Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

Geographical distribution and adaptive variation of VRN-A3 alleles in worldwide polyploid wheat (Triticum spp.) species collection.

MAIN CONCLUSION: The distribution of early flowering alleles of VRN-A3 was found to be biased to low latitudes, and these alleles may contribute to environmental adaptability to low latitudes in cultivated emmer wheat. In wheat (Triticum spp.), the flowering time is an important trait for successful seed production and yield by adapting to the regional environment. An early flowering allele of VRN-A3 with 7- and 25-bp insertions in the promoter region (Vrn-A3a-h1) has recently been reported from the analysis of an emmer wheat (Triticum turgidum L. ssp. dicoccum) accession, TN26. This early flowering allele of VRN-A3 might be associated with the regional adaptation of wheat. In this study, we elucidated its geographic distribution to assess the importance of the early flowering allele of VRN-A3 in worldwide wheat collection. From sequence analysis, we identified six VRN-A3 alleles with the 7- and 25-bp insertions, namely, Vrn-A3a-h2, Vrn-A3a-h3, Vrn-A3a-h4, Vrn-A3a-h5, Vrn-A3a-h6, and Vrn-A3c-h2 from wild emmer wheat, while we identified two VRN-A3 alleles with these insertions, Vrn-A3a-h2 and Vrn-A3c-h1 from cultivated tetraploid and hexaploid wheat species in addition to Vrn-A3a-h1. Among VRN-A3 alleles distributed in cultivated wheat, we found that Vrn-A3a-h2 promoted early heading, whereas Vrn-A3c-h1 did not affect heading time. Our analysis showed that the distribution of early flowering alleles of VRN-A3 dominated in cultivated emmer wheat in Ethiopia and India, which actually showed an early flowering phenotype. This implied that the early flowering alleles of VRN-A3 contribute to adaptability to a low-latitude environment in cultivated emmer wheat. We could not find durum (T. turgidum L. ssp. durum) and bread wheat (T. aestivum L. ssp. aestivum) accessions with these early flowering alleles. Our findings indicated that Vrn-A3a-h1 and Vrn-A3a-h2 were useful for breeding of early flowering cultivars in durum and bread wheat varieties.

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.