MIG-seq (Multiplexed ISSR 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 SSRs through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and 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.

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.

Grafting-induced flowering is a key phenomenon to understand systemic floral induction caused by florigen. It can also be used as 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 didn’t induce 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 stable floral induction of the grafted cabbage, and this can be accomplished by increasing FT transcription and the leaf area of the rootstock.

A non-flowering natural cabbage mutant among the open-pollinated line 'T15' was found 42 years ago. The mutant was named 'nfc' (non-flowering cabbage) and has been propagated vegetatively by cuttings. 'nfc' hardly flowers during the spring season even after plenty of low-temperature periods. This study characterized the non-flowering trait of 'nfc' and assumed the mechanism. In the first experiment, we investigated the flowering characteristics of 'T15' and 'nfc' over three years. Throughout the 3-year cultivation period, all 'T15' plants flowered, while the flowering ratios of 'nfc' propagated by cuttings at the 1st, 2nd, and 3rd year were 0%, 32%, and 4%, respectively. In the last two years, other traits were also investigated in detail. The flowering dates of 'nfc' flowering plants were later than those of 'T15', and the average numbers of flowering shoots per flowering plant of 'nfc' were lower than those of 'T15'. Moreover, the terminal bud of 'nfc' flowering plants continued to grow vegetatively, even when their lateral shoots flowered. In the second experiment, to verify the hypothesis that 'nfc' is a chimeric plant, we investigated protoplast-regenerated plants' flowering characteristics from mesophyll protoplasts of 'nfc'. We obtained colonies derived from different protoplasts of 'nfc' and regenerated plants from each colony (nfcPP lines). Suppose the 'nfc' is a chimeric plant, in that case, protoplast-regenerated plants' flowering characteristics should be the same for each colony they derived. However, both flowering and non-flowering plants appeared in the same nfcPP lines derived from a single colony. From this, we concluded that 'nfc' is not a chimeric plant on flowering characteristics. These results indicate that 'nfc' is a mutant that maintains its flowering ability, but its flowering is strongly suppressed by some factors other than its structural characteristics.

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