Keeping green leaf color at the time of harvest is one of the important traits for breeding of Brassica oleracea var. capitata f. alba, and this trait is related to low anthocyanin contents. To understand the differential accumulation of anthocyanins in cabbage, we selected high anthocyanin accumulators (HAAs) and low anthocyanin accumulator (LAAs) of cabbages and examined the anthocyanin content and the expression of anthocyanin biosynthesis-related genes. Among many genes investigated, BoDFR1 was found to be closely related to anthocyanin accumulation, even under low temperature (LT) conditions. BoDFR1 sequence analyses between HAAs and LAAs revealed that there is a single nucleotide polymorphism (SNP) (1118T/A) in the coding sequence, which substitutes one amino acid from Leu261 to His261; we named BoDFR1 with His261 substitution as BoDFR1v. This amino acid substitution did not affect dihydroflavonol 4-reductase (DFR) activity and substrate specificity, but the polymorphism showed tight association to the BoDFR1 expression, i.e., high level expression of BoDFR1 and low level expression of BoDFR1v under LT conditions. The high levels of BoDFR1 expression were due to the high levels of BoMYB114 and BobHLH expressions combined with low level expression of BoMYBL2, a repressor MYB. On the other hand, low levels of BoDFR1v expression seemed to be related to very low level expressions of BoMYB114 and BobHLH combined with a high level expression of BoMYBL2. It seems that different expression levels of these regulatory genes for MBW (MYB-bHLH-WD40) complex between HAAs and LAAs regulate BoDFR expression and anthocyanin accumulation. Using a single nucleotide polymorphism (SNP) between BoDFR1 and BoDFR1v, molecular markers for PCR and high resolution melt analyses were developed and validated to distinguish between HAAs and LAAs. Combined use of the BoDFR1 SNP marker with other stress markers, such as a cold tolerant marker, will greatly improve cabbage breeding.

For sustainable crop cultivation in the face of global warming, it is important to unravel the genetic mechanisms underlying plant adaptation to a warming climate and apply this information to breeding. Thermomorphogenesis and ambient temperature signaling pathways have been well studied in model plants, but little information is available for vegetable crops. Here, we investigated genes responsive to warming conditions from two Brassica rapa inbred lines with different geographic origins: subtropical (Kenshin) and temperate (Chiifu). Genes in Gene Ontology categories “response to heat”, “heat acclimation”, “response to light intensity”, “response to oxidative stress”, and “response to temperature stimulus” were upregulated under warming treatment in both lines, but genes involved in “response to auxin stimulus” were upregulated only in Kenshin under both warming and minor-warming conditions. We identified 16 putative high temperature (HT) adaptation-related genes, including 10 heat-shock response genes, 2 transcription factor genes, 1 splicing factor gene, and 3 others. BrPIF4, BrROF2, and BrMPSR1 are candidate genes that might function in HT adaptation. Auxin response, alternative splicing of BrHSFA2, and heat shock memory appear to be indispensable for HT adaptation in B. rapa. These results lay the foundation for molecular breeding and marker development to improve warming tolerance in B. rapa.

We determined the specificity of mutations induced by the CRISPR-Cas9 gene-editing system in tobacco (Nicotiana benthamiana) alleles and subsequent genetic stability. For this, we prepared 248 mutant plants using an Agrobacterium-delivered CRISPR-Cas9 system targeting α-1,3-fucosyltransferase 1 (FucT1) and β-1,2-xylosyltransferase1 (XylT1) genes, for which the mutation rates were 22.5% and 25%, respectively, with 20.5% for both loci. Individuals with wild-type (WT) alleles at the NbFucT1 locus in T₀ were further segregated into chimeric progeny (37–54%) in the next generation, whereas homozygous T₀ mutants tended to produce more (~70%) homozygotes than other bi-allelic and chimeric progenies in the T₁ generation. Approximately 81.8% and 77.4% of the homozygous and bi-allelic mutations in T₀ generation, respectively, were stably inherited in the next generation, and approximately 50% of the Cas9-free mutants were segregated in T₂ generation. One homozygous mutant (Ta 161-1) with a +1 bp insertion in NbFucT1 and a −4 bp deletion in NbXylT1 was found to produce T₂ progenies with the same alleles, indicating no activity of the integrated Cas9 irrespective of the insertion or deletion type. Our results provide empirical evidence regarding the genetic inheritance of alleles at CRISPR-targeted loci in tobacco transformants and indicate the potential factors contributing to further mutagenesis.

Global warming accelerates the development of high temperature (HT)- and high humidity (HH)-tolerant varieties. This is further facilitated by the identification of HTHH-tolerant genes and the development of molecular markers based on these genes. To identify genes involved in HTHH tolerance in cabbage (Brassica oleracea var. capitata), we performed RNA-seq analysis of two inbred lines, BN1 (HTHH-tolerant) and BN2 (HTHH-susceptible), and selected trehalose 6- phosphate phosphatase I-2 (BoTPPI-2) as one of the HTHH-tolerant-associated genes. We also developed a segregating F₂ population from a cross between BN1 and BN2. RNA-seq results showed that BoTPPI-2 transcript levels were high in the HTHH-tolerant inbred line BN1, but not detectable in the HTHH-susceptible inbred line BN2. The expression pattern of BoTPPI-2 was not related to the expression of heat shock-related genes. Soft rot resistance, used as an indicator of HTHH tolerance, was higher in BN1 than in BN2. F₂ individuals similar to BN1 with respect to phenotype appeared to be HTHH-tolerant, whereas BN2-types were susceptible to HTHH. Analysis of the genomic DNA revealed the presence of a long terminal repeat (LTR; ca. 4.6 kb) in the ninth intron of the BoTPPI-2_BN2 allele, thereby suppressing its transcription and exhibiting HTHH phenotype. Except for the LTR insertion, the sequence of BoTPPI-2_BN2 was almost identical to that of BoTPPI-2_BN1. On the basis of the LTR and BoTPPI-2 sequences, we developed a molecular marker to identify HTHH-tolerant genotypes and validated its efficiency using F₂ individuals, inbred lines, and cultivars from diverse sources. The marker explained the genetic basis of HTHH tolerance in at least 80%, but not 100%, of the cabbage genotypes. Thus, additional markers associated with HTHH tolerance are needed for perfect selection.