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.

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-Wide Identification of Loci Associated With Phenology-Related Traits and Their Adaptive Variations in a Highbush Blueberry Collection.

Genetic variation in phenological traits is the key in expanding production areas of crops. Southern highbush blueberry (SHB) is a blueberry cultivar group adapted to warmer climates and has been developed by multiple interspecific hybridizations between elite northern highbush blueberry (NHB) (Vaccinium corymbosum L.) and low-chill Vaccinium species native to the southern United States. In this study, we employed a collection of diverse SHB accessions and performed a genome-wide association study (GWAS) for five phenology-related traits [chilling requirement (CR), flowering date, ripening date, fruit development period, and continuous flowering] using polyploid GWAS models. Phenology-related traits showed higher heritability and larger correlation coefficients between year replications, which resulted in the detection of robust phenotype-genotype association peaks. Notably, a single association peak for the CR was detected on Chromosome 4. Comparison of genotypes at the GWAS peaks between NHB and SHB revealed the putative introgression of low-chill and late-flowering alleles into the highbush genetic pool. Our results provide basic insights into the diversity of phenological traits in blueberry and the genetic establishment of current highbush cultivar groups.