【正文】 6 and gene prediction for Anthocyanins content in kernel of maize. Master39。s degree candidate: Wang Xu Supervisor: Professor Cai Yilin Major: Crop Geics and Breeding Direction: Maize Molecular Breeding Chongqing ? China May 20xx 獨(dú)創(chuàng)性聲明 學(xué)位論文題目： 玉米籽粒花色苷含量主效 QTLAC6 的精細(xì)定位及基因預(yù)測 本人提交的學(xué)位論文是在導(dǎo)師指導(dǎo)下進(jìn)行的研究工作及取得的研究成果。本人授權(quán)西南大學(xué)研究生院（籌）可以將學(xué)位論文的全部或部分內(nèi)容編入有關(guān)數(shù)據(jù)庫進(jìn)行檢索，可以采用影印、縮印或掃描等復(fù)制手段保存、匯編學(xué)位論文。黑玉米富含花色苷，對人體具有多種生理生化功能。前人已將控制花色苷含量的主效 QTLAC6 定位在第 6 染色體的分子標(biāo)記 S8umc1014 之間。在MuSBC4F3 群體的 精細(xì) 的 遺傳圖譜中，每個(gè)分子標(biāo)記之間的平均遺傳距離是 cM， 在MoSBC4F3 群體的 精細(xì) 的 遺傳圖譜中，每個(gè)分子標(biāo)記之間的平均遺傳距離是 cM。其中 ssr9 與 umc1014的物理距離為 255KB， S73 與 umc1014 的物理距離 為 114KB。從其他研究者已經(jīng)分離的花色苷含量相關(guān)基因在染色體中的分布情況來看，在第 6 染色體上也正好是這個(gè)基因，所以， pl transcription factor 極有可能就是候選基因。 Abstract III Fine mapping of the main effect QTLAC6 and gene prediction for Anthocyanin content in kernel of maize. Candidate: Wang Xu Major: Crop Geics and Breeding Supervisor: Professor Cai Yilin Abstract With the improvement of living standard and the adjustment of the diet, the nutritional quality of maize is increasingly high required. Composition and content of the pigment are closely related to the nutritional quality of maize kernel. The pigment position changes with the different colors of maize kernel. As one of the important nutritional quality, anthocyanin plays an important role in improving human health and preventing disease. Maize is not only an important raw material for the development of natural health food, but also is widely used in cosmetics, medicine and industries. Black maize is rich in anthocyanins, which possesses a variety of physiological and biochemical functions to the human body. Hence, black maize is a good material for anthocyanins study. The understanding of the geic rule of anthocyanins provide a new reference for the application of the functional quality in natural health food, cosmetics, medicine and other fields. Meanwhile, it gives some reference for maize anthocyanins relative basic research and molecular marker assisted breeding. Black maize SDM (super dark maize) is a specific maize inbred line created by maize research institute of southwest university. In this study, SDM was used as mon male parent to create two populations MoS from the cross Mo17 SDM and MuS from Mu6 SDM for studying the geic rule of anthocyanin. QTLAC6, the main effect QTL controlling of anthocyanin content, located in the mon interval of S8umc1014 on chromosome 6. To continue the previous research, BF 4C3 was used for further positioning QTLAC6. The progeny from one plant of the last generation of near isogenic line was used to fine mapping AC6. Then, the genome sequences of the nearby positioning area were analysised and genes within this region were annotated. The main results were as follows: 西南大學(xué)碩士學(xué)位論文 IV 1. The construction of fine geic map of BC4F3 population By selecting the existing SSR polymorphism markers and the new developed polymorphism markers nearby the target QTL regions, seven pairs and eight pairs of SSR polymorphism markers were used to constract fine linkage map for MuSBC4F3 population and MoSBC4F3 population, respectively. The average geic distance between each molecular marker was cM in the fine geic map of MuS BC4F3 population and cM. in MoSBC4F3 population. 2. Fine mapping of the main effect QTLAC6 of Anthocyanins content Two near isogenic line segregation populations of MuSBC4F3 and MoSBC4F3 were used to restrict the main effect QTLAC6 in the interval of S8umc1014 and umc1014mmc0523 respectively, which were close to each other, sharing the mon umc1014 molecular marker. Selfing generation BC4F3 of single plant of last generation was used to fine mapping AC6. In MuS population and MoS population, 10 single plants were screened respectively. According to the marker genotype and phenotype of the single plant, AC6 was defined in molecular marker ssr9umc1014 and S73umc1014, and the two regions overlapped. The physical distance between ssr9 and umc1014 was 255 KB, while the distance between S73 and umc1014 was 114 KB. 3. Gene annotation and prediction of the positioning region , b2ghome were used for analysis of locking region and the annotation of genes. A total of 49 transposons, 29 retrotransposons, 8 assumptions protein, 31 gene were found. Further information parative analysis by using NCBI ( reveals that within the positioning area, pl transcription factor was a heme oxygenase gene, participating in maize pigment regulation. From the results of other researchers who had separated related gene of anthocyanins content, pl was also located on chromosome 6. Therefore, pl transcription factor was most likely the candidate gene. The gene was not cloned with a variaty of amplification conditions in the process of gene cloning. The main reason maybe that an unkown length DNA sequence was inserted into pl transcription factor in mutant materials SDM, resulting that the gene was not cloned in SDM. Key wards： maize； anthocyanin content； QTL fine mapping； gene prediction. 第 1 章 文獻(xiàn)綜述 1 第 1 章 文獻(xiàn)綜述 QTL 精細(xì)定位策略及研究進(jìn)展 QTL 精細(xì)定位 一般情況下， QTL 的初步定位只能將其定位到染色體的一個(gè)區(qū)段范圍之內(nèi)，標(biāo)記區(qū)間的大小通常為 1030cM（ Kearsey and Farquhar 1998），在這么大的區(qū)間內(nèi)很難確定 QTL 的具體位置，也很難確定該區(qū)間內(nèi)是否只包含一個(gè) QTL，或者是幾個(gè) QTL 共同作用，從而無法將控制某性狀的基因確定出來。為了克服關(guān)聯(lián)分析假陽性較高的缺點(diǎn)， Pritchard 結(jié)合群體結(jié)構(gòu)估計(jì)與關(guān)聯(lián)分析提出了新方法（ Pritchard and Rosenberg 1999）， Yu 等人也提出了混合模型方法（ Yu et al. 20xx）。其中高代回交系為沒有進(jìn)行選擇的輪回選擇與回交系，在作物精細(xì)定位中最為常用。 遺傳背景一致，精細(xì)定位準(zhǔn)確，可估計(jì)基因間上位性效應(yīng)。 群體容易構(gòu)建，而且可精細(xì)定位到 1cM的范圍內(nèi)。 群體構(gòu)建時(shí)間長，工作較繁瑣。 QTL 精細(xì)定位策略 大量研究表明，影響 QTL 初級(jí)定位精確度和靈敏度的最重要因素是群體的大小。因此，為了使數(shù)量性狀的遺傳基礎(chǔ)被更精確地了解，在初級(jí)定位的基礎(chǔ)之上，還必須對 QTL 進(jìn)行高分辨率的精細(xì)定位，也就是在目標(biāo) QTL 區(qū)域上建立更高的分辨率的分子標(biāo)記圖譜，并分析目標(biāo) QTL 與標(biāo)記之間的連鎖關(guān)系。不過，這種方法特別的費(fèi)工費(fèi)時(shí)，而且只能用在效應(yīng)比較大的 QTL 的精細(xì)定位。 全基因組 QTL 的精細(xì)定位 為系統(tǒng)地開展 QTL 的精細(xì)定