Genome sequencing-based coverage analyses facilitate high-resolution detection of deletions linked to phenotypes of gamma-irradiated wheat mutants.

BACKGROUND: Gamma-irradiated mutants of Triticum aestivum L., hexaploid wheat, provide novel and agriculturally important traits and are used as breeding materials. However, the identification of causative genomic regions of mutant phenotypes is challenging because of the large and complicated genome of hexaploid wheat. Recently, the combined use of high-quality reference genome sequences of common wheat and cost-effective resequencing technologies has made it possible to evaluate genome-wide polymorphisms, even in complex genomes. RESULTS: To investigate whether the genome sequencing approach can effectively detect structural variations, such as deletions, frequently caused by gamma irradiation, we selected a grain-hardness mutant from the gamma-irradiated population of Japanese elite wheat cultivar "Kitahonami." The Hardness (Ha) locus, including the puroindoline protein-encoding genes Pina-D1 and Pinb-D1 on the short arm of chromosome 5D, primarily regulates the grain hardness variation in common wheat. We performed short-read genome sequencing of wild-type and grain-hardness mutant plants, and subsequently aligned their short reads to the reference genome of the wheat cultivar "Chinese Spring." Genome-wide comparisons of depth-of-coverage between wild-type and mutant strains detected ~ 130 Mbp deletion on the short arm of chromosome 5D in the mutant genome. Molecular markers for this deletion were applied to the progeny populations generated by a cross between the wild-type and the mutant. A large deletion in the region including the Ha locus was associated with the mutant phenotype, indicating that the genome sequencing is a powerful and efficient approach for detecting a deletion marker of a gamma-irradiated mutant phenotype. In addition, we investigated a pre-harvest sprouting tolerance mutant and identified a 67.8 Mbp deletion on chromosome 3B where Viviparous-B1 and GRAS family transcription factors are located. Co-dominant markers designed to detect the deletion-polymorphism confirmed the association with low germination rate, leading to pre-harvest sprouting tolerance. CONCLUSIONS: Short read-based genome sequencing of gamma-irradiated mutants facilitates the identification of large deletions linked to mutant phenotypes when combined with segregation analyses in progeny populations. This method allows effective application of mutants with agriculturally important traits in breeding using marker-assisted selection.

Wheat yellow mosaic virus resistant line, 'Kitami-94', developed by introgression of two resistance genes from the cultivar 'Madsen'.

'Kitahonami' is a soft red winter wheat (Triticum aestivum L.) cultivar that has high yield, good agronomic performance and good quality characteristics. It currently accounts for 73% of the wheat cultivation area of Hokkaido the northern island in Japan and 42% of Japan's overall wheat cultivation. However, this cultivar is susceptible to Wheat yellow mosaic virus (WYMV). WYMV has become widespread recently, with serious virus damage reported in Tokachi and Ohotsuku districts, which are the main wheat production areas in Hokkaido. Here, we report a new wheat breeding line 'Kitami-94', which was developed over four years by repeated backcrossing with 'Kitahonami' using DNA markers for WYMV resistance linked to the Qym1 and Qym2 from 'Madsen'. Basic maps of Qym1 and Qym2 were created and used to confirm that 'Kitami-94' reliably carried the two resistance genes. 'Kitami-94' demonstrated WYMV resistance, and had agronomic traits and quality equivalent to 'Kitahonami' except for higher polyphenol oxidase activity and lower thousand grain weight. 'Kitami-94' may be useful for elucidating the mechanism of WYMV resistance in the background of 'Kitahonami', and for developing new cultivars.

Normalization method for canopy temperature as an indirect indicator of yield potential in wheat breeding programs.

Canopy temperature (CT) is often related to potential yield and is a possible yield indicator in breeding programs. However, it is difficult to evaluate genetic variations of CT accurately in large-scale investigations, such as breeding programs, because CT is strongly affected by environmental conditions. In this study, to precisely evaluate these genetic variations, we determined the environmental factors that affect CT measurement and proposed a convenient normalization method to minimize their influence. We measured the CT of CT-high or CT-low cultivars in the field under various conditions. We found that as the sun and shade levels were alternated, the CT changed within seconds; the position in the field also critically affected the CT. However, even under these conditions, the differences between cultivars became clearer if CT was normalized by neighboring lines. Additionally, we revealed that CT measurements between 12:00 and 15:00 maximized the difference between cultivars. Using our normalization technique under the favorable conditions specified can help breeders select high-yield lines using CT in breeding programs.