發(fā)布時(shí)間：2018-04-20 13:50

  本文選題：不結(jié)球白菜 + 分蘗性狀��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：不結(jié)球白菜(Brassica rapa ssp. chinensis Makino)原產(chǎn)中國,是十字花科蕓薹屬中重要的蔬菜作物之一。不結(jié)球白菜可以分為五大類,分別是: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.分蘗菜 var. multiceps Hort.。其中分蘗菜表現(xiàn)為特殊的多頭分枝特性,在營養(yǎng)生長階段即有大量側(cè)枝形成,狀如分蘗,又叫多頭菜。本研究主要以分蘗菜品種'馬耳頭'和普通白菜品種'蘇州青'為研究材料,對分蘗菜的多頭分枝特性進(jìn)行觀察,解剖,測定內(nèi)源激素含量變化,探討其發(fā)生機(jī)理。構(gòu)建F2分離群體,利用高通量測序技術(shù)進(jìn)行分蘗性狀相關(guān)的候選基因的挖掘。研究內(nèi)容如下:1不結(jié)球白菜分呭性狀的特點(diǎn)不結(jié)球白菜的多數(shù)品種在開花期會有大量側(cè)枝從莖基部(一級分枝)及葉腋之間(二級分枝)抽出,一般在營養(yǎng)生長期無側(cè)枝形成。但不結(jié)球白菜變種之一的分蘗菜在營養(yǎng)生長階段即有側(cè)芽從短縮莖基部和成葉間的葉腋處長出,進(jìn)一步伸長生長發(fā)育成為由多片小葉組成的分枝。形態(tài)學(xué)觀察表明,營養(yǎng)生長達(dá)到一定階段(10-12片),不結(jié)球白菜分蘗菜中的側(cè)芽開始形成并長出,直到每一個(gè)葉腋處都有一個(gè)側(cè)芽并最終長成分蘗,具有一片成葉對應(yīng)一個(gè)分呭的特點(diǎn)。組織切片觀察顯示,'蘇州青'和'馬耳頭'都有腋分生組織的形成,但形成時(shí)期略有不同。'蘇州青'中腋分生組織形成后進(jìn)入休眠狀態(tài)而'馬耳頭'中腋分生組織形成后無休眠過程直接繼續(xù)發(fā)育并進(jìn)一步形成分呭。生殖生長開始后,'蘇州青'中的腋芽結(jié)束休眠也開始向外生長,形成側(cè)枝后開花結(jié)果。成株期部分營養(yǎng)品質(zhì)含量測定結(jié)果顯示,分蘗菜的硝態(tài)氮、可溶性蛋白和Vc含量都顯著高于普通白菜。2激素對不結(jié)球白菜分呭性狀的影響利用酶聯(lián)免疫反應(yīng)法(ELISA)測定'馬耳頭'和'蘇州青'內(nèi)源激素含量結(jié)果顯示:生長素主要參與了'蘇州青'中側(cè)芽的休眠,抑制側(cè)芽向外生長,是'蘇州青'中頂端優(yōu)勢現(xiàn)象起主要作用的激素;細(xì)胞分裂素在'馬耳頭'分蘗發(fā)生過程中起主要促進(jìn)作用。對'馬耳頭'的根部外施細(xì)胞分裂素(6-BA)和獨(dú)腳金內(nèi)酯(GR24),處理6天后發(fā)現(xiàn),獨(dú)腳金內(nèi)酯抑制側(cè)芽的發(fā)生和生長而細(xì)胞分裂素促進(jìn)側(cè)芽的發(fā)生和伸長。Q-PCR結(jié)果顯示,細(xì)胞分裂素響應(yīng)基因BcRR4和BcRR5在'馬耳頭'中的增加量顯著高于'蘇州青'中的增加量,說明'馬耳頭'對細(xì)胞分裂素的響應(yīng)更敏感;BcBRC1基因在'馬耳頭'中的表達(dá)量顯著低于'蘇州青'中的表達(dá)量,說明在“細(xì)胞分裂素-獨(dú)腳金內(nèi)酯調(diào)控莖分枝”這一抑制分蘗發(fā)生的調(diào)控途徑中,'馬耳頭'響應(yīng)機(jī)制劣于'蘇州青'。3兩雜交組合6個(gè)遺傳群體的數(shù)量遺傳學(xué)分析利用分蘗親本'馬耳頭'、'如皋毛菜'和不分蘗親本'蘇州青'構(gòu)建遺傳群體(P1、P2、F1、F2、BC1和BC2),對分蘗數(shù)和葉片數(shù)進(jìn)行主基因+多基因的混合遺傳模型分析。結(jié)果顯示不結(jié)球白菜分蘗數(shù)和葉片數(shù)均符合E-1混合遺傳模型,受2對加性-顯性-上位性主基因+加性-顯性多基因控制。分蘗性狀不僅受兩對主基因控制,也受更多微效多基因的遺傳影響。F2中主基因加多基因的遺傳率達(dá)到61.58%～96.83%,說明分蘗數(shù)和葉片數(shù)主要由遺傳因素控制,環(huán)境對分蘗數(shù)和葉片數(shù)的影響穩(wěn)定而微效。遺傳率的高低決定選擇世代的早晚,高遺傳率說明早期選擇是有效的。4利用F2群體進(jìn)行BSA-簡化基因組測序分析及SSR分子標(biāo)記開發(fā)測定1000株F2群體的分蘗數(shù),選取分蘗數(shù)分別為0、1-2、5-6、8-9和≥10各50株抽提DNA,形成五份DNA混池并利用限制性內(nèi)切酶EcoRI+Mse I酶切后測序。利用分布在全基因組的SNP標(biāo)記對五種DNA池進(jìn)行基因型鑒定,篩選在若干DNA混池之間存在多態(tài)性差異的標(biāo)記(Marker),根據(jù)這些標(biāo)記進(jìn)一步確定與分蘗相關(guān)的候選區(qū)域,對區(qū)域內(nèi)基因進(jìn)行注釋和q-PCR篩選,進(jìn)一步確定用于做功能驗(yàn)證的基因。根據(jù)簡化基因組測序的結(jié)果進(jìn)行與分蘗性狀相關(guān)的分子標(biāo)記的開發(fā),得到9對SSR分子標(biāo)記。其中有8對能在親本中擴(kuò)增出較為清晰的條帶,有效引物比例為90%,其中編號為Bra028643、Bra023312、Bra002958和Bra002957的基因?qū)��?yīng)的4對引物在親本中表現(xiàn)出穩(wěn)定的多態(tài)性,SSR標(biāo)記引物開發(fā)成功率為50%。5不結(jié)球白菜分蘗性狀轉(zhuǎn)錄組學(xué)分析將'蘇州青'和'馬耳頭'兩個(gè)時(shí)期的根(R)、莖尖(SAM)和葉片(L)共計(jì)12個(gè)樣本進(jìn)行了無參轉(zhuǎn)錄組測序并組裝成一套參考序列。獲得了總長為120,205,788 nt的119,237個(gè)Unigenes,其中得到注釋的基因共計(jì)104,086 (87.29%)。獲得了32,065個(gè)SSR標(biāo)記位點(diǎn)。KEGG數(shù)據(jù)庫注釋得到3,768 (6.25%)個(gè)涉及到激素信號調(diào)控途徑的基因。將非分蘗期(Stage0)和分蘗期(Stage 1)樣本中的基因進(jìn)行差異性表達(dá)分析后發(fā)現(xiàn),在兩個(gè)種質(zhì)之間及同一品種的不同組織部位之間存在巨大的差異性表達(dá)基因(DGEs)。在'馬耳頭'的根、莖尖和葉片的DGEs中共統(tǒng)計(jì)得到分屬于30個(gè)基因家族的612個(gè)轉(zhuǎn)錄因子。組裝的結(jié)果提供了一個(gè)覆蓋范圍比例相當(dāng)大的轉(zhuǎn)錄組數(shù)據(jù),可以用于與分蘗相關(guān)的差異表達(dá)基因和功能基因定位的進(jìn)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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S634.3

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