Detection And Analyses Of Plant Type Related Genes In Non-heading Chinese Cabbage

Brassica rapa crops belong to Cruciferae Brassica plants, including Chinese cabbage(Brassica rapa ssp. pekinensis), non-heading chinese cabbage (B. rapa ssp. chinensis;NHCC) and turnip (B. rapa ssp. rapa) and originated from China, have a long history of cultivation and variety richness. Among them, non-heading Chinese cabbage is a popular vegetable and widely planted in the region along the Yangtze River. NHCC has rich morphological diversity, and contains several varieties. Among these several varieties, the plant type is different, including non-upright, half-upright and upright which contained un-girdling and girdling. However, the genetic mechanism underlying the high plant type diversity in the crop remains unknown. In our study, we conduct a research on the girdling trait in non-heading Chinese cabbage. The girdling related genes are detected by SLAF-seq,RNA-seq and MeDIP-seq in F2 population and "Qinglan". Based on the results, we selected Aux/IAA, ARF and SnRK2 genes to analyze and clone which belonged to Auxin and ABA signal translation pathways in Chinese cabbage and NHCC, respectively. In this paper, the main research results are as follows:1. We defined the girdling trait is a quantitative character and relative to plant height and length of petiole through the investigation in F2 populations crossed by 'Suzhouqing×Wutacai' and 'Qinglan×Wutacai' . The girdling ratio which equal to the ratio of the width of waist and Width of turnips were normally distribution. We found that the forming of girdling is accompanied by plant growth and low temperature accumulating plays a key role during this process that the plants under 18?-24? treatment were non-girdling.2. We constructed an analysis on the plant type trait through specific-locus amplified fragment sequencing in a population consisting of 1500 F2 individuals derived from the cross of two varieties with extreme plant type traits. We obtained 78,723,756 reads in sequencing and the percentages of GC were about 42%. Among the genome,62978 SLAFs were uniformly distributed on the chromosomes with average sequence depth of 414.26-fold. Finally, 3 association regions were located on chromosome A05 and A09 with 8810 SNPs and 148 genes. According to the RNA-seq data of different non-heading Chinese cabbage varieties, 25 candidate genes were found. Among these genes, there were 11 F-box genes. In before and after girdling samples, the expression of candidate genes changed strongly.3. We constructed a conjoint analysis to identify the candidate genes using RNA-seq and MeDIP-seq for before and after girdling samples in NHCC. Approximately 103,846,926 paired-end reads were obtained by RNA-seq and 70% of them were mapped on the reference genome. Among these genes,there were 1560 up-regulated genes and 1228 down-regulated genes. On other hand, through MeDIP-seq 223,380,998 paired-end reads with length of 49bp were obtained. 78% of these reads were mapped on the reference genome and the Methylation level of whole genome was improved in after girdling sample.We totally obtained 123 genes which were methylated in the genebody region and 387 genes which the methylation occurred on the promoter region. Among them, the numbers and percentage of genes, which included in metabolic pathways, biosynthesis of secondary metabolites pathways, plant hormone signal transduction and plant-pathogen interaction pathways, were large. Finally, we selected 14 candidate gene from plant hormone signal transduction pathway and confirmed them by real-time PCR.4. Aux/IAAs and ARFs are the candidate genes of conjoint analysis of RNA-seq and MeDIP-seq. All 33 of the ARFs, including 1 AL (a type of ARF-like protein), and 53 Aux/IAAs were identified in the B. rapa genome. The genes mainly diverged approximately 13 million years ago. After the split, no Aux/IAA was completely lost, and they were more preferentially retained than ARFs. In land plants, compared to ARFs, which increased in stability, Aux/IAAs expanded more rapidly and were under more relaxed selective pressure. Moreover, BraIAAs were expressed in a more tissue-specific fashion than BraARFs and demonstrated functional diversification during gene duplication under different treatments,which enhanced the cooperative interaction of homologs to help plants adapt to complex environments. In addition, ALs existed widely and had a closer relationship with ARFs.5. One of the candidate genes, SnRK2.6, belongs to the SnRK2 gene family. We found that this gene family was preferentially retained in Brassicas after the Brassica-Arabidopsis thaliana split. Next, we cloned and sequenced 13 SnRK2 from both cDNA and DNA libraries of stress-induced Pak-cho. Most of the BcSnRK2s have eight exons and could be divided into three groups. The results of an analysis of the expression patterns of the BcSnRK2 genes showed that BcSnRK2 group ? genes were robustly induced by ABA treatments. Most of the BcSnRK2 genes were activated by low temperature, and the BcSnRK2.6 genes responded to both ABA and low temperature. They may be global regulators that function at the intersection of multiple signaling pathways to play important roles in Pak-choi morphogenesis.