Genomics-Enabled Analysis of Puroindoline b2 Genes Identifies New Alleles in Wheat and Related Triticeae Species

Kernel hardness is a key trait of wheat seeds, largely controlled by two tightly linked genes Puroindoline a and b (Pina and Pinb). Genes homologous to Pinb, namely Pinb2, have been studied. Whether these genes contribute to kernel hardness and other important seed traits remains inconclusive. Using the high-quality bread wheat reference genome, we show that PINB2 are encoded by three homoeologous loci Pinb2 not syntenic to the Hardness locus, with Pinb2-7A locus containing three tandem copies. PINB2 proteins have several features conserved for the Pin/Pinb2 phylogenetic cluster but lack a structural basis of significant impact on kernel hardness. Pinb2 are seed-specifically expressed with varied expression levels between the homoeologous copies and among wheat varieties. Using the high-quality genome information, we developed new Pinb2 allele specific markers and demonstrated their usefulness by 1) identifying new Pinb2 alleles in Triticeae species; and 2) performing an association analysis of Pinb2 with kernel hardness. The association result suggests that Pinb2 genes may have no substantial contribution to kernel hardness. Our results provide new insights into Pinb2 evolution and expression and the new allele-specific markers are useful to further explore Pinb2’s contribution to seed traits in wheat.

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.Key words: puroindolines, GSP-1, chromosome 5D, BAC library, tauschii.

The hardness locus in Australian wheat lines

The genetic factors that determine grain hardness in Australian wheat (Triticum aestivum L.) germplasm were investigated by studying the grain from 4 crosses (160–180 lines per cross). Although not all the crosses were between hard and soft wheats, the doubled haploid lines derived from the crosses showed significant variation in hardness as assessed either by the Single-Kernel Characterisation System (SKCS 4100) or the scanning electron microscopy appearance of cut surfaces. The wheat cultivars used in the study were Cranbrook, Halberd, CD87, Katepwa, Sunco, Tasman, Egret, and Sunstar and of these only Egret is normally regarded as soft. The quantitative information from the SKCS 4100 was integrated into the genetic map information established for the Cranbrook Halberd, CD87 Katepwa, and Sunco Tasman crosses. For the Egret Sunstar cross, the limited information available from the positioning of genetic markers was used to specifically examine the linkage between the hardness trait and the Pina-D1 (puroindoline) genetic locus on 5DS, and very close linkage was established. The Egret Sunstar cross was also used to develop a more rigorous rheologically based analysis of the raw SKCS crush response data. In addition, the cut surface of the lines was analysed and most (98%) of the samples showed a genetic linkage between the appearance of the cut surface (related to vitreousness) and the SKCS hardness index. Among the other crosses only Cranbrook Halberd showed linkage of the hardness trait to the previously identified hardness locus (ha) located on 5DS as defined by DNA markers for the Pina-D1 locus, and the microsatellite wmc233. The statistical association was shown to be highly significant, with approximately 30% of the variation accounted for by the 5DS region. Another region on chromosome 4D showed a significant association in the Cranbrook Halberd cross. The CD87 Katepwa cross did not show any consistent associations between the SKCS measures and chromosome region, whereas in the Sunco Tasman cross a highly significant association only on chromosome 4B (accounting for 20% of the variation) was suggested. The Sunco Tasman cross showed an overlap of the chromosome region that accounted for variation in both grain weight and hardness and this influence of grain weight on hardness was independently confirmed by a detailed qualitative rheological analysis of the crush response profiles for the Egret Sunstar lines. It is evident from the study that, in Australian wheat lines, there are some major effects on grain hardness that are not associated with the classical ha locus located on 5DS.