【作者】 黄琪；

【导师】 蒋立希； 华水金；

【作者基本信息】 浙江大学 ， 农艺与种业， 2023， 硕士

【摘要】 甘蓝型油菜（Brassica napus.L）是我国重要的油料作物,油菜籽是我国重要的植物油和蛋白饲料的来源之一。随着国内市场对植物油和饲料蛋白原料需求的增加,油菜籽的供不应求问题变得日益严重,因此,提高油菜的生产潜力是十分紧迫的任务。在油菜中,花瓣面积与单株油菜节能减耗、群体光能利用以及生态抑病等密切相关,小花瓣性状在这些方面具有优势。虽然植物花瓣面积的研究已有很多报道,但是关于油菜花瓣面积的研究却不多见。这项研究对295份甘蓝型油菜核心种质材料的花瓣面积进行了全基因组关联分析,揭示了一些与花瓣面积呈显著相关的SNP位点,并开发了一个可以在育种上加以利用的CAPS分子标记。此外,通过结合转录组分析,我们还发掘了一系列调控甘蓝型油菜花瓣面积大小的候选基因。主要研究结果如下:1.甘蓝型油菜花瓣面积的全基因组关联分析对2021年、2022年统计的295份核心种质的花瓣面积数据进行统计分析,结果显示花瓣面积表型变异广泛。在2021年和2022年分别为130.79-595.79mm²和147.34-496.41 mm²;平均值分别为233.78 mm²和239.95 mm²;变异系数分别为25.45%和19.20%。对2021年、2022年以及两年BLUP（Best linear unbiased prediction）花瓣面积分别进行全基因组关联分析,共得到位于A03、A04、A05、C03和Ann染色体上202个显著SNP位点,202个显著SNP位点的上下游75 kb范围内共关联到602个候选基因。2.CAPS分子标记的开发202个显著SNP位点中24个SNP位点在3个实验结果中均出现。在24个SNP中,13个SNP至少在2个实验结果中-log₁₀P-value大于5.5。13个SNP中4个SNP与花瓣面积显著相关且能够引起限制性内切酶识别序列的变化。本研究选择Chr A04＿88230用于CAPS分子标记的开发,并验证了该标记能有效地区分大、小花瓣基因型。3.大、小花瓣基因型的表型差异和转录组分析利用扫描电镜观察大、小花瓣基因型的花瓣细胞,结果显示细胞数目与花瓣面积正相关且小花瓣基因型花瓣的细胞数目要显著少于大花瓣基因型。同样地,在转录组分析中,与大花瓣基因型相比,小花瓣基因型中与细胞增殖正向相关的GO项明显下调,说明小花瓣基因型中细胞增殖活动弱于大花瓣基因型。因此,细胞增殖活动的差异是造成花瓣面积大小差异的主要原因。4.甘蓝型油菜花瓣面积调控基因鉴定通过转录组和GWAS分析相结合,我们得到了23个调控花瓣面积的关键基因。其中Bna A04g00710D是PIP1-1的同源基因,该基因已在玫瑰中被证明正向调节细胞扩张。对Bna A04g00710D上的SNP进行分析,我们发现Bna A04g00710D上的4个SNP变异情况的不同组合导致花瓣面积表型在甘蓝型油菜不同生态型群体间的显著分化。更多还原

【Abstract】 Rapeseed（Brassica napus L.）is an important oil crop in China and one of the important sources of vegetable oil and protein feed.As the domestic demand for vegetable oil and protein feed continues to increase,the problem of inadequate supply of rapeseed has become increasingly severe.Therefore,it is an urgent task to improve the production potential of rapeseed.During the growth and development process of rapeseed,petal size is closely related to factors such as energy conservation,group photosynthesis,and ecological disease control.Small petal traits have advantages in these areas.Although there have been many studies on plant petal size,research on rapeseed petal size is relatively rare.In this study,we conducted a genome-wide association analysis（GWAS）of petal size in 295 core accessions of B.napus,revealing some significant SNP loci related to petal size and developed a CAPS molecular marker that can be used in rapeseed breeding project.By combining transcriptome analysis,we identified a series of candidate genes that regulated the petal size of B.napus.The main research results are summarized below.1.Genome-wide association analysis of petal size in B.napusStatistics analysis of petal size data obtained from 2021 to 2022 indicated that extensive phenotypic variation of petal size in 295 core accessions of B.napus.The size of total petals in 2021 and 2022 are 130.79-595.79 and 147.34-496.41 mm²;the mean is 233.78 and 239.95 mm²;the coefficients of variation（CV）are 25.45%and19.20%.Genome-wide association was performed using the phenotypic data of 2021,2022 and the BLUP results data,and a total of 202 significantly related SNPs were identified.These SNPs are distributed on the A03,A04,A05,C03 and Ann chromosomes.A total of 602 candidate genes were associated with 202 significant SNPs in the upstream and downstream 75 kb range.2.Development of CAPS molecular markersAmong 202 significant SNPs,24 SNPs appeared in all the three experimental results.Among the 24 SNPs,13 SNPs had-log₁₀P-value greater than 5.5 in at least 2experimental results.Among the 13 SNPs,4 SNPs were significantly correlated with petal size and could induce the change of restriction enzyme recognition sequence.In this study,Chr A04＿88230 was selected for the development of CAPS molecular markers,and it was verified that Chr A04＿88230 could effectively distinguish big and small petal genotypes.3.Phenotypic differences and transcriptome analysis of big and small petal genotypesScanning electron microscopy（SEM）was used to observe the petal cells of big and small petal genotypes.The results showed that the number of cells was positively correlated with petal size,and the number of cells of small petal genotypes was significantly less than that of big petal genotypes.Similarly,in transcriptome analysis,the GO items positively associated with cell proliferation was significantly down-regulated in small petal genotypes compared to big petal genotypes,indicating that cell proliferation activity was weaker in small petal genotypes than in big petal genotypes.Therefore,the differences in cell proliferation activity as the main cause of the difference in petal size.4.Identification of genes regulating petal size in B.napusBy combining transcriptome and GWAS analysis,we identified 23 key genes that regulate petal size.Among them,Bna A04g00710D is a homologous gene of PIP1-1,which has been shown to positively regulate cell expansion in rose.By analyzing SNPs on Bna A04g00710D,we found that different combinations of four SNP variations on Bna A04g00710D resulted in significant differentiation of petal size phenotypes among different ecotype populations in B.napus.更多还原