Development of chickpea near-isogenic lines for Fusarium wilt.

Four pairs of near-isogenic lines (NILs) of chickpea with resistance/susceptibility to Fusarium oxysporum f. sp. ciceris (Foc) have been developed in this study. These lines were produced by searching in advanced recombinant inbred lines (RILs) that are segregating for Foc race 5 based on a phenotypic screening. The sequence tagged microsatellite (STMS) marker TA59, closely linked to wilt resistance genes on linkage group 2 (LG2) of the chickpea map, was used to assist the selection of resistant or susceptible genotypes. The NILs were also characterized for disease reaction to Foc races 1A, 2, 3 and 4. Resistance, susceptibility and slow wilting reactions were found in these NILs. Our results suggest that more than one gene controls the resistance to race 5. Combination of the major gene foc-5 linked to TA59 with other gene/s appears to be required to complete resistance, and the absence of these unknown genes leads to slow wilting reactions. The independent differential responses to races 2 and 3 observed in three NILs could be explained as recombination events. This result suggests that foc-2 and foc-3 are delimiting points at opposite ends of a genomic region that includes the remaining foc genes and the TA59 marker. This set of NILs has great potential for studying the genetics and mechanisms of wilt resistance. In addition, the NIL RIP8-94-11 can be used as differential line for Foc race 3; it showed a clear resistance reaction to race 3 and susceptibility to the other Foc races.

Cloning and molecular characterization of B-hordeins from Hordeum chilense (Roem. et Schult.).

One of the main limitations of cereal breeding is the lack of genetic variability within cultivated crops. Hordeum chilense is a wild relative of Hordeum vulgare, which has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. Among the agronomic traits of these new amphiploids, the allelic variation in the endosperm storage proteins and their influence on breadmaking and malting quality are of special interest. B-hordeins are sulfur rich prolamins, which account for 70-80% of the total hordein fraction in barley. In this work, rapid amplification of cDNA ends by PCR (RACE-PCR) has been used for the cloning of the full-length open reading frame (ORF) of six sequences of B3-hordeins from two lines of H. chilense. Two consensus sequences of 813 and 822 bp for the H1 and H7 lines, respectively, were determined by alignment of all the sequences generated. Between both lines, differences involving single base changes, which could correspond to single nucleotide polymorphisms (SNP), insertions and deletions were observed. Of these differences, only six out of the 13 within the ORF caused a change of amino acid. Two insertions/deletions of 9 and 12 bp were also observed between both lines. The derived amino acid sequences showed a similar structure to the B-hordeins from cultivated barley and other prolamins. The repetitive region is based on the repetition of the motif PQQPFPQQ. The copy number of the B3-hordeins was estimated as a minimum of nine and five copies for the H1 and H7 lines, respectively. The expression profile of the B-hordeins through the developing endosperm is also described in this work. This study of the storage proteins of H. chilense is a useful contribution to the knowledge of the genetic diversity available in wild relatives of cultivated barley. In addition, the origin of the different prolamins can be better understood with an in-depth knowledge of its wild equivalent.

Cloning and characterization of a gamma-3 hordein mRNA (cDNA) from Hordeum chilense (Roem. et Schult.).

Hordeum chilense is a wild relative of H. vulgare, cultivated barley, that has been successfully used in the synthesis of amphiploids by crossing with Triticum spp. These amphiploids-named generically x Tritordeum-have been tested under field conditions, and one of them, the hexaploid tritordeum obtained following chromosome doubling of the hybrid H. chilense x T. turgidum, shows traits of interest inherited from the barley parent. Of great interest is the allelic variation observed in the endosperm storage proteins and their influence on the breadmaking and malting quality of tritordeum. We report here two mRNA (cDNA) sequences for a gamma-3 hordein from two accession lines of H. chilense, H1 and H7, and their characterization by quantitative real time (QRT)-PCR in the developing endosperm. Sequences were obtained by rapid amplification of cDNA ends and "edge-to-edge" amplification of open reading frames from cDNA of H. chilense. Eight putative single nucleotide polymorphisms and one codon insertion were identified in the sequences of the H1 and H7 gamma-3 hordeins. The deduced amino acid sequences showed similar features to that of the gamma-3 hordein and gamma-gliadins from barley and wheat, respectively. While the repetitive motif (PQQQPF) is similar to that of the gamma-3 hordein from H. vulgare, there are 19 motif repeats in H. vulgare, whereas H. chilense shows 15 tandem repeats. The transcription of the genes encoding for the gamma-3 hordein were monitored by QRT-PCR: in both lines maximum transcription occurred 12 days after flowering.