Variability for restriction fragment lengths and phylogenies in lentil

Summary Thirty accessions of domesticated (Lens culinaris ssp. culinaris) and wild (L. culinaris ssp. orientalis, L. culinaris ssp. odemensis, L. nigricans ssp. ervoides and L. nigricans ssp. nigricans) lentil were evaluated for restriction fragment length polymorphisms (RFLPs) using ten relative low-copy-number probes selected from partial genomic and cDNA libraries of lentil. Nei's average gene diversity was used as a measure of genetic variability for restriction fragment lengths within subspecies and a dendrogram was constructed from genetic distance estimates between subspecies. The wild lentils L. culinaris ssp. orientalis and L. culinaris ssp. odemensis showed the greatest variability for restriction fragment lengths and were closely positioned to domesticated lentil in the dendrogram. Little variability for restriction fragment lengths was observed within accessions of L. nigricans ssp. ervoides and L. nigricans ssp. nigricans. This observation is consistent with a previously published proposal that nigricans may have been independently domesticated. Estimates of genetic variability based on RFLPs tended to be greater than estimates from isozymes.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

Comparison of crossability, RAPD, SDS-PAGE and morphological markers for revealing genetic relationships within and among Lens species

The phylogenetic relationships among (sub)-species in the genus Lens have been reviewed based on recent published reports. There was both a substantial level of agreement and disagreement between reports based on different analytical procedures and different plant germ plasms. Lens culinaris ssp. orientalis appeared as the wild progenitor of the cultivated lentils. A gene flow from L. odemensis and L. ervoides during lentil crop evolution was suggested. Morphological characters (quantitative and qualitative) showed a different taxonomic pattern in the genus Lens. The use of nuclear and biochemical markers (RFLPs, RAPDs, seed-protein electrophoresis) appeared to be the most consistent and reliable methods for determining genetic relationships. It is suggested that these techniques be used in combination for taxonomic analysis of the genus Lens.     

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.On sabbatical leave from HP Agricultural University, Palampur 176 062, India     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

Hybridization in the genus Lens by means of embryo culture

Summary The cultivated lentil L. culinaris and the wild lentil L. ervoides are reproductively isolated from one another due to their hybrid embryo breakdowns. Using embryo culture, vegetatively normal hybrids were obtained. One specific hybrid, heterozygous for a reciprocal translocation, had about 50% gamete viability and produced aborted and viable embryos in a 11 ratio. In the F₂, vegetatively normal and highly fertile plants were selected. With the aid of embryo culture techniques, L. ervoides can be included in the wild gene pool of the cultivated lentil.     

In vitro propagation ofLens species and their F1 interspecific hybrids

As an initial step in establishing interspecific hybridization to broaden the genetic basis of lentils [Lens culinaris ssp.culinaris (Medikus) Williams], a set of experiments was carried out to produce an efficient in vitro protocol for propagation of lentil and two of its wild relatives (Lens ervoides andLens culinaris ssp.orientalis). The objective of the experiments was to optimize the media (Murashige and Skoog) to regenerate shootsin vitro from nodal segments without a callogenic phase. The number of shoots per explant, the number of nodes per shoot and shoot length showed that species differences, gibberellic acid and benzyladenine levels had the largest effects, with only minor interaction effects. The experiments therefore identified a standard protocol which gave the optimum levels of growth regulators, Murashige and Skoog (MS) salts and sucrose concentrations for maximum plant regeneration from the nodal segment of these species. The medium recommended for optimal shoot regeneration without a callogenic stage contained 2.89 μM GA₃ in combination with 1.11 μM BA in MS medium lacking sucrose. The optimal medium for root induction on these shoots had the MS medium supplemented with 5.37 μM NAA. Final successful establishment of regenerated plants was completed by the transfer to a third medium containing half-strength MS salts.     

Classification and Characterization of Species within the Genus Lens Using Genotyping-by-Sequencing (GBS)

Lentil (Lens culinaris ssp. culinaris) is a nutritious and affordable pulse with an ancient crop domestication history. The genus Lens consists of seven taxa, however, there are many discrepancies in the taxon and gene pool classification of lentil and its wild relatives. Due to the narrow genetic basis of cultivated lentil, there is a need towards better understanding of the relationships amongst wild germplasm to assist introgression of favourable genes into lentil breeding programs. Genotyping-by-sequencing (GBS) is an easy and affordable method that allows multiplexing of up to 384 samples or more per library to generate genome-wide single nucleotide Polymorphism (SNP) markers. In this study, we aimed to characterize our lentil germplasm collection using a two-enzyme GBS approach. We constructed two 96-plex GBS libraries with a total of 60 accessions where some accessions had several samples and each sample was sequenced in two technical replicates. We developed an automated GBS pipeline and detected a total of 266,356 genome-wide SNPs. After filtering low quality and redundant SNPs based on haplotype information, we constructed a maximum-likelihood tree using 5,389 SNPs. The phylogenetic tree grouped the germplasm collection into their respective taxa with strong support. Based on phylogenetic tree and STRUCTURE analysis, we identified four gene pools, namely L. culinaris/L. orientalis/L. tomentosus, L. lamottei/L. odemensis, L. ervoides and L. nigricans which form primary, secondary, tertiary and quaternary gene pools, respectively. We discovered sequencing bias problems likely due to DNA quality and observed severe run-to-run variation in the wild lentils. We examined the authenticity of the germplasm collection and identified 17% misclassified samples. Our study demonstrated that GBS is a promising and affordable tool for screening by plant breeders interested in crop wild relatives.     

Searching chromosomal landmarks in Indian lentils through EMA-based Giemsa staining method

Lentil is one of the oldest protein-rich food crop with only one cultivated and six wild species. India is one important cultivator, producer and consumer of lentils and possesses a large number of germplasms. All species of lentil show 2n?=?14 chromosomes. The primary objective of the present paper is to search chromosomal landmarks through enzymatic maceration and air drying (EMA)-based Giemsa staining method in five Indian lentil species not reported elsewhere at a time. Additionally, gametic chromosome analysis, tendril formation and seed morphology have been studied to ascertain interspecific relationships in lentils. Chromosome analysis in Lens culinaris, Lens orientalis and Lens odemensis revealed that they contain intercalary sat chromosome and similar karyotypic formula, while Lens nigricans and Lens lamottei showed presence of terminal sat chromosomes not reported earlier. This distinct morphological feature in L. nigricans and L. lamottei may be considered as chromosomal landmark. Meiotic analysis showed n?=?7 bivalents in L. culinaris, L. nigricans and L. lamottei. No tendril formation was observed in L. culinaris, L. orientalis and L. odemensis while L. nigricans and L. lamottei developed very prominent tendrils. Based on chromosomal analysis, tendril formation and seed morphology, the five lentil species can be separated into two distinct groups. The outcome of this research may enrich conventional and biotechnological breeding programmes in lentil and may facilitate an easy and alternative method for identification of interspecific hybrids.     

Allozyme divergence and evolution in the genusLens

The genusLens includes 5 taxonomic species:L. culinaris is cultivated andL. orientalis, L. odemensis, L. ervoides, andL. nigricans are wild. All the species are annual and almost exlusively selfers. The wild lentils are distributed over a large geographical area and form small disjunct populations which are composed of a small number of plants. 67Lens populations were assayed electrophoretically for 9 enzyme systems; 15 enzymic genes with 37 alleles were identified. The genetic distances (D) measured between the pairs of populations indicated a significantly greater similarity between populations belonging to the same taxonomic species. Assuming the populations represent a random sample of the variability in each of the species the genetic distances (D) between the 5 taxa were calculated. The shortest genetic distance was found betweenL. orientalis andL. culinaris. Another significant feature of the data is the apparent isolation ofL. nigricans from the other 4 species. The genetic distances between theLens species are compared to the patterns of crossability barriers between them.     

From the cradle of agriculture a handful of lentils: History of domestication

Literature on lentil domestication is reviewed, particularly considering archeobotanical, phylogenetic, and molecular evidence. Lentils are one of the oldest crops cultivated and domesticated by man. Carbonized small lentil seeds have been found in several archaeological remains starting from the Neolithic. It is probable, however, that the most ancient remains refer to wild lentils; this is difficult to ascertain since seed size was probably selected after the establishment of a domesticated lentil. It is general opinion that cultivation occurred before domestication, but for how long is still an open question. It is now well accepted that the domestication of lentils was accomplished in the Near East, in an area called “the cradle of agriculture”. The genus Lens is very small, containing only 6 taxa. A wide range of morphological and molecular evidence supports the idea that the lentil wild progenitor is Lens culinaris ssp. orientalis. On the other hand, the most distantly related species within the genus appears to be L. nigricans, whose domestication was also attempted without success. The first characters involved in lentil domestication were pod dehiscence and seed dormancy. These traits are under a simple genetic control, and therefore mutants must have been fixed in a relatively short time. These and other morphological traits possibly involved in lentil domestication have been mapped in several linkage maps. However, generally these maps are not easily integrated since they are based on a limited number of markers. Newer maps, mainly built on different kinds of molecular markers, have been more recently produced. A consensus map is needed to fill the gap in lentil breeding and, at the same time, endow with deeper information on the genetics of lentil domestication, giving new insight into the origins of this crop, which present fragmented knowledge is unable.      

Genetic Diversity in Lens Species Revealed by EST and Genomic Simple Sequence Repeat Analysis

Low productivity of pilosae type lentils grown in South Asia is attributed to narrow genetic base of the released cultivars which results in susceptibility to biotic and abiotic stresses. For enhancement of productivity and production, broadening of genetic base is essentially required. The genetic base of released cultivars can be broadened by using diverse types including bold seeded and early maturing lentils from Mediterranean region and related wild species. Genetic diversity in eighty six accessions of three species of genus Lens was assessed based on twelve genomic and thirty one EST-SSR markers. The evaluated set of genotypes included diverse lentil varieties and advanced breeding lines from Indian programme, two early maturing ICARDA lines and five related wild subspecies/species endemic to the Mediterranean region. Genomic SSRs exhibited higher polymorphism in comparison to EST SSRs. GLLC 598 produced 5 alleles with highest gene diversity value of 0.80. Among the studied subspecies/species 43 SSRs detected maximum number of alleles in L. orientalis. Based on Nei’s genetic distance cultivated lentil L. culinaris subsp. culinaris was found to be close to its wild progenitor L. culinaris subsp. orientalis. The Prichard’s structure of 86 genotypes distinguished different subspecies/species. Higher variability was recorded among individuals within population than among populations.     

Genetic mapping of an ancient translocation in the genus Lens

Summary Segregation of 18 marker genes was monitored in selfed progeny of a Lens culinaris × L. ervoides hybrid; five linkage groups were mapped, one of which contained a reciprocal translocation break-point that differentiates between the parents. Four markers were found to be linked to the translocation break-point: Aco-1 and Pgm-2 on one side and Gs and Got-2 on the other. The gene pairs on both sides of the translocation are not linked in L. culinaris or in L. orientalis. The L. ervoides gene order was also found in L. odemensis but with significantly reduced map distances. Analysis of monogenic segregations in a number of Lens inter-specific crosses revealed some consistent patterns of deviations from the expected Mendelian ratios. The factors responsible for these unequal segregations, genotypic effects on recombination frequencies, negative interference, and the possible ancient origin of the translocation are discussed.     

Protein subunits and amino acid composition of wild lentil

Three wild species of lentil, Lens orientalis, L. ervoides and L. nigricans were investigated for protein subunits of the albumin protein fraction (APF), globulin protein fraction (GPF) and for protein and free amino acid composition. The APF and GPF formed 12.7–16.8 % and 34.7–49.0 %, respectively, of the meal nitrogen. SDS-PAGE showed APF to contain 15 to 20 major and a similar number of minor protein subunits ranging in M_r at least from 14 400 to 94 000. The GPF was also heterogenous and contained some subunits having M_r similar to APF subunits but none < 15 000. The three wild lentil species were distinguishable by their protein subunit composition. The protein amino acid composition of the wild species was identical and similar to that of the cultivated lentil. The wild species, like the cultivated species (L. culinaris), contained major amounts of free arginine, glutamic and aspartic acids, serine and a number of unidentified amino acids. L. orientalis, L. nigricans and the cultivated lentil contained two acidic and two basic unidentified amino acids. However, L. ervoides was distinctly different in that it contained only the two acidic plus one neutral unidentified amino acid, but none of the two basic unidentified amino acids.     

Paternal plastid DNA can be inherited in lentil

Restriction fragment analysis was used to study the inheritance of chloroplast DNA (cpDNA) in F₁ progeny from crosses between Lens culinaris ssp. orientalis and L. culinaris ssp. culinaris. Twenty-five combinations of 11 restriction enzymes and three heterologous probes from Petunia hybrida cpDNA were used to screen six accessions of L.c. culinaris and one accession of L. c. orientalis for restriction fragment length polymorphisms (RFLPs). No variation in cpDNA was observed within the subspecies L. c. culinaris, but the L. c. orientalis accession was unambiguously distinguished from all six L. c. culinaris accessions by two RFLPs. Of ten F₁ progeny from L. c. orientalis x L. c. culinaris crosses, nine had only maternal cpDNA restriction fragments but one F₁ plant inherited cpDNA fragments from both parents. Nuclear DNA inheritance was biparental in all ten F₁ progeny.     

Linkages between restriction fragment length,isozyme, and morphological markers in lentil

Summary A genetic linkage map of lentil comprising 333 centimorgans (cM) was constructed from 20 restriction fragment length, 8 isozyme, and 6 morphological markers segregating in a single interspecific cross (Lens culinaris × L. orientalis). Because the genotypes at marker loci were determined for about 66 F₂ plants, linkages are only reported for estimates of recombination less than 30 cM. Probes for identification of restriction fragment length polymorphisms (RFLPs) were isolated from a cDNA and EcoRI and PstI partial genomic libraries of lentil. The cDNA library gave the highest frequency of relatively low-copy-number probes. The cDNAs were about twice as efficient, relative to random genomic fragments, in RFLP detection per probe. Nine markers showed significant deviations from the expected F₂ ratios and tended to show a predominance of alleles from the cultigen. Assuming a genome size of 10 Morgans, 50% of the lentil genome could be linked within 10 cM of the 34 markers and the map is of sufficient size to attempt mapping of quantitative trait loci.     

The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

 Lens includes L. culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples. Received May 8, 2000 Accepted October 26, 2001     

Chloroplast DNA variation and evolution in the genus Lens Mill

 Chloroplast DNA (cpDNA) restriction site diversity was assessed by 21 enzyme/probe combinations in 30 accessions of six Lens species, including the recently recognized L. lamottei and L. tomentosus. A total of 118 fragments were scored and 26 restriction site mutations were identified. The cpDNA restriction pattern supports circumscribing L. lamottei and L. tomentosus as independent species. The value of the data for reconstructing phylogeny in the genus is discussed. The cpDNA of all 13 accessions of the lentil’s wild progenitor, L. culinaris subsp. orientalis, differed from that of the single lentil cultivars used in this study. This diversity indicates that other populations of this subspecies from Turkey and Syria examined by Mayer and Soltis (1994) are potentially the founder members of lentil. Examination of L. lamottei×L. nigricans hybrids between accessions having different restriction patterns showed paternal plastid inheritance in L. nigricans. Received: 2 July 1996 / Accepted: 19 July 1996     

Lentil domestication: On the quality of evidence and arguments

The current views on lentil domestication are based on biological attributes of the wild progenitorLens culinaris ssp. orientalis and on assumptions which have never been tested. Seed dormancy, a major factor in the adaptation of ssp.orientalis to its natural habitat, makes it inappropriate for cultivation, because poor germination causes seed yield following cultivation to be equal to the amount of sown seeds. Higher yield, resulting from the evolution of a non-dormant type can be obtained only after five or six cycles of unprofitable cultivation. It is doubtful that incipient farmers would have undertaken such an endeavor without preexisting knowledge that non-dormant types could eventually be obtained. Experiments involving the sowing of wild lentil would have been much more successful if the non-dormant types were present in appreciable quantities in the seed stock. Establishment of that type in the natural population would have required a period of seven to eight years with favorable growing conditions allowing the non-dormant type to become widespread in the population, followed by massive predation by man reducing the hazard of a population explosion. The close similarity between isozyme profiles of the cultivated lentil and its wild progenitor indicates that lentil cultivation was attempted with seeds derived from different populations and in different areas.