under different agro-ecological conditions for various qualitative and quantitative traits. We found that certain accessions of L. ervoides and L. nigricans does not flower properly during normal winter season and these accessions were planted again in summer himalayan nursery at Sangla in Himachal Pradesh during long days. The study revealed that remarkable variability with respect to genetic and non-genetic factors exists within the species and would be useful to the geneticists and breeders to plan phenotyping of wild Lens genepool. Phenotyping Procedure

Genetic relationships among wild Lens Mill. species revealed by SDS-PAGE

Summary: Seed storage proteins of 18 lentil accessions of the following Lens taxa: L. culinaris, L. orientalis, L. odemensis, L. nigricans, L. ervoides, L. lamottei, L. tomentosus and L. lenticula, were analyzed by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS–PAGE). All visible polypeptide bands were scored for presence or absence and data were used for dendrogram construction by UPGMA method. Three clusters were formed at the dendrogram: the fi rst one integrated accessions of L. orientalis and L. odemensis; the second cluster joined L. culinaris и L. tomentosus accessions, and related to them L. lamottei species, the third cluster included L. nigricans and L. ervoides accessions. Species affi liation of accessions L. orientalis к-2861, L. nigricans к-2859 and L. nigricans к-2860 was confi rmed. Taxonomic position of L. lenticula k-2858 accession was not determined exactly.

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.

Identification and isolation of lectin nucleotide sequences and species relationships in the genus Lens (Miller)

Genes for lectin, a component of legume storage proteins, were identified and characterised in two lentil cultivars ( Lens culinaris ssp. culinaris) and six wild relatives. In each taxon no differences were found among the two or three lectin clones sequenced, while differences were observed among lectin genes isolated from the different taxa. All of the clones analysed contained an insert of 828 bp and showed a high similarity with the nucleotide sequence of Pisum sativum seed lectin, PSL1. The deduced amino acid lectin sequences in all taxa were 275 amino acids long, and their multiple alignment showed that most of the variation among them occurred in regions which are not important for metal- and sugar-binding. The data from Southern blot analysis indicated the presence of only one lectin gene in all Lens taxa except L. tomentosus. Phylogenetic analyses carried out on the lectin sequences showed the existence of two main clusters and clearly indicated that L. nigricans falls outside the two groups.

Population genetic structure in Lens taxa revealed by isozyme and RAPD analysis

An understanding of the genetic structure of populations is vital for the formation of optimum collection, conservation and utilization strategies for plant genetic resources. This is of particular importance in the case of in-situ conservation, a strategy gaining in popularity. The population genetic structures of five wild lentil taxa, Lens culinaris subsp. orientalis, L. odemensis, L. ervoides, L. nigricans and L. lamottei were investigated using isozyme electrophoresis and random amplified polymorphic DNA (RAPD). Approximately 20 plants from each of 5 populations per taxon were screened for variation at 11 isozyme loci and using three RAPD primers. Levels of variation were generally low, although considerable variation existed in the levels of diversity found within populations of L. culinaris subsp. orientalis and L. lamottei. Comparison of the results obtained in this study with the results obtained in a previous study indicate that this is a trend occurring across all species. It implies that levels of diversity within populations must be measured and considered prior to targeting of specific populations for in-situ conservation. Analysis of molecular variance of both isozyme and RAPD data revealed that between 78% and 99% of the variation was attributable to between-population differences. Isozyme results from L. lamottei populations were, however, contradictory. Possible explanations for this difference are discussed.

AFLP analysis of the diversity and phylogeny of Lens and its comparison with RAPD analysis

AFLP and RAPD marker techniques have been used to evaluate and study the diversity and phylogeny of 54 lentil accessions representing six populations of cultivated lentil and its wild relatives. Four AFLP primer combinations revealed 23, 25, 52 and 48 AFLPs respectively, which were used to partition variation within and among Lens taxa. The results of AFLP analysis is compared to previous RAPD analysis of the same material. The two methods provide similar conclusions as far as the phylogeny of Lens is concerned. The AFLP technique detected a much higher level of polymorphyism than the RAPD analysis. The use of 148 AFLPs arising from four primer combinations was able to discriminate between genotypes which could not be distinguished using 88 RAPDs. The level of variation detected within the cultivated lentil with AFLP analysis indicates that it may be a more efficient marker technology than RAPD analysis for the construction of genetic linkage maps between carefully chosen cultivated lentil accessions.

Relationships among cultivated and wild lentils revealed by RAPD analysis

RAPD markers were used to distinguish between six different Lens taxa, representing cultivated lentil and its wild relatives. Twenty-four arbitrary sequence 10-mer primers were identified which revealed robust and easily interpretable amplification-product profiles. These generated a total of 88 polymorphic bands in 54 accessions and were used to partition variation within and among Lens taxa. The data showed that, of the taxa examined, ssp. orientalis is most similar to cultivated lentil. L. ervoides was the most divergent wild taxon followed by L. nigricans. The genetic similarity between the latter two species was of the same magnitude as between ssp. orientalis and cultivated lentil. In addition, species-diagnostic amplification products specific to L. odemensis, L. ervoides and L. nigricans were identified. These results correspond well with previous isozyme and RFLP studies. RAPDs, however, appear to provide a greater degree of resolution at a sub-species level. The level of variation detected within cultivated lentils suggests that RAPD markers may be an appropriate technology for the construction of genetic linkage maps between closely related Lens accessions.