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.

Upregulation of tandem duplicated BoFLC1 genes is associated with the non-flowering trait in Brassica oleracea var. capitata.

KEY MESSAGE: Tandem duplicated BoFLC1 genes (BoFLC1a and BoFLC1b), which were identified as the candidate causal genes for the non-flowering trait in the cabbage mutant 'nfc', were upregulated during winter in 'nfc'. The non-flowering natural cabbage mutant 'nfc' was discovered from the breeding line 'T15' with normal flowering characteristics. In this study, we investigated the molecular basis underlying the non-flowering trait of 'nfc'. First, 'nfc' was induced to flower using the grafting floral induction method, and three F2 populations were generated. The flowering phenotype of each F2 population was widely distributed with non-flowering individuals appearing in two populations. QTL-seq analysis detected a genomic region associated with flowering date at approximately 51 Mb on chromosome 9 in two of the three F2 populations. Subsequent validation and fine mapping of the candidate genomic region using QTL analysis identified the quantitative trait loci (QTL) at 50,177,696-51,474,818 bp on chromosome 9 covering 241 genes. Additionally, RNA-seq analysis in leaves and shoot apices of 'nfc' and 'T15' plants identified 19 and 15 differentially expressed genes related to flowering time, respectively. Based on these results, we identified tandem duplicated BoFLC1 genes, which are homologs of the floral repressor FLOWERING LOCUS C, as the candidate genes responsible for the non-flowering trait of 'nfc'. We designated the tandem duplicated BoFLC1 genes as BoFLC1a and BoFLC1b. Expression analysis revealed that the expression levels of BoFLC1a and BoFLC1b were downregulated during winter in 'T15' but were upregulated and maintained during winter in 'nfc'. Additionally, the expression level of the floral integrator BoFT was upregulated in the spring in 'T15' but hardly upregulated in 'nfc'. These results suggest that the upregulated levels of BoFLC1a and BoFLC1b contributed to the non-flowering trait of 'nfc'.

Quantitative Analysis of Florigen for the Variability of Floral Induction in Cabbage/Radish Inter-generic Grafting.

Grafting-induced flowering is a key phenomenon to understand systemic floral induction caused by florigen. It can also be used as a breeding technique enabling rapid seed production of crops with long generation times. However, the degree of floral induction in grafted plants is often variable. Moreover, it is difficult in some crop species. Here, we explored the factors promoting variability in the grafting-induced flowering of cabbage (Brassica oleracea L. var. capitata), an important vegetable crop with a long generation time, via the quantitative analysis of florigen accumulation. Significant variability in the flowering response of grafted cabbage was observed when rootstocks of different genotypes were used. As reported previously, B. oleracea rootstocks did not induce the flowering of grafted cabbage plants, but radish (Raphanus sativus L.) rootstocks unstably did, depending on the accessions used. Immunoblotting analysis of the FLOWERING LOCUS T (FT) protein, a main component of florigen, revealed that floral induction was quantitatively correlated with the level of accumulated FT protein in the grafted scion. To identify rootstock factors that cause variability in the floral induction of the grafted scion, we investigated FT protein accumulation and flowering response in grafted scions when the transcription levels of FT and the leaf area of rootstocks were altered by vernalization, daylength and leaf trimming treatments. We concluded that increasing the total amount of FT protein produced in the rootstock is important for the stable floral induction of the grafted cabbage, and this can be accomplished by increasing FT transcription and the leaf area of the rootstock.

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.

Non-vernalization Flowering and Seed Set of Cabbage Induced by Grafting Onto Radish Rootstocks.

Cabbage (Brassica oleracea var. capitata) requires a long-term low-temperature exposure for floral induction, causing a delay in the breeding cycle. The objective of this study is to develop a method to induce flowering in cabbage without low-temperature treatment, using a grafting method. We conducted grafting experiments using two flower-induced Chinese kale cultivars (B. oleracea var. alboglabra) and seven radish cultivars/accessions as rootstocks and investigated the flowering response of grafted cabbage scions without low-temperature treatment. "Watanabe-seiko No.1" cabbage, when grafted onto the two Chinese kale cultivars, did not formed flower buds. Flowering was successfully induced in "Watanabe-seiko No.1" by grafting onto three out of the seven tested radish cultivars, and in "Kinkei No.201" and "Red cabbage" by grafting onto one tested radish cultivar. In "Watanabe-seiko No.1," the earliest flower bud appearance was observed at 29 days after grafting. Seeds were also obtained from the three cabbage cultivars that flowered by grafting. Gene expression analysis of "Watanabe-seiko No.1" cabbage scions which formed flower buds by grafting, revealed high expression of the homolog of the floral integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BoSOC1), at the time of flower bud appearance. However, in the same leaf samples, we observed low expression of two homologs of florigen, FLOWERING LOCUS T (BoFT.C2 and BoFT.C6). In addition, two homologs of the floral repressor FLOWERING LOCUS C (BoFLC3 and BoFLC4), which are known to be down-regulated before flower bud differentiation in the vernalization pathway, were highly expressed, indicating that grafting onto radish induces cabbage flowering independently of the vernalization pathway. The expression level of the radish FT homolog (RsFT) in "Rat's tail-G2," which had highly induced flowering in the grafted cabbage scion, was higher than in the other radish cultivars. However, although "Rat's tail-CH" effectively induced flowering in the cabbage scion, the expression of RsFT was low in this cultivar. In this study, floral induction of non-vernalized cabbage cannot be explained by the expression levels of RsFT in rootstock plants, alone. The flowering of non-vernalized cabbage would be induced by transmissible agents from rootstocks and not by the expression of cabbage FT, BoFT, from the scion itself.