Positional cloning of a gene responsible for high grain protein content in tetraploid wheat

Abstract

High Grain Protein Content (GPC) is a desirable trait in breadmaking and pasta wheat varieties because of its positive effects on quality and nutritional value. However, selection for GPC is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. The long-term goal of this project is to provide a better understanding of the genes controlling GPC in wheat. The specific objectives of this project were: a) to develop a high-density genetic map of the GPC gene in tetraploid wheat, b) to construct a T. turgidum Bacterial Artificial Chromosome (BAC) library, c) to construct a physical map of the GPC gene and identify a candidate for the GPC gene. A gene with a large effect on GPC was detected in Triticum turgidum var. dicoccoides and was previously mapped in the short arm of chromosome 6B. To define better the position of the Gpc-B1 locus we developed homozygous recombinant lines with recombination events within the QTL region. Except for the 30-cM region of the QTL these RSLs were isogenic for the rest of the genome minimizing the genetic variability. To minimize the environmental variability the RSLs were characterized using 10 replications in field experiments organized in a Randomized Complete Block Design, which were repeated three times. Using this strategy, we were able to map this QTL as a single Mendelian locus (Gpc-B1) on a 2.6-cM region flanked by RFLP markers Xcdo365 and Xucw67. All three experiments showed that the lines carrying the DIC allele had an average absolute increase in GPC of 14 g/kg. Using the RFLP flanking markers, we established the microcolinearity between a 2.l-cM region including the Gpc-B1 gene in wheat chromosome 6BS and a 350-kb region on rice chromosome 2. Rice genes from this region were used to screen the Triticeae EST collection, and these ESTs were used to saturate the Gpc-B1 region with molecular markers. With these new markers we were able to map the Gpc-B1 locus within a 0.3-cM region flanked by PCR markers Xucw83 and Xucw71. These flanking markers defined a 36-kb colinear region with rice, including one gene that is a potential candidate for the Gpc-B1 gene. To develop a physical map of the Gpc-B1 region in wheat we first constructed a BAC library of tetraploid wheat, from RSL#65 including the high Gpc-B1 allele. We generated half- million clones with an average size of l3l-kb (5.1 X genome equivalents for each of the two genomes). This coverage provides a 99.4% probability of recovering any gene from durum wheat. We used the Gpc-BI flanking markers to screen this BAC library and then completed the physical map by chromosome walking. The physical map included two overlapping BACs covering a region of approximately 250-kb, including two flanking markers and the Gpc-B1 gene. Efforts are underway to sequence these two BACs to determine if additional wheat genes are present in this region. Weare also developing new RSLs to further dissect this region. We developed PCR markers for flanking loci Xucw79andXucw71 to facilitate the introgression of this gene in commercial varieties by marker assisted selection (httQ://maswheat.ucdavis.edu/ orotocols/HGPC/index.hlm). Using these markers we introgressed the Gpc-B1 gene in numerous pasta and common wheat breeding lines.