本文選題：QTL定位 + RNAi　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：小麥面粉顏色相關(guān)性狀包括面粉亮度(L*)、紅度(a*)、黃度(b*)及黃色素含量(YPC),是影響面制品色澤的重要因素,已成為小麥品質(zhì)改良的重要內(nèi)容。對(duì)面粉顏色性狀研究多集中于QTL定位、基因克隆及功能標(biāo)記開發(fā),而關(guān)鍵基因Psyl功能驗(yàn)證及遺傳調(diào)控機(jī)理研究還鮮有報(bào)道。由于作圖群體及分子標(biāo)記局限性,大量面粉顏色性狀基因未被發(fā)現(xiàn)。本研究利用小麥90K SNP芯片,對(duì)藁城8901/周麥16RIL體和247份小麥品種進(jìn)行QTL定位和全基因組關(guān)聯(lián)分析,揭示面粉顏色相關(guān)性狀的分子基礎(chǔ)；采用RNAi、RNA-Seq和TILLING技術(shù)相結(jié)合的實(shí)驗(yàn)策略,研究影響面粉顏色關(guān)鍵基因Psyl功能及遺傳調(diào)控機(jī)理。主要結(jié)果如下：1.應(yīng)用小麥90K SNP芯片,分析藁城8901/周麥16 RIL群體,構(gòu)建高密度遺傳圖譜,對(duì)面粉顏色相關(guān)性狀進(jìn)行QTL定位及候選基因發(fā)掘。整個(gè)遺傳圖譜包含2205個(gè)骨架標(biāo)記,覆蓋普通小麥21條染色體,總長(zhǎng)度為2859.2 cM,每條染色體平均長(zhǎng)度為136.2 cM,標(biāo)記間平均遺傳距離為1.3cM。共檢測(cè)到52個(gè)影響面粉顏色性狀的QTL,位于第1、2、5、7同源群染色體和3B、4A、4B、6B染色體上,單個(gè)QTL解釋5.3-32.2%的表型變異。與前人研究相比,發(fā)現(xiàn)5個(gè)影響a。,2個(gè)影響b*,1個(gè)影響L*和1個(gè)影響YPC的新QTL。發(fā)現(xiàn)7個(gè)面粉顏色性狀的候選基因,一個(gè)為Pinb-D1基因,其余6個(gè)基因均與萜類化合物骨架合成途徑相關(guān)。由此可見,基于90K SNP芯片構(gòu)建高密度遺傳圖譜是進(jìn)行復(fù)雜數(shù)量性狀QTL定位及候選基因發(fā)掘的有效工具。2.利用小麥90K SNP芯片,對(duì)247份普通小麥品種進(jìn)行面粉顏色相關(guān)性狀全基因組關(guān)聯(lián)分析,共檢測(cè)到19個(gè)顯著關(guān)聯(lián)位點(diǎn),包含1個(gè)影響a*和1個(gè)影響L*的新位點(diǎn)。發(fā)現(xiàn)6個(gè)影響面粉顏色性狀的候選基因,即Pinb-D1、Psy-A1、Psy-B1和NADP-依賴性蘋果酸酶、細(xì)胞質(zhì)型蘋果酸脫氫酶及抗壞血酸過氧化物酶編碼基因,證實(shí)應(yīng)用90K SNP芯片對(duì)復(fù)雜數(shù)量性狀進(jìn)行全基因組關(guān)聯(lián)分析的高效性和可靠性。3.RNAi轉(zhuǎn)基因植株籽Psy1表達(dá)顯著降低(54-76%),YPC顯著下降(26-35%),驗(yàn)證Psyl對(duì)小麥籽粒黃色素合成具有重要影響。利用RNA-Seq技術(shù)對(duì)3個(gè)RNAi效果較好的轉(zhuǎn)基因株系(273-2A、275-3A和279-1A)進(jìn)行轉(zhuǎn)錄組測(cè)序,發(fā)現(xiàn)次級(jí)代謝途徑和核心代謝途徑上的多個(gè)候選基因協(xié)同響應(yīng)Psy1基因表達(dá)下調(diào)。應(yīng)用TILLING技術(shù)對(duì)Psyl功能分析顯示,天冬氨酸富集區(qū)(DXXXD)是PSYl重要功能結(jié)構(gòu)域,其附近保守核苷酸通過調(diào)控基因表達(dá)、酶活性及選擇性剪接等方式影響黃色素合成。RNAi轉(zhuǎn)基因植株及EMS突變體Psy1基因表達(dá)分析結(jié)果均顯示,花后14天是小麥籽粒Psyl基因表達(dá)遺傳調(diào)控的關(guān)鍵時(shí)期。優(yōu)異EMS突變體為小麥面粉顏色及營(yíng)養(yǎng)品質(zhì)的遺傳改良提供重要種質(zhì)資源。
[Abstract]:The relative characters of wheat flour color including the brightness of wheat flour, the degree of red, the content of yellow pigment and the content of yellow pigment YPC are the important factors affecting the color and color of flour products, which have become the important content of wheat quality improvement. The studies on color traits of flour were mainly focused on QTL mapping, gene cloning and functional marker development. However, the functional verification of key gene Psyl and the mechanism of genetic regulation were rarely reported. Due to the limitations of mapping population and molecular markers, a large number of flour color trait genes were not found. In this study, wheat 90K SNP microarray was used to localize and analyze the whole genome of Gaocheng 8901 / Zhoumai 16RIL body and 247wheat cultivars to reveal the molecular basis of flour color related traits, and the experimental strategy of combining RNA-Seq with TILLING technique was used. To study the function and genetic regulation mechanism of flour color key gene Psyl. The main results are as follows: 1. Wheat (Triticum aestivum) 90K SNP microarray was used to analyze the population of 8901 / Zhoumai 16 RIL in Gaocheng, and to construct a high-density genetic map, and to map the color related traits of wheat flour by QTL and explore the candidate genes. The whole genetic map contains 2205 skeleton markers covering 21 chromosomes of common wheat, the total length is 2859.2 cm, the average length of each chromosome is 136.2 cm, and the average genetic distance between markers is 1.3 cm. A total of 52 QTLs affecting the color traits of flour were detected, which were located on the chromosomes of homologous group 1 and chromosome 3BX 4A4 B, and the phenotypic variation of 5.3-32 2% was explained by a single QTL. Compared with previous studies, it was found that five effects, two effects, one effect on L * and one new QTL affecting YPC were found. Seven candidate genes for flour color traits were found, one of which was Pinb-D1 gene, and the other 6 genes were all related to the skeleton synthesis pathway of terpenes. Therefore, constructing high density genetic map based on 90K SNP chip is an effective tool for QTL mapping and candidate gene discovery of complex quantitative traits. Using wheat 90K SNP microarray, a total of 19 significant loci were detected for the whole genome association analysis of wheat flour color related traits, including one new locus affecting a * and one new affecting L *. Six candidate genes affecting flour color traits, namely Pinb-D1 Psy-A1 Psy-B1 and NADP-dependent malonase, cytoplasmic malate dehydrogenase and ascorbic acid peroxidase encoding genes were found. It was confirmed that the high efficiency and reliability of the whole genome association analysis of complex quantitative traits with 90K SNP microarray. 3. The expression of Psy1 in seeds of RNAi transgenic plants was significantly decreased by 54-760.YPC significantly decreased from 26 to 355.It was proved that Psyl had an important effect on the synthesis of yellow pigment in wheat grains. The transcriptome sequencing of three transgenic lines with good RNAi effect was carried out by RNA-Seq. It was found that several candidate genes in the secondary metabolic pathway and the core metabolic pathway responded to the down-regulation of Psy1 gene expression. The functional analysis of Psyl by TILLING showed that Aspartic acid rich region (DXXXD) is an important functional domain of PSYl, and its nearby conserved nucleotides regulate gene expression. The effects of enzyme activity and selective splicing on the expression of Psy1 gene in xanthine synthesis. RNAi transgenic plants and EMS mutants showed that 14 days after anthesis was the key period for genetic regulation of Psyl gene expression in wheat grains. Excellent EMS mutants provide important germplasm resources for genetic improvement of wheat flour color and nutritional quality.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S512.1

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