不结球白菜分蘖性状的发生机理及其候选基因挖掘

发布时间：2018-04-20 13:50

本文选题：不结球白菜 + 分蘖性状　；参考：《南京农业大学》2016年博士论文

【摘要】：不结球白菜(Brassica rapa ssp. chinensis Makino)原产中国,是十字花科芸薹属中重要的蔬菜作物之一。不结球白菜可以分为五大类,分别是:1.普通白菜var.communis (Tsen etLee) Hanelt, 2.塌菜 var. rosularis (TsenetLee)Hanelt, 3.菜薹 var.parfaachinensis (L.H. Bailey) Hanelt, 4.薹菜 var.tai-tsai Hort.,5.分蘖菜 var. multiceps Hort.。其中分蘖菜表现为特殊的多头分枝特性,在营养生长阶段即有大量侧枝形成,状如分蘖,又叫多头菜。本研究主要以分蘖菜品种'马耳头'和普通白菜品种'苏州青'为研究材料,对分蘖菜的多头分枝特性进行观察,解剖,测定内源激素含量变化,探讨其发生机理。构建F2分离群体,利用高通量测序技术进行分蘖性状相关的候选基因的挖掘。研究内容如下:1不结球白菜分呭性状的特点不结球白菜的多数品种在开花期会有大量侧枝从茎基部(一级分枝)及叶腋之间(二级分枝)抽出,一般在营养生长期无侧枝形成。但不结球白菜变种之一的分蘖菜在营养生长阶段即有侧芽从短缩茎基部和成叶间的叶腋处长出,进一步伸长生长发育成为由多片小叶组成的分枝。形态学观察表明,营养生长达到一定阶段(10-12片),不结球白菜分蘖菜中的侧芽开始形成并长出,直到每一个叶腋处都有一个侧芽并最终长成分蘖,具有一片成叶对应一个分呭的特点。组织切片观察显示,'苏州青'和'马耳头'都有腋分生组织的形成,但形成时期略有不同。'苏州青'中腋分生组织形成后进入休眠状态而'马耳头'中腋分生组织形成后无休眠过程直接继续发育并进一步形成分呭。生殖生长开始后,'苏州青'中的腋芽结束休眠也开始向外生长,形成侧枝后开花结果。成株期部分营养品质含量测定结果显示,分蘖菜的硝态氮、可溶性蛋白和Vc含量都显著高于普通白菜。2激素对不结球白菜分呭性状的影响利用酶联免疫反应法(ELISA)测定'马耳头'和'苏州青'内源激素含量结果显示:生长素主要参与了'苏州青'中侧芽的休眠,抑制侧芽向外生长,是'苏州青'中顶端优势现象起主要作用的激素;细胞分裂素在'马耳头'分蘖发生过程中起主要促进作用。对'马耳头'的根部外施细胞分裂素(6-BA)和独脚金内酯(GR24),处理6天后发现,独脚金内酯抑制侧芽的发生和生长而细胞分裂素促进侧芽的发生和伸长。Q-PCR结果显示,细胞分裂素响应基因BcRR4和BcRR5在'马耳头'中的增加量显著高于'苏州青'中的增加量,说明'马耳头'对细胞分裂素的响应更敏感;BcBRC1基因在'马耳头'中的表达量显著低于'苏州青'中的表达量,说明在“细胞分裂素-独脚金内酯调控茎分枝”这一抑制分蘖发生的调控途径中,'马耳头'响应机制劣于'苏州青'。3两杂交组合6个遗传群体的数量遗传学分析利用分蘖亲本'马耳头'、'如皋毛菜'和不分蘖亲本'苏州青'构建遗传群体(P1、P2、F1、F2、BC1和BC2),对分蘖数和叶片数进行主基因+多基因的混合遗传模型分析。结果显示不结球白菜分蘖数和叶片数均符合E-1混合遗传模型,受2对加性-显性-上位性主基因+加性-显性多基因控制。分蘖性状不仅受两对主基因控制,也受更多微效多基因的遗传影响。F2中主基因加多基因的遗传率达到61.58%～96.83%,说明分蘖数和叶片数主要由遗传因素控制,环境对分蘖数和叶片数的影响稳定而微效。遗传率的高低决定选择世代的早晚,高遗传率说明早期选择是有效的。4利用F2群体进行BSA-简化基因组测序分析及SSR分子标记开发测定1000株F2群体的分蘖数,选取分蘖数分别为0、1-2、5-6、8-9和≥10各50株抽提DNA,形成五份DNA混池并利用限制性内切酶EcoRI+Mse I酶切后测序。利用分布在全基因组的SNP标记对五种DNA池进行基因型鉴定,筛选在若干DNA混池之间存在多态性差异的标记(Marker),根据这些标记进一步确定与分蘖相关的候选区域,对区域内基因进行注释和q-PCR筛选,进一步确定用于做功能验证的基因。根据简化基因组测序的结果进行与分蘖性状相关的分子标记的开发,得到9对SSR分子标记。其中有8对能在亲本中扩增出较为清晰的条带,有效引物比例为90%,其中编号为Bra028643、Bra023312、Bra002958和Bra002957的基因对应的4对引物在亲本中表现出稳定的多态性,SSR标记引物开发成功率为50%。5不结球白菜分蘖性状转录组学分析将'苏州青'和'马耳头'两个时期的根(R)、茎尖(SAM)和叶片(L)共计12个样本进行了无参转录组测序并组装成一套参考序列。获得了总长为120,205,788 nt的119,237个Unigenes,其中得到注释的基因共计104,086 (87.29%)。获得了32,065个SSR标记位点。KEGG数据库注释得到3,768 (6.25%)个涉及到激素信号调控途径的基因。将非分蘖期(Stage0)和分蘖期(Stage 1)样本中的基因进行差异性表达分析后发现,在两个种质之间及同一品种的不同组织部位之间存在巨大的差异性表达基因(DGEs)。在'马耳头'的根、茎尖和叶片的DGEs中共统计得到分属于30个基因家族的612个转录因子。组装的结果提供了一个覆盖范围比例相当大的转录组数据,可以用于与分蘖相关的差异表达基因和功能基因定位的进一步分析。
[Abstract]:Brassica rapa ssp. chinensis Makino is one of the most important vegetable crops in the Brassica genus of the family Cruciferae. The non heading Chinese cabbage can be divided into five categories: 1. common Chinese cabbage var.communis (Tsen etLee) Hanelt, 2. var. rosularis (TsenetLee) and 3. vegetable stalk ) Hanelt, 4. bolting var.tai-tsai Hort., 5. tillering vegetable var. multiceps Hort.., among which the tiller vegetables are characterized by a special multi branch branch. In the vegetative stage, there are a large number of lateral branches, such as tillers and multi headed vegetables. This study mainly used the tiller variety 'malt' and the common cabbage variety 'Suzhou green' as the research material and the tillering. The characteristics of the multi head branch of the vegetable were observed, dissected, the changes in the content of endogenous hormones were measured, and the mechanism of its occurrence was investigated. The F2 separation group was constructed and the candidate genes related to the tillering traits were excavated by high throughput sequencing technology. The contents of the study are as follows: 1 the characteristics of non heading Chinese cabbage are characterized by non heading Chinese cabbage in the flowering period. A large number of lateral branches are drawn from the base of the stem (first grade branch) and the axillary (grade two branching), usually in the vegetative period. But the tiller of one of the non heading Chinese cabbage varieties has a lateral bud growing from the base of the short stem and the leaf axil between the leaves in the vegetative stage. Morphological observation showed that the vegetative growth reached a certain stage (10-12 slices). The lateral buds in the non heading Chinese cabbage tiller began to form and grow out until each leaf axil had a lateral bud and eventually became tiller, with a leaf corresponding to a separate characteristic. The tissue section showed that the axillary points of 'Suzhou green' and 'malt' head 'were both. The formation of raw tissue, but the formation period is slightly different. The axillary meristem is formed after the formation of the axillary meristem in Suzhou green, while the axillary meristem is formed after the formation of the axillary meristem in the 'malt head'. The results showed that the nitrate nitrogen, soluble protein and Vc content of the tiller cabbage were significantly higher than the effect of.2 hormone on the characteristics of non heading Chinese cabbage. The results of the determination of 'malt head' and 'Suzhou green' endogenous hormones by enzyme linked immunosorbent assay (ELISA) showed that the main parameters of auxin were the main reference. With the dormancy of lateral buds in 'Suzhou green' and inhibition of lateral bud growth, it is a hormone that plays a major role in the top dominance phenomenon in 'Suzhou green'; cytokinin plays a major role in the formation of the 'malt head' tiller. The application of cytokinin (6-BA) and GR24 to the roots of 'malt' is found after 6 days of treatment. The.Q-PCR results showed that the increase in the cytokinin response gene BcRR4 and BcRR5 in the 'malt head' was significantly higher than that in 'Suzhou green', indicating that the 'malt head' was more sensitive to the cytokinin response, and the BcBRC1 gene was in the 'malt head'. The expression amount was significantly lower than that in 'Suzhou green', indicating that the response mechanism of 'malt head' was inferior to the quantitative genetic analysis of 'Suzhou green'.3 two hybrid combinations' by the quantitative genetic analysis of the 6 genetic populations of the "Suzhou green two hybrid". The tiller parent 'Suzhou green' constructed genetic population (P1, P2, F1, F2, BC1 and BC2). The number of tillering and the number of leaves was analyzed by the mixed genetic model of the main gene + polygene. The results showed that the number of tillers and the number of leaves in the non heading Chinese cabbage were all conformed to the E-1 mixed genetic model, and were controlled by 2 pairs of additive explicit epistatic main genes + additive dominant gene. Not only controlled by two main genes, but also influenced by the genetic influence of more genes and multiple genes, the heritability of the main gene multiplex gene in.F2 reached 61.58% ~ 96.83%. It indicated that the number of tillers and the number of leaves were mainly controlled by genetic factors. The influence of the environment on the number of tillers and the number of leaves was stable and micro. The high and low heritability determined the early and late generations of the selection. The transmission rate indicates that early selection is an effective.4 using F2 population to carry out BSA- genome sequencing analysis and SSR molecular markers to determine the number of 1000 F2 populations, and select the number of 50 strains of DNA, five DNA mixing pools and the restriction endonuclease EcoRI+Mse I enzyme digestion. Genotypic SNP markers were used to genotypic identification of five DNA pools, and a marker (Marker) was screened for polymorphic differences between several DNA pools. According to these markers, the candidate regions related to the tillers were further identified, and the genes in the region were annotated and q-PCR screened to further determine the genes used for functional verification. 9 pairs of SSR molecular markers were developed for the molecular markers associated with tiller traits. 8 of them were able to amplify a clearer band in their parents, with an effective primer ratio of 90%, of which 4 pairs of primers corresponding to the genes of Bra028643, Bra023312, Bra002958 and Bra002957 were stable in their parents. The successful rate of SSR marker primer development was 50%.5 non heading Chinese cabbage tiller character transcriptional analysis, the root (R) of 'Suzhou green' and 'malt' head '(R), stem tip (SAM) and leaf (L) were sequenced and assembled into a set of reference sequences. 119237 Unig of the total length of 120205788 NT were obtained. Enes, 104086 (87.29%) of the annotated genes were obtained. 3768 (6.25%) genes involved in the regulation of hormone signaling were obtained from the.KEGG database annotation of 32065 SSR markers. The genes in the non tillering stage (Stage0) and the tiller period (Stage 1) samples were found in the differential expression analysis, and between two germplasm and the same. There was a huge differential expression gene (DGEs) between the different tissue parts of a variety. The DGEs in the roots of the 'malt head', the tip of the stem and the leaves of the leaves were counted to be divided into 612 transcription factors belonging to the 30 gene families. The results of the assembly provided a fairly large scale of transcriptional data that could be used for the tiller related difference. Further analysis of the location of genes and functional genes.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S634.3

【参考文献】