DNA Markers for Identifying waxy Mutations and Improving Noodle Quality in Wheat

Approximately 70% of wheat endosperm consists of starch, and variations in the quality and quantity of starch affect the processing characteristics of wheat flour. Amylose content in particular has a major effect on Asian noodle quality, and selection of wheat lines with slightly lower amylose levels is an important goal in Japanese wheat breeding programs. Accurately measuring amylose content by direct methods such as colorimetric assays was found to be problematic, suggesting there was a need for a more efficient and accurate method of screening for reduced amylose content. Therefore, we characterized mutations in the wheat waxy genes, which control amylose synthesis, and developed DNA markers for the identification of null waxy alleles. In this review, we describe the development of these markers and outline their utility for wheat breeding programs. Discipline: Plant breeding Additional key words: breeding, codominant marker, marker-assisted selection, starch M. Saito, P. Vrinten & T. Nakamura 110 JARQ 44 (2) 2010 Recently, a large number of genes and quantitative trait loci (QTLs) related to important agronomic traits such as disease resistance, pest resistance and quality of wheat flour have been described. This information has allowed rapid progression in the development of DNA markers able to distinguish lines with desirable phenotypes based on plant genotypes. Due to the hexaploid nature of wheat, three copies of most genes are present, and recessive mutations are difficult to detect phenotypically. Therefore, selection using DNA markers able to exactly identify the required genotype is particularly important in wheat breeding. In this review, we summarize the development of DNA markers for distinguishing waxy alleles, and outline the introduction of these markers into wheat breeding programs in Japan. The waxy mutations in wheat The successful separation of the three waxy proteins, Wx-A1, Wx-B1 and Wx-D1, by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowed the identification of waxy mutants lacking one or two waxy proteins. The waxy mutations were first investigated at the molecular level using waxy wheat derived from crosses between Kanto 107, which lacks Wx-A1 and WxB1 proteins, and Bai Huo, which lacks Wx-D1 protein. A 23-bp deletion and a 4-bp insertion was detected in the null Wx-A1 allele (Fig. 1a), while a 588-bp deletion and a 12-bp insertion occurred in the null Wx-D1 allele (Fig. 1c), and the entire coding region was deleted in the Wx-B1 null allele (Fig. 1b). PCR-based markers for each null allele of waxy wheat Based on nucleotide sequences of the wild-type and null waxy alleles, primer sets capable of identifying the genotype at each waxy locus were designed (Figs. 1 and 2). Since these primer sets can work under a single set of conditions, mutations at the Wx-A1, Wx-B1 and Wx-D1 loci can be identified in a single reaction using multiplex PCR. These primers can identify mutations occurring in Wx-A1 and Wx-B1 genes of wheat lines not only from Japan but also from the major wheat producing countries Australia, Canada and the USA. Therefore, the origins of the waxy mutations in lines from these countries appear to be identical to the mutations in Kanto 107, suggesting the markers will be useful in wheat breeding programs around the world. Molecular comparison of waxy null alleles The PCR markers described above were used to characterize waxy mutations in 168 wheat lines from 20 countries. Although most cultivars lacking Wx-A1 protein had the same mutation found in Kanto 107, lines from Turkey produced a fragment 173 bp longer than the fragment from wild-type alleles. This newly identified mutation was caused by the insertion of a transposable element into exon 4 of the Wx-A1 gene. The transposable element was 165 bp in size, with 12-bp terminal inverted repeats on each end. An 8-bp target site duplication consisting of Wx-A1 sequence flanked each end of the insertion. It is noteworthy that this insertion was conserved only in lines from Turkey, while the more common deletion described above was found in lines from several areas of the world, suggesting that the insertion event occurred comparatively recently. In all lines where the Wx-B1 protein was absent, the Wx-B1 allele appeared to be identical to the mutation carried by Kanto 107, suggesting that the null Wx-B1 mutation has a single origin. From the geographical distribution pattern of null Wx-B1 alleles, we speculated that this mutation arose in western Asia and spread to other areas of Asia, with modern plant breeding programs facilitating the distribution of the allele to lines bred in Australia, Europe and North America. Several spontaneous mutations caused by different mechanisms than those described above have been identified in Wx-B1 and -D1 alleles. Since these mutations were not found in multiple cultivars, they may have arisen relatively recently. Further molecular analysis of these mutations will provide additional insight into the origins and geographical distribution of mutations at the Wx proteins AM (%) Wx-A1 Wx-B1 Wx-D1