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.

Introduction of a diverse genetic background of Pyrus into Malus through intergeneric hybridization.

Wide hybridizations across species and genera have been employed to enhance agriculturally important traits in crops. Within the tribe Maleae of the Rosaceae family, different genera and species exhibit several traits useful for increasing diversity and gene pool through hybridization. This study aimed to develop and characterize intergeneric hybrid individuals between Malus and Pyrus. Through seed germination, shoot multiplication, and rooting in vitro, acclimatized seedlings showing vegetative growth on their own roots were obtained from crosses of Malus × domestica pollinated by Pyrus communis, P. bretschneideri, and the Pyrus interspecific hybrid (P. communis × P. pyrifolia). Comparative analysis of leaf morphology, flow cytometry, and molecular genotyping confirmed the hybrid status of the individuals. Genome-wide genotyping revealed that all the hybrid individuals inherited genomic fragments symmetrically from the Malus and Pyrus parents. To the best of our knowledge, this is the first report on the development of intergeneric hybrid seedlings between Malus × domestica and P. bretschneideri. Furthermore, the Pyrus interspecific hybrid individual served as a bridge plant for introducing the genetic background of P. pyrifolia into Malus × domestica. The results of this study provided a crucial foundation for breeding through intergeneric hybridization between Malus and Pyrus, facilitating the incorporation of valuable traits from diverse gene pools.

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.

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.

The early flowering trait of an emmer wheat accession (Triticum turgidum L. ssp. dicoccum) is associated with the cis-element of the Vrn-A3 locus.

KEY MESSAGE: We identified a novel allele of the Vrn-A3 gene that is associated with an early flowering trait in wheat. This trait is caused by a cis-element GATA box in Vrn-A3. To identify novel flowering genes in wheat, we investigated days from germination to heading (DGH) in tetraploid wheat accessions. We found that the tetraploid variety Triticum turgidum L. ssp. dicoccum (TN26) harbors unknown genes that surpass the earliness effect of the early flowering allele Ppd-A1a harbored by TN28 (T. turgidum L. ssp. turgidum conv. pyramidale). Using recombinant inbred lines resulting from a cross between TN26 and TN28, we performed a quantitative trait locus (QTL) analysis for DGH. We identified a QTL for earliness in TN26 on chromosome 7AS, the chromosome on which Vrn-A3 is located. By sequence analysis for the Vrn-A3 locus in both TN26 and TN28, we identified a 7-bp insertion that included a cis-element GATA box sequence at the promoter region of the Vrn-A3 locus of TN26. Based on an expression analysis using sister lines for Vrn-A3, we suggest that the early flowering trait of TN26 was caused by the GATA box in Vrn-A3. In addition, we identified tetraploid wheat as a useful genetic resource for wheat breeding.

A simple method for measuring pollen germination rate using machine learning.

The pollen germination rate decreases under various abiotic stresses, such as high-temperature stress, and it is one of the causes of inhibition of plant reproduction. Thus, measuring pollen germination rate is vital for understanding the reproductive ability of plants. However, measuring the pollen germination rate requires much labor when counting pollen. Therefore, we used the Yolov5 machine learning package in order to perform transfer learning and constructed a model that can detect germinated and non-germinated pollen separately. Pollen images of the chili pepper, Capsicum annuum, were used to create this model. Using images with a width of 640 pixels for training constructed a more accurate model than using images with a width of 320 pixels. This model could estimate the pollen germination rate of the F2 population of C. chinense previously studied with high accuracy. In addition, significantly associated gene regions previously detected in genome-wide association studies in this F2 population could again be detected using the pollen germination rate predicted by this model as a trait. Moreover, the model detected rose, tomato, radish, and strawberry pollen grains with similar accuracy to chili pepper. The pollen germination rate could be estimated even for plants other than chili pepper, probably because pollen images were similar among different plant species. We obtained a model that can identify genes related to pollen germination rate through genetic analyses in many plants.

Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.).

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.