Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild self-incompatible species related to cultivated barley

Abstract

Gametophytic self-incompatibility (GSI) in the grasses is controlled by a distinct two-locus genetic system governed by the multiallelic loci S and Z. We have employed diploid Hordeum bulbosum as a model species for identifying the self-incompatibility (SI) genes and for elucidating the molecular mechanisms of the two-locus SI system in the grasses. In this study, we attempted to identify S haplotype-specific cDNAs expressed in pistils and anthers at the flowering stage in H. bulbosum, using the AFLP-based mRNA fingerprinting (AMF, also called cDNA-AFLP) technique. We used the AMF-derived DNA clones as markers for fine mapping of the S locus, and found that the locus resided in a chromosomal region displaying remarkable suppression of recombination, encompassing a large physical region. Furthermore, we identified three AMF-derived markers displaying complete linkage to the S locus, although they showed no significant homology with genes of known functions. Two of these markers showed expression patterns that were specific to the reproductive organs (pistil or anther), suggesting that they could be potential candidates for the S gene.

Molecular phylogeny of the genus Hordeum using thioredoxin-like gene sequences

“…The HTL gene is closely linked to the S locus (at 0 cM), one of the two self-incompatibility loci, in H. bulbosum (Kakeda et al 2000(Kakeda et al , 2008. The homologous gene (bm2) in P. coerulescens (Aveneae) was also confirmed for complete linkage to the S locus (Li et al 1994, Bian et al 2004.…”

“…The same two-locus SI system is also observed in other grasses and is distributed within the subfamily Pooideae, covering the tribes Triticeae, Aveneae and Poeae (Langridge and Baumann 2008). In the course of mapping SI loci, a common DNA marker was found to be closely linked to the S locus in H. bulbosum (Kakeda et al 2000(Kakeda et al , 2008 and Phalaris coerulescens (Li et al 1994, Bian et al 2004. The marker sequence was predicted to encode a thioredoxin hlike protein and was thus named HTL (Hordeum thioredoxinlike) in H. bulbosum (Kakeda et al 2000).…”

“…Sequence comparison of these orthologs showed that the coding region is highly conserved (93.9-98.4% nucleotide identity) among the four species whereas the 5′-untranslated region (5′-UTR) is quite variable (48.4-85.8%) between the two Hordeum species and Secale or Phalaris (Kakeda et al 2000). The HTL gene was confirmed to be a single-copy gene exhibiting complete linkage to the S locus by a detailed mapping study using 626 individuals in H. bulbosum (Kakeda et al 2008). These features of the HTL gene appear to be appropriate for phylogenetic analysis, because a high level of interspecific sequence variation could be present in the variable region while stable amplification from different species would be expected using PCR primers designed for the conserved regions.…”

“…Primers were designed based on the sequences conserved between the HTL gene from H. bulbosum (Kakeda et al 2000(Kakeda et al , 2008 and the homologous gene (Bm2) reported in P. coerulescens (Li et al 1994): Trx-F(5′-CGCRRAATATT CCACKTCCC-3′) and Trx-R(5′-YTGGTCCCAGTCCTCT TTGG-3′). PCR was performed in a reaction mixture of 20 μl containing approximately 100 ng of genomic DNA, 1x Pfx reaction buffer, 1 mM MgSO 4 , 0.3 mM dNTPs, 0.3 μM of each primer and 0.5 unit of Pfx DNA polymerase (Invitrogen).…”

Expression and functional analysis of the barley Nud gene using transgenic rice

“…DNA blot analysis was performed as described previously ( Kakeda et al 2008). Ten micrograms of genomic DNA from each plant was digested with HindIII, blotted and hybridized to PCR-amplified probe sequences of Nud (amplified with primers Nud-F and Nud-R) and Os06ERF (amplified with primers Os06ERF-F and Os06ERF-R) (see Table 1 for primer sequences).…”