| Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino), originated in China, is a popular and widely planted vegetable. It contains 5 varieties, including var.communis (Tsen et Lee) Hanelt, var. rosularis (Tsen et Lee) Hanelt, var. parachinensis (L.H.Bailey) Hanelt, var.tai-tsai Hort and var. multiceps Hort. Among them, only the variety of var. multiceps Hort. shows special multiple shoot branching in the vegetative stage. This distinctive pattern of shoot branching is also called tillering trait in non-heading Chinese cabbage. In this study, the multiple shoot branching cultivar, 'Maertou' and common cutivar, 'Suzhouqing' were chosen as the main materials. Their morphological and microscopic characteristics were observed to study the development mechanism of shoot branching in NHCC. Furthermore, we conducted the genomic sequencing by SLAF-seq using F2 population, and found that candidate genes related in multiple shoot branching and developed special SSR markers. Moreover, de novo transcriptome sequencing of'Suzhouqing' and 'Maertou' were analyzed for further study of shoot branching trait. The main results are as follows:1 Characteristics of shoot branching (tillering) in var. multiceps Hort. of NHCCMost of the varieties in Non-heading Chinese cabbage show typical shoot branching in reproductive stage after vegetative growth. Usually, there is no shoot branching visible in the vegetative stage. However, the variety var. multiceps Hort. is characterized by a condensed stem at early growth stages, and several dozen leaves grow from the axil at the mature stage when it is still in the vegetative stage. The axillary buds outgrowth at 10-12 leaves stage, then axillary buds outgrowth considerable and develop into branch shoot until the leaf number reaches maximum. Finally, each leaf grows to a tiller bud. On one hand,the variety var. multiceps Hort. produce tillers from nodes of non-elongated internodes pre-flowering like most ecotypes; on the other hand, it also produces branches from nodes flanked by elongated internodes, post-flowering, that resemble branches of eudicots. The microscopic observation of longitudinal sections showed that the axillary meristems and axillary bud formation occured both in 'Suzhouqing' and 'Maertou',but the formation time and the occurrence frequency were significantly different in two materials. The axillary bud retained dormancy in 'Suzhouqing,. However, the axillary bud had grown out and developed into branch shoot since the axillary bud formed in 'Maertou'? The lateral bud was also visible in 'Suzhouqing, when the vegetative development transferred into reproductive development, and then the lateral bud developed into branching and flowering.We also evaluated the representative contents of nutritional qualitied in NHCC. The soluble proteins and vitamin C were significantly higher in 'Maertou' than those in 'Suzhouqing'.2 The endogenous hormones effects on shoot branchingAnalysis of endogenous hormone contents during plant development stages showed IAA played an important role in the axillary bud inhibition and dormancy in 'Suzhouqing'.IAA maintained lateral bud dormancy and repressed outgrowth to form shoot branching in the vegetative stage, showing apical dominance in 'Suzhouqing'. By contrast, there was no inhibition in 'Maertou'. The ZR level in 'Maertou'was higher than that in 'Suzhouqing',implying that cytokinin was more active in axillary bud outgrowth and developing into branch shoots in 'Maertou'. The data for lateral bud number and length after 6 days treated by 6-BA, GR24 or their compounds demonstrated that 6-BA promoted axillary bud outgrowth and elongation, whereas GR24 treatment did not promote any amount of bud elongation and even inhibited bud outgrowth. The qPCR results indicated the changing folds of CTK-responsive genes like BcRR4 and BcRR5 in 'Maertou' significantly higher than those in the 'Suzhouqing'. 'Maertou' was more sensitive and directly responsive to exogenous cytokinin (6-BA) than 'Suzhouqing'. The relative expression of BcBRCl in'Suzhouqing' was higher than that in 'Maertou', indicating that the 'SL-CTK branching regulatory pathway' in 'Maertou' was weaker than that in 'Suzhouqing'.3 Heritability and gene effects analysis using joint segregation analysis (JSA)Three cultivars including 'Maertou','Rugaomaocai' and 'Suzhouqing',were crossed to study gene effects and heritability involved in the development of tiller and leaf. This study revealed that a significant positive correlation existed between tiller number and leaf number. The joint segregation analysis in six generation populations,P1,P2, F,,F2, B1 and B2, indicating the influence of major genes mixed with polygenes. The results revealed the traits of tiller number and leaf number in non-heading Chinese cabbage were controlled by two major genes and polygenes with dominant, additive and epistasis effects, respectively.The major gene heritability and polygenic heritability in F2 were varing from 61.58% to 96.83%, indicating that tiller formation in non-heading Chinese cabbage was mainly influenced by genetic factor, while the environmental impacts were stably tiny. As the generation of non-heading Chinese cabbage was mainly determined by the heritability,selection in early stages may be useful.4 F2 population genomic sequencing by SLAF-seq and SSR developmentThis study used a F2 population of 1,000 seedlings which had been obtained by crossing with ?'Maertou' and ?'Suzhouqing'. First, we observed tiller numbers of seedlings in this F2 population, and we divided into five groups based on the tiller numbers that refers to 0, 1-2, 5-6, 8-9 and ?10 respectively. Fifty plants were selected representing for each level, and their DNA were extracted from mixed samples, forming five DNA mixed pools. The restriction enzymes of Eco RI and Mse I were used to cleave the DNA,and the DNA library were sequenced. According to the SNP markers which were relevant to the tiller regions, there were more than 1,000 candidate genes. Annotated these genes and validated by qPCR, for identified genes for further functional verification. Based on SLEF-seq genomic information using strictly filter parameters, we determined 9 SSR markers linked to tillering trait. There were 8 SSR markers in parents, which could be amplified and produced clear DNA bands, suggesting an effective primers ratio with 90%.Four SSR primers produced steady DNA polymorphism in parents, representing 50% in the total primers.5 De novo transcriptome sequencing and analysisTwelve sequencing libraries of 'Suzhouqing‘ and 'Maertou' were sequenced using Illumina Hiseq 2000 to investigate differential gene expression between leaf, shoot axillary meristem and root at two stages. A total of 119,237 unigenes were detected, accounting to around 120 million nucleotides. BLAST analysis against different public databases identified the total annotation unigenes were 104,086 (87.29%). A total of 32,065 SSR loci were identified. The differential expression genes compared with stage0 and stage 1 showed there were wide discrepancies in gene expression in each tissue of the two varieties. The KEGG annotation showed 3,768 out of 60,308 genes (6.25%) were annotated as being involved in hormone signal transduction. The transcriptome assemblies with high quality in this study identified potential genes involved in tillering in 'Maertou', and provided a molecular level comparison between the tillering varieties and non-tillering varieties. |
PDF Full Text Request