Evidence that the N-terminal extension of the vicieae convicilin genes evolved by intragenic duplications and trinucleotide expansions.

This study was aimed to identify lentil (Lens culinaris subsp. culinaris) convicilin genes and to carry out a comparative analysis of these genes in the tribe Vicieae. Convicilins differ from vicilins, a related group of plant seed storage proteins, mainly by the presence of an additional sequence of amino acids in the sequence corresponding to the first exon, referred as the N-terminal extension. A single gene for convicilin, a component of legume seed storage proteins, was identified in the cultivated lentil. In this species, the N-terminal extension is formed by a stretch of 126 amino acids of which 59.2% are charged amino acids: 29.6% glutamic acid, 3.2% aspartic acid, 14.4% arginine, 8.8% lysine, and 3.2% histidine. This lentil convicilin sequence is similar to the sequence of convicilins in other species of the tribe Vicieae. However, the size of the N-terminal extension clearly differs among convicilins. Sequence comparison and phylogenetic analyses including convicilin and vicilin of Vicieae species indicated that the differentiation between vicilins and convicilins predated the differentiation of the two vicilin gene families (47- and 50-kDa vicilins), and that the N-terminal extension evolved mainly by a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA.     

Production and characterization of a mouse monoclonal antibody specific for lentil lectin

A murine IgA monoclonal antibody (MoAb) was produced against the widely used glucose/mannose-specific two-chain mitogen from Lens culinaris (lentil) belonging to the Vicieae tribe of the Leguminosae family. The MoAb designated, 98, F-10, was found to be specific for lentil lectin when tested in dot blotting against 22 different native lectins. The antigenic specificity was also tested against subunits of 13 completely sequenced legume lectins separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose filters. The MoAb showed a strong reaction only against the lentil heavy subunit. Comparison of the amino-acid sequences revealed 13 amino-acid residues which might be involved in the epitope reactive with this antibody. The MoAb did not react with synthetic peptides from the heavy subunit of lentil.     

Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species.

Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins.     

Leveraging genomic resources of model species for the assessment of diversity and phylogeny in wild and domesticated lentil

Advances in comparative genomics have provided significant opportunities for analysis of genetic diversity in species with limited genomic resources, such as the genus Lens. Medicago truncatula expressed sequence tags (ESTs) were aligned with the Arabidopsis thaliana genome sequence to identify conserved exon sequences and splice sites in the ESTs. Conserved primers (CPs) based on M. truncatula EST sequences flanking one or more introns were then designed. A total of 22% of the CPs produced polymerase chain reaction amplicons in lentil and were used to sequence amplicons in 175 wild and 133 domesticated lentil accessions. Analysis of the sequences confirmed that L. nigricans and L. ervoides are well-defined species at the DNA sequence level. Lens culinaris subsp. odemensis, L. culinaris subsp. tomentosus, and L. lamottei may constitute a single taxon pending verification with crossability experiments. Lens culinaris subsp. orientalis is the progenitor of domesticated lentil, L. culinaris subsp. culinaris (as proposed before), but a more specific area of origin can be suggested in southern Turkey. We were also able to detect the divergence, following domestication, of the domesticated gene pool into overlapping large-seeded (megasperma) and small-seeded (microsperma) groups. Lentil domestication led to a loss of genetic diversity of approximately 40%. The approach followed in this research has allowed us to rapidly exploit sequence information from model plant species for the study of genetic diversity of a crop such as lentil with limited genomic resources.     

Bowman–Birk inhibitors in Lens: identification and characterization of two paralogous gene classes in cultivated lentil and wild relatives

In order to investigate the genetic structure of lentil Bowman–Birk inhibitors (BBIs), primers were designed on pea BBI sequences. The sequences obtained from lentil DNA, using these primers, indicate that lentil possesses at least two paralogous genes. Protein sequences translated in silico from lentil DNA sequences suggest that the two coded proteins are highly similar to Pisum trypsin inhibitor TI1 and TI6 BBIs, respectively. In fact, both are double-headed inhibitors, one class showing the presence of a trypsin- and a chymotrypsin-reactive site, the other showing two trypsin-inhibition sites, similar to pea TI1 and TI6, respectively. The same primers were used to amplify sequences from the DNA of other Lens species. The results strongly support that all species of Lens possess the same classes of BBI coding genes, orthologous to those identified in the cultivated lentil. Lens nigricans showed the most diverging sequences both at the nucleotide and the amino acid level. The similarity of the two gene classes identified in the genus Lens to those of Pisum and the observations that the patterns of expression of the Lens genes are equivalent to those of pea orthologous genes, possibly imply that BBIs in Lens are coded by gene classes with similar genome organization and function to those of pea. Finally, a phyletic analysis, based on the comparison of sequences obtained from other species belonging to the Vicieae tribe of the Fabaceae family, strongly suggests that all Vicieae could have a similar genome organization and function for BBI genes, and that this could be a general rule in all the Fabaceae family.Publication of the Institute of Plant Genetics N. 50     

Evolutionary relationships among Ascochyta species infecting wild and cultivated hosts in the legume tribes Cicereae and Vicieae

Evolutionary relationships were inferred among a worldwide sample of Ascochyta fungi from wild and cultivated legume hosts based on phylogenetic analyses of DNA sequences from the ribosomal internal transcribed spacer regions (ITS), as well as portions of three protein-coding genes: glyceraldehyde-3-phosphate-dehydrogenase (G3PD), translation elongation factor 1-alpha (EF) and chitin synthase 1 (CHS). All legume-associated Ascochyta species had nearly identical ITS sequences and clustered with other Ascochyta, Phoma and Didymella species from legume and nonlegume hosts. Ascochyta pinodes (teleomorph: Mycosphaerella pinodes [Berk. & Blox.] Vestergen) clustered with Didymella species and not with well characterized Mycosphaerella species from other hosts and we propose that the name Didymella pinodes (Berk. & Blox.) Petrak (anamorph: Ascochyta pinodes L.K. Jones) be used to describe this fungus. Analysis of G3PD revealed two major clades among legume-associated Ascochyta fungi with members of both clades infecting pea ("Ascochyta complex"). Analysis of the combined CHS, EF and G3PD datasets revealed that isolates from cultivated pea (P. sativum), lentil (Lens culinaris), faba bean (Vicia faba) and chickpea (Cicer arietinum) from diverse geographic locations each had identical or similar sequences at all loci. Isolates from these hosts clustered in well supported clades specific for each host, suggesting a co-evolutionary history between pathogen and cultivated host. A. pisi, A. lentis, A. fabae and A. rabiei represent phylogenetic species infecting pea, lentil, faba bean and chickpea, respectively. Ascochyta spp. from wild relatives of pea and chickpea clustered with isolates from related cultivated hosts. Isolates sampled from big-flower vetch (Vicia grandiflora) were polyphyletic suggesting that either this host is colonized by phylogenetically distinct lineages of Ascochyta or that the hosts are polyphyletic and infected by distinct evolutionary lineages of the pathogen. Phylogenetic species identified among legume-associated Ascochyta spp. were fully concordant with previously described morphological and biological species.     

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.     

5S rDNA genome regions of Lens species.

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.     

Inter-primer binding site retrotransposon and inter-simple sequence repeat diversity among wild Lens species

Even though lentil has been an important food legume for centuries, genetic studies in lentil are still in their infancy. Genetic diversity and relationships among wild Lens species from Turkey has seldom been investigated. Additionally, a limited number of simple sequence repeat (SSR) markers have been developed for use in breeding and genetic studies of lentil crop. In this study, molecular characterization of 50 accessions mostly from Turkey, belonging to 6 wild and 1 cultivated Lens species, was performed using newly developed inter-primer binding site (iPBS) retrotransposons and inter-SSR (ISSR) markers. The 10 iPBS primers generated a total of 151 scorable bands, of which 150 were polymorphic (99.3%) with an average of 15.0 polymorphic fragments per primer. The 10 ISSR primers detected 138 scorable bands showing 100% polymorphism, with an average of 13.5 bands per primer. The average polymorphism information content (PIC) value for ISSR markers (0.97) was higher than that for iPBS markers (0.90). Lens orientalis was found to be the most diverse species, raising the possibility of wide crosses with cultivated species Lens culinaris. Cultivated varieties also showed high level of polymorphism, at 82.92% and 51.92% with ISSR and iPBS markers, respectively. Lens lamottei and Lens tomentosus were found as the least polymorphic species using both marker systems. The grouping of accessions and species within clusters were almost similar when iPBS and ISSR graphs were compared. Our data also suggested the role of iPBS-retrotransposons as ‘a universal marker’ for molecular characterization of wild and cultivated Lens species.     

A comparative study of convicilin storage protein gene sequences in species of the tribe Vicieae.

Convicilins, a set of seed storage proteins, differ from vicilins, a related group of seed storage proteins, mainly because of the presence of the N-terminal extension, an additional sequence of amino acids in the sequence corresponding to the first exon. Convicilins have been described only in species of the legume tribe Vicieae. One or two genes for convicilins have been identified in most species of this tribe. The genus Pisum is the main exception, since two genes have been identified in most of its species. Thirty-four new convicilin gene sequences from 29 different species (Lathyrus, Lens, Pisum, and Vicia spp.) have been analyzed here. Convicilin gene sequences are generally organized in 6 exons, but in some instances one of the internal introns (2nd or 4th) is lost. In these 29 species, the N-terminal extension is formed by a stretch of 99 to 196 amino acids particularly rich in polar and charged amino acids (on average, it contains 29.43% glutamic acid and 15.38% arginine residues). This N-terminal extension has the characteristics of an intrinsically unstructured region (IUR), one of the categories of protein "degenerate sequences". A comparative analysis indicates that the N-terminal extension sequence has evolved faster than the surrounding sequence, which is common to all vicilins, and it evolved mainly through a series of duplications of short internal sequences and triplet expansions, the predominant one being GAA. This agrees with the evolution of IURs, which is faster than the evolution of surrounding sequences and is mainly due to replication slippage and unequal crossover recombination. Alternative maximum-likelihood trees of phylogenetic relationships among the 29 Vicieae species based on the convicilin exon sequences are presented and discussed.     

ITS sequence analysis and phylogenetic inference in the genus Lens mill

The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA from cultivated lentil (Lens culinaris subsp. culinaris) and its wild relatives was isolated and analysed for nucleotide sequence variation. Sequence divergence values ranged from no polymorphism within single species and between the cultigen and one accession of its wild progenitor (L. culinaris subsp. orientalis) to 14 base substitutions between L. nigricans and L. lamottei. Jukes and Cantor distance ranged from 0 to 1.79 %. Phylogenetic analysis confirmed the divergence of L. nigricans from all species, and the closeness of cultivated lentil to its wild progenitor, although two gene pools could possibly be identified in subsp. orientalis. Based on this study, the two recently recognized species, L. lamottei and L. tomentosus were separated from the other species. Each wild species showed peculiar autapomorphies and, in general, did not display much variation among accessions. The trees using chickpea as an outgroup formed two main clusters, one constituted by L. nigricans only and the other including the remaining taxa. Within this larger group, small subclades could be identified.     

Preparation of the lectin B component from the hemagglutinin of Lens culinaris]

Out of a mixture of both lectins of the lentil (Lens culinaris) the component B may be separated rapidly and easily because of the different temperature sensitivity of haptene protected isolectins A and B.     

Two crystal forms of the lentil lectin diffract to high resolution.

The legume lectins are an important class of polysaccharide-binding proteins with a wide range of biochemical and immunological applications. Two high-resolution crystal forms are obtained for the lentil (Lens culinaris) lectin: a monoclinic P21 and an orthorhombic P212121. The unit cell dimensions for the monoclinic form are a = 58.0 A, b = 56.0 A, c = 82.1 A, beta = 104.4 degrees, while for the orthorhombic form a = 56.4 A, b = 74.6 A, c = 124.9 A. The asymmetric unit contains one dimer in both cases. The crystals diffract to 1.7 A resolution using synchrotron radiation. Preliminary data have been collected to 2.3 A on both crystal forms using a conventional X-ray source.     

Evidence for domestication of Lens nigricans (M.Bieb.) Godron in S Europe

Evidence for domestication of Lens nigricans (M.Bieb.) Godron in S Europe. Small disjunct populations of the wild lentil Lens nigricans were found in secondary and man-made habitats throughout S Europe. Much larger populations of this species were found in primary habitats in SE Spain, S Italy and along the Adriatic coast of Yugoslavia. The majority of the L. nigricans populations were cytogenetically alike. One population differed from them by one reciprocal translocation, and another two by four chromosomal rearrangements. The L. nigricans accessions from S Europe were invariably cross-incompatible with L. nigricans of the Middle-East and with the cultivated lentil L. culinaris. The latter two were cross-compatible and their hybrids partially fertile. The existence of small L. nigricans populations in man-made habitats was interpreted as escapes from cultivation. Being cross-incompatible with L. culinaris these L. nigricans populations apparently were derived from a domesticated form of L. nigricans.     

Interaction of DNA with cationic liposomes: ability of transfecting lentil protoplasts.

The vesicle made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were multilamellar and rather homogeneous in shape as seen by transmission electron microscopy. Upon addition of circular DNA plasmids of different lengths to the liposomes, the formation of vesicle clusters around the DNA filament was observed, with dimensions dictated by the ratio DNA/lipid. These liposomes were able to transfect lentil (Lens culinaris) protoplasts inside the cells two different reporter genes, chloramphenicol-acetyltransferase and beta-glucuronidase. The activity of these two enzymes could be found in the cell lysates after 24 h from the incubation of protoplasts with the lipid-DNA complexes.     

Detection of ribosomal DNA sites in lentil and chickpea by fluorescent in situ hybridization.

Hybridization sites of an rDNA probe coding for the 18S, 5.8S, and 26S genes were detected on lentil and chickpea somatic chromosomes using fluorescent in situ hybridization. One pair of hybridization sites was detected in cultivated lentil Lens culinaris L. and wild lentil L. orientalis (Boiss.) Hand.-Mazz., and in both the hybridization sites of the ribosomal probe correspond to the secondary constriction. In cultivated chickpea Cicer arietinum three pairs of rDNA sites were detected and in the wild C. reticulatum two pairs were detected. The karyotypic relationship between the cultivated C. arietinum and its wild progenitor C. reticulatum is discussed.     

Purification and primary structure of novel lipid transfer proteins from germinated lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis>) seeds

A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92-93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116-118-residue proteins with 24-25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.