Molecular Evaluation of the Effects of FLC Homologs and Coordinating Regulators on the Flowering Responses to Vernalization in Cabbage (Brassica oleracea var. capitata) Genotypes.

The flowering loci of cabbage must be understood to boost their productivity. In this study, to clarify the flowering mechanisms of cabbage, we examined the three flowering repressors BoFLC1, 2 and 3, and the flowering regulators BoGI, BoCOOLAIR, and BoVIN3 of early (CAB1), middle (CAB3), and late (CAB5) flowering cabbage genotypes. Analysis of allele-specifically amplified genomic DNA and various sequence alignments demonstrated that maximal insertions and deletions influenced cabbage flowering behavior, notably in CAB3 and CAB5. Phylogenetic studies showed that BoFLC1, 2, and 3 in the CAB1, 3, and 5 genotypes had the highest homologies to other Brassica species, with CAB3 and 5 the most similar. Although CAB3 and CAB5 have comparable genetic patterns, flowering repressors and flowering regulators were investigated individually with and without vernalization to determine their minor flowering differences. The expression investigation revealed that vernalized CAB5 downregulated all BoFLC genes compared to CAB3 and, in contrast, CAB3 exhibited upregulated BoCOOLAIR. We hypothesized that the CAB3 BoFLC locus' additional insertions may have led to BoCOOLAIR overexpression and BoFLC downregulation. This study sheds light on cabbage genotypes-particularly those of CAB1 and CAB5-and suggests that structural variations in BoFLC2 and 3 bind flowering regulators, such as COOLAIR, which may affect cabbage flowering time.

Simultaneously mapping loci related to two plant architecture traits by phenotypic recombination BSA/BSR in peanut (Arachis hypogaea L.).

KEY MESSAGE: We developed a new method phenotypic recombination BSA/BSR (PR-BSA/BSR), which could simultaneously identify the candidate genomic regions associated with two traits in a segregating population. Bulked segregant analysis sequencing (BSA-seq) has been widely used for identifying the genomic regions affecting a certain trait. In this study, we developed a modified BSA/bulked segregant RNA-sequencing (BSR-seq) method, which we named phenotypic recombination BSA/BSR (PR-BSA/BSR), to simultaneously identify candidate genomic regions associated with two traits in a segregating population. Lateral branch angle (LBA) and flower-branch pattern (FBP) are two important traits associated with the peanut plant architecture because they affect the planting density and light use efficiency. We generated an F6 population (with two segregating traits) derived from a cross between the inbred lines Pingdu9616 (erect and sequential; ES-type) and Florunner (spreading and alternating; SA-type). The selection of bulks with extreme phenotypes was a key step in this study. Specifically, 30 individuals with recombinant phenotypes [i.e., spreading and sequential (SS-type) and erect and alternating (EA-type)] were selected to generate two bulks. The transcriptomes of individuals were sequenced and then the loci related to LBA and FBP were simultaneously detected via a ΔSNP-index strategy, which involved the direction of positive and negative peaks in the ∆SNP-index plot. The LBA-related locus was mapped to a 6.82 Mb region (101,743,223-108,564,267 bp) on chromosome 15, whereas the FBP-related locus was mapped to a 2.16 Mb region (117,682,534-119,846,824 bp) on chromosome 12. Furthermore, the marker-based classical QTL mapping method was used to analyze the PF-F6 population, which confirmed our PR-BSA/BSR results. Therefore, the PR-BSA/BSR method produces accurate and reliable data.

Genetic Dissection of CRISPR-Cas9 Mediated Inheritance of Independently Targeted Alleles in Tobacco α-1,3-Fucosyltransferase 1 and ß-1,2-Xylosyltransferase 1 Loci.

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 ß-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.