Association genetics studies on frost tolerance in wheat (Triticum aestivum L.) reveal new highly conserved amino acid substitutions in CBF-A3, CBF-A15, VRN3 and PPD1 genes.

BACKGROUND: Understanding the genetic basis of frost tolerance (FT) in wheat (Triticum aestivum L.) is essential for preventing yield losses caused by frost due to cellular damage, dehydration and reduced metabolism. FT is a complex trait regulated by a number of genes and several gene families. Availability of the wheat genomic sequence opens new opportunities for exploring candidate genes diversity for FT. Therefore, the objectives of this study were to identity SNPs and insertion-deletion (indels) in genes known to be involved in frost tolerance and to perform association genetics analysis of respective SNPs and indels on FT. RESULTS: Here we report on the sequence analysis of 19 candidate genes for FT in wheat assembled using the Chinese Spring IWGSC RefSeq v1.0. Out of these, the tandem duplicated C-repeat binding factors (CBF), i.e. CBF-A3, CBF-A5, CBF-A10, CBF-A13, CBF-A14, CBF-A15, CBF-A18, the vernalisation response gene VRN-A1, VRN-B3, the photoperiod response genes PPD-B1 and PPD-D1 revealed association to FT in 235 wheat cultivars. Within six genes (CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1) amino acid (AA) substitutions in important protein domains were identified. The amino acid substitution effect in VRN-A1 on FT was confirmed and new AA substitutions in CBF-A3, CBF-A15, VRN-B3, PPD-B1 and PPD-D1 located at highly conserved sites were detected. Since these results rely on phenotypic data obtained at five locations in 2 years, detection of significant associations of FT to AA changes in CBF-A3, CBF-A15, VRN-A1, VRN-B3, PPD-B1 and PPD-D1 may be exploited in marker assisted breeding for frost tolerance in winter wheat. CONCLUSIONS: A set of 65 primer pairs for the genes mentioned above from a previous study was BLASTed against the IWGSC RefSeq resulting in the identification of 39 primer combinations covering the full length of 19 genes. This work demonstrates the usefulness of the IWGSC RefSeq in specific primer development for highly conserved gene families in hexaploid wheat and, that a candidate gene association genetics approach based on the sequence data is an efficient tool to identify new alleles of genes important for the response to abiotic stress in wheat.

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1.

KEY MESSAGE: SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate.

Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance.

Genetic analyses of the host-pathogen system Turnip yellows virus (TuYV)-rapeseed (Brassica napus L.) and development of molecular markers for TuYV-resistance.

The aphid transmitted Turnip yellows virus (TuYV) has become a serious pathogen in many rapeseed (Brassica napus L.) growing areas. Three-years' field trials were carried out to get detailed information on the genetics of TuYV resistance derived from the resynthesised B. napus line 'R54' and to develop closely linked markers. F(1) plants and segregating doubled-haploid (DH) populations derived from crosses to susceptible cultivars were analysed using artificial inoculation with virus-bearing aphids, followed by DAS-ELISA. Assuming a threshold of E (405) = 0.1 in ELISA carried out in December, the results led to the conclusion that pre-winter inhibition of TuYV is inherited in a monogenic dominant manner. However, the virus titre in most resistant lines increased during the growing period, indicating that the resistance is incomplete and that the level of the virus titre is influenced by environmental factors. Bulked-segregant marker analysis for this resistance locus identified two closely linked SSR markers along with six closely linked and three co-segregating AFLP markers. Two AFLP markers were converted into co-dominant STS markers, facilitating efficient marker-based selection for TuYV resistance. Effective markers are particularly valuable with respect to breeding for TuYV resistance, because artificial inoculation procedures using virus-bearing aphids are extremely difficult to integrate into practical rapeseed breeding programs.