No abstract

Usage of high-throughput sequencing approaches allow for the generation and characterization of reference transcriptome datasets that support gene-based marker discovery, which in turn can be used to build genetic maps among other purposes. We have obtained a transcriptome assembly including 49,453 genes for the lentil ( Lens culinaris Medik.) cultivar Alpo using RNAseq methodology. This transcriptome was used as reference to obtain 6,306 quality polymorphic markers (SNPs and short indels) analyzing genotype data from a RIL population at F 7 generation derived from the interspecific cross between L . culinaris cv. Alpo and L . odemensis accession ILWL235. L . odemensis is a wild species included in the secondary gene pool and can be used as a source for gene introgression in lentil breeding programs. Marker data were used to construct the first genetic interspecific map between these two species. This linkage map has been used to precisely identify regions of the CDC-Redberry lentil draft genome in which the candidate genes for some qualitative traits (seed coat spotting pattern, flower color, and stem pigmentation) could be located. The genome regions corresponding to a significant single quantitative trait locus (QTL) controlling “time to flowering” located in chromosome 6 and three QTLs regulating seed size and positioned in chromosomes 1 and 5 (two QTLs) were also identified. Significant QTLs for Ascochyta blight resistance in lentil were mapped to chromosome 6 in the genome region or close to it where QTLs for Ascochyta blight resistance have previously been reported.

Powdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.

As part of a project on lentil molecular genetics, the sequence of the 18S-25S ribosomal RNA gene intergenic spacer (IGS) of Lens culinaris Medik. was determined. DNA was cloned after polymerase chain reaction (PCR) amplification. The spacer of 2939 bp was composed of nonrepetitive sequences and four tandem arrays of repeated sequences, named A to D. C and D arrays were formed by the repetition of very short consensus sequences. Similarity was found between lentil and other legume species, in particular those of the Vicieae tribe. A transcription initiation site, putative sites of termination and processing, and promoter-enhancer sequences were detected by computer-aided searches. These sites resemble motifs conserved in the IGS sequences of other plant species. The conservation of motifs in the otherwise highly variable plant IGS sequences points to the relevance of these motifs as functional sequences.

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.