【作者】 王书；

【导师】 徐新福；

【作者基本信息】 西南大学 ， 作物栽培学与耕作学， 2016， 硕士

【摘要】 我国畜牧业迅速发展,导致饲草紧缺,尤其在冬春季节尤为严重,一定程度上制约了其可持续发展。油菜生长速度快、营养体产量高,不仅是重要的油料作物,其植株还可以用作饲草。油菜具有较强的耐寒性,适宜在冬春季节种植,能有效地缓解冬春季饲草缺乏的情况。种植饲用油菜还能提高土壤肥力并获得较高种植效益。为更好地对饲用油菜进行高效利用以及选育最佳营养成分含量的饲用油菜,分析其营养成分含量、测定并评估其营养物质在动物体内的利用效率和饲养效果,并掌握控制其营养品质性状的相关基因显得尤为重要。本试验研究群体由300份甘蓝型油菜材料组成,去除生长不正常的材料,田间取样的有279份。采用概略养分分析法和Van Soest法对样品的水分、粗蛋白、粗脂肪、中性洗涤纤维、酸性洗涤纤维、酸性洗涤木质素、酸不溶灰分、纤维素、半纤维素和无氮浸出物这10个饲用营养品质性状进行测定与分析;利用广泛分布于甘蓝型油菜全基因组的大量SNP标记,对该自然群体进行群体结构、亲缘关系以及连锁不平衡(LD)分析;基于群体的LD状态,利用SNP标记对饲用品质性状进行初步全基因组关联分析。主要结果如下:1.饲用品质相关性状的表型分析:本研究中的油菜群体在水分、粗蛋白、粗脂肪、中性洗涤纤维、酸性洗涤纤维、酸性洗涤木质素、酸不溶灰分、纤维素、半纤维素、无氮浸出物的含量共10个与油菜饲用品质相关的性状都存在广泛的表型变异,并符合典型的数量性状的特点,多个性状之间存在显著或极显著的相关性。2.群体结构、亲缘关系分析:本研究采用3791个SNP标记对300份材料组成的自然群体分别进行群体结构分析与亲缘关系分析。分析结果将该群体划分为P1、P2两个类群。其中P1类群占26.2%,包含79份材料;P2类群占73.8%,包含221份材料。亲缘关系分析结果显示,约有57.63%的材料的亲缘关系值为0,亲缘关系值小于0.05的比例占71.78%,总体上亲缘关系值在0.5以内的材料达到了总比例的99.15%,表明该群体材料之间的亲缘关系较弱,对关联分析的影响较小。3.连锁不平衡分析:A、C亚基因组的LD(r2)均随着物理距离的增加而下降,且两个亚基因组的衰减程度不同。当r2的阈值设为0.2时,A亚基因组的平均衰减距离约为150Kb,而C亚基因组的平均衰减距离约为750Kb,说明白菜被更广泛地运用于甘蓝型油菜的育种中。4.油菜饲用品质各性状的关联分析:在Q+K模型下,利用TASSEL5.1软件的MLM模型程序,对群体材料各性状的表型值分别进行全基因组SNP关联分析,共检测出50个标记位点与目标性状呈极显著关联,这些标记分布在不同的染色体上。其中与粗蛋白含量相关联的标记位点有2个;与粗脂肪含量相关联的位点有3个;与中性洗涤纤维含量相关联的标记位点有4个;与酸性洗涤纤维相关联的标记有4个;与酸性洗涤木质素相关联的位点有1个;与酸不溶灰分含量相关联的标记有6个;与半纤维素相关联的标记位点有10个;与纤维素相关联的标记有15个;与无氮浸出物含量相关联的标记有5个。5.候选基因预测:通过分析显著SNP位点的LD区域与甘蓝型油菜对应的区间序列,共筛选出62个候选基因。其中,7个基因GDH2、GRP17、ADC2、AT1G31150、AT5G06839、AT3G22980和AT5G06800与粗蛋白含量有关,通过影响氨基酸的合成与基因的转录或翻译,从而影响蛋白质的合成;9个基因与粗脂肪含量相关:OLEO1、AT1G54570、ACBP4、AT3G04360、LTL1、LPP3、AT1G56670、AT5G03610和AT3G05180,它们编码的蛋白质通过参与脂类的各种生理活动来调节脂类的代谢与累积;11个基因与粗纤维含量有关:其中XND1、CESA6、CSLA07和CESA8通过编码纤维素合酶影响纤维素的含量;MUCI10、AGP17、AGP12、AGP1、AT4G38940、AT1G55270、VGT1所编码的蛋白质通过影响糖类的合成与运输,从而影响半纤维素的含量;39个基因与酸不溶灰分有关:FSD3、FTRA1、FRO4、ZIP6、KT1等基因,其编码的蛋白质主要通过影响矿质元素的吸收来调节灰分的含量;6个基因与无氮浸出物含量相关:RCA、EULS3、SWEET9、PGP1、AT2G39470和AT5G54800,它们编码的蛋白通过影响光合作用或影响糖类和淀粉的代谢来影响无氮浸出物的含量。更多还原

【Abstract】 The rapid development of animal husbandry in China results in a shortage of forage grass, especially during winter and spring, which restricts its sustainable development to a certain extent. Because of its fast growth and high biological output, rape is not only an important oil crop, but also a kind of forage grass. Rape is suitable to be grown in winter and spring with its strong cold resistance, which can effectively alleviate the forage scarcity in such seasons. Growing forage rape can also improve soil fertility and obtain high planting benefit. In order to make efficient use of forage rape and to select and breed the kind with best nutrient content, it is important to analyze its nutrient content with its utilization efficiency in animals as well as feeding effect, and to grasp the genes that control the traits related to feeding value.Based on the population consisting of 300 kinds of rape(Brassica napus L.) with 279 kinds selected except for those which grew abnormally, the research measured and analyzed the content of moisture, CP, EE, NDF, ADF, ADL, AIA, hemicellulose, fiber and NFE with proximate analysis and Van Soest’s method. Analyze the population structure, kinship and linkage disequilibrium(LD) with quantities of SNP markers, then make genome-wide SNP association analysis based on the LD status of the population. The main results were summed up as follows:1. Phenotypic analysis of traits related to feeding value: The research population got extensive phenotypic variations in the 10 target traits related to feeding value, which was in line with the typical characteristics of quantitative traits. Between each two traits, significant or extremely significant phenotypic correlation was observed mostly.2. Analysis of population structure and relative kinship: The study divided the population of 300 materials into two groups named P1 and P2 with 3791 SNP markers. P1 group accounts for 26.2% with 79 materials while P2 73.8% with 221 materials. The results of kinship analysis showed that of all the materials, 57.63% got the kinship value of 0 and 71.78% got it less than 0.05. The kinship value within 0.5 for 99.15% materials in total indicated the weak relationship, which could cause little influence on the association analysis.3. Analysis of linkage disequilibrium: By calculating the r2 value of pairwise markers in the same chromosome, the study simulated the trend lines of LD and found that r2 value declined rapidly while the physical distance increased. It showed that when the threshold value of r2 set as 0.2, the decay of the LD is around 150 Kb in A genome while 750 Kb in C genome, indicating that Brassica rapa. was more widely used in Brassica napus L. breeding.4. Association analysis of the traits related to feeding value: Making a genome-wide SNP association analysis with the MLM model program in TASSEL5.1 software under the Q+K model, the study detected 50 SNP markers associated with the target traits significantly. The markers distribute on different chromosomes, of which, 2 were associated with CP content, 3 associated with EE content, 4 associated with NDF content, 4 associated with ADF content, 1 associated with ADL content, 6 associated with AIA content, 10 associated with hemicellulose content, 15 associated with fiber content and 5 associated with NFE content.5. Prediction of candidate genes: The study analyzed the homologous genome sequences of B. napus with the SNP markers annotated according to the genome information of Arabidopsis thaliana, selecting out 62 candidate genes. Among which, 7 genes i.e. GDH2, GRP17, ADC2, AT1G31150, AT5G06839, AT3G22980 and AT5G06800 are associated with CP content, influencing gene transcription, translation and the biosynthesis of amino acids thus affecting protein synthesis. 9 genes are associated with EE content i.e. OLEO1, AT1G54570, ACBP4, AT3G04360, LTL1, LPP3, AT1G56670, AT5G03610 and AT3G05180, encoding proteins which participate in the physiology activity of lipid to regulate lipid metabolism and accumulation. 11 genes are associated with CF content, in which, XND1, CESA6, CSLA07 and CESA820 affect fiber content by encoding cellulose synthase, other 7 genes i.e. MUCI10, AGP17, AGP12, AGP1, AT4G38940, AT1G55270 and VGT1 affect hemicellulose content through making an influence on the synthesis and transport of saccharides. 39 genes are associated with AIA content such as FSD3, FTRA1, FRO4, ZIP6, KT1, and they encode proteins that mainly adjust the content of ash by affecting the absorption of mineral elements. 6 genes are associated with NFE content i.e. RCA, EULS3, SWEET9, PGP1, AT2G39470 and AT5G54800, encoding proteins which influence the content of NFE by the means of affecting photosynthesis and the synthesis of sugar and starch. The selected candidate genes will lay the foundation of further study in molecular breeding and gene cloning for the traits related to feeding value of rape.更多还原