It is important to detect specific genes expressed in the guard cells, which control gas exchange and play key roles in response to drought and salt stresses. Due to the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell specific genes. We reported an optimized protocol of in situ RT-PCR by using an FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be specially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method for locating expression in the guard cells in Brassica rapa. Moreover, we proved the guard cell specific expression of Bra001929 in the epidermis indicating its' applicability as a marker gene for guard cells of Brassica rapa.

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China. To increase the availability of DNA markers in B. rapa, we developed insertion-deletion (InDel) markers utilizing high-resolution melting (HRM) curve analysis. We designed primers for 252 InDels (≥ 3 bp) evenly distributed in the genome and tested gene polymorphisms with eight accessions. In total, 208 markers were specifically amplified, and 148 InDels with polymorphism were genotyped successfully using HRM. We further analyzed the correlation with InDel size, GC number, and predicted the difference in T_m values (∆T_m) using 208 markers with specific amplification. We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-∆T_m, but not clearly correlated with the length of InDel. When the GC number within InDel was ≥ 8, the successful rate exceeded 90.0%. When the predicted-∆T_m reached 0.5 ℃, the success rate was greater than 90.0%, and when it was ≥ 0.6 ℃, the rate climbed to 100.0%, indicating their role as the optimal parameter for successful development of an applicable InDel marker. The polymorphic InDel markers can be easily genotyped using HRM. They are of great value in genetic analysis, construction of linkage map, and molecular marker-assisted selection in B. rapa.

Self-incompatibility (SI), which has recurred during the evolution of plants, is one of the most important cross-pollination mating systems. Three S-loci have been reported in Brassicaceae, namely, Arabidopsis lyrata (Al), Brassica (Br), and Leavenworthia alabamica (La) S-loci. Here, through multi-genomic comparative analysis of 20 species, we revealed that the most ancient S-locus was formed prior to the divergence of Brassicaceae lineage I and II. It was retained and inherited by Arabidopsis, as the Al S-locus in Brassicaceae lineage I. Furthermore, we found that the Br S-locus, which has been widely used in the breeding of Brassica crops to generate hybrid seeds, was formed through segmental translocation (ST) in the hexaploid ancestor of Brassica in Brassicaceae lineage II. The Br S-locus was evolved through a ST from one of the triplicated ancestral S-locus paralogs in the Brassica hexaploidy ancestor, while the other two S-locus paralogs were lost. Together with the previous discovery that the La S-locus was formed through a secondary origin in Brassicaceae lineage I, we conclude the monophyletic origin of Al and Br S-loci and clarify the evolutionary route of S-loci in the Brassicaceae family. Our findings will contribute to evolutionary studies and breeding applications of the S-locus in Brassicaceae.

Ethiopian mustard (Brassica carinata) is mainly grown as a leafy vegetable and oilseed crop. Sinigrin is predominant glucosinolate in the leaves of Ethiopian mustard. It is hydrolyzed by enzyme myrosinase to produce bioactive product specifically allyl-isothiocyanates. Due to sinigrin content, Ethiopian mustard has a mild flavor and it is a healthy addition to human diet. However, sinigrin concentration depends on genotype. Understanding variations of plant morphology and sinigrin content may be the first step for crop improvement. According to the analyses of various plant characteristics among ninety nine Ethiopian mustard accessions, we found significant differences in plant height, chlorophyll content, and petiole length. In addition, sinigrin content was determined using ultra-performance liquid chromatography tandem quadrupole mass spectrometers/tunable ultraviolet detector (UPLC-TQS/TUV). We found a significant difference in sinigrin content among leaves of 94 Ethiopian mustard accessions. Except for petiole length, the sinigrin content was negatively correlated with leaf area, leaf width, number of primary branches, and plant height. This information will help Ethiopian mustard breeders in screening breeding lines for new accessions.

Long terminal repeat retrotransposons (LTR-RTs) are a predominant group of plant transposable elements (TEs) that are an important component of plant genomes. A large number of LTR-RTs have been annotated in the genomes of the agronomically important oil and vegetable crops of the genus Brassica. Herein, full-length LTR-RTs in the genomes of Brassica and other closely related species were systematically analyzed. The full-length LTR-RT content varied greatly (from 0.43% to 23.4%) between different species, with Gypsy-like LTR-RTs constituting a primary group across these genomes. More importantly, many annotated LTR-RTs (from 10.03% to 33.25% of all detected LTR-RTs) were found to be enriched in localized hotspot regions. Furthermore, all of the analyzed species showed evidence of having experienced at least one round of a LTR-RT burst, with Raphanus sativus experiencing three or more. Moreover, these relatively ancient LTR-RT amplifications exhibited a clear expansion at specific time points. To gain a further understanding of this timing, Brassica rapa, B. oleracea, and R. sativus were examined for the presence of syntenic regions, but none were present. These findings indicate that these LTR-RT burst events were not inherited from a common ancestor, but instead were species-specific bursts that occurred after the divergence of Brassica species. This study further exemplifies the complexities of TE amplifications during the evolution of plant genomes and suggests that these LTR-RT bursts play an important role in genome expansion and divergence in Brassica species.