Identification of the causal mutation in early heading mutant of bread wheat (Triticum aestivum L.) using MutMap approach.

In bread wheat (Triticum aestivum L.), fine-tuning the heading time is essential to maximize grain yield. Photoperiod-1 (Ppd-1) and VERNALIZATION 1 (Vrn-1) are major genes affecting photoperiod sensitivity and vernalization requirements, respectively. These genes have predominantly governed heading timing. However, Ppd-1 and Vrn-1 significantly impact heading dates, necessitating another gene that can slightly modify heading dates for fine-tuning. In this study, we developed an early heading mutant from the ethyl methanesulfonate-mutagenized population of the Japanese winter wheat cultivar "Kitahonami." MutMap analysis identified a nonsense mutation in the clock component gene Wheat PHYTOCLOCK 1/LUX ARRHYTHMO (WPCL-D1) as the probable SNP responsible for the early heading mutant on chromosome 3D. Segregation analysis using F2 and F3 populations confirmed that plants carrying the wpcl-D1 allele headed significantly earlier than those with the functional WPCL-D1. The early heading mutant exhibited increased expression levels of Ppd-1 and circadian clock genes, such as WPCL1 and LATE ELONGATED HYPOCOTYL (LHY). Notably, the transcript accumulation levels of Ppd-A1 and Ppd-D1 were influenced by the copy number of the functional WPCL1 gene. These results suggest that a loss-of-function mutation in WPCL-D1 is the causal mutation for the early heading phenotype. Adjusting the functional copy number of WPCL1 will be beneficial in fine-tuning of heading dates. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01478-5.

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.

Unleashing floret fertility in wheat through the mutation of a homeobox gene.

Floret fertility is a key determinant of the number of grains per inflorescence in cereals. During the evolution of wheat (Triticum sp.), floret fertility has increased, such that current bread wheat (Triticum aestivum) cultivars set three to five grains per spikelet. However, little is known regarding the genetic basis of floret fertility. The locus Grain Number Increase 1 (GNI1) is shown here to be an important contributor to floret fertility. GNI1 evolved in the Triticeae through gene duplication. The gene, which encodes a homeodomain leucine zipper class I (HD-Zip I) transcription factor, was expressed most abundantly in the most apical floret primordia and in parts of the rachilla, suggesting that it acts to inhibit rachilla growth and development. The level of GNI1 expression has decreased over the course of wheat evolution under domestication, leading to the production of spikes bearing more fertile florets and setting more grains per spikelet. Genetic analysis has revealed that the reduced-function allele GNI-A1 contributes to the increased number of fertile florets per spikelet. The RNAi-based knockdown of GNI1 led to an increase in the number of both fertile florets and grains in hexaploid wheat. Mutants carrying an impaired GNI-A1 allele out-yielded WT allele carriers under field conditions. The data show that gene duplication generated evolutionary novelty affecting floret fertility while mutations favoring increased grain production have been under selection during wheat evolution under domestication.

Association mapping and validation of QTLs for flour yield in the soft winter wheat variety Kitahonami.

The winter wheat variety Kitahonami shows a superior flour yield in comparison to other Japanese soft wheat varieties. To map the quantitative trait loci (QTL) associated with this trait, association mapping was performed using a panel of lines from Kitahonami's pedigree, along with leading Japanese varieties and advanced breeding lines. Using a mixed linear model corrected for kernel types and familial relatedness, 62 marker-trait associations for flour yield were identified and classified into 21 QTLs. In eighteen of these, Kitahonami alleles showed positive effects. Pedigree analysis demonstrated that a continuous pyramiding of QTLs had occurred throughout the breeding history of Kitahonami. Linkage analyses using three sets of doubled haploid populations from crosses in which Kitahonami was used as a parent were performed, leading to the validation of five of the eight QTLs tested. Among these, QTLs on chromosomes 3B and 7A showed highly significant and consistent effects across the three populations. This study shows that pedigree-based association mapping using breeding materials can be a useful method for QTL identification at the early stages of breeding programs.