柑橘高密度遗传连锁图谱的构建及落叶性状的QTL定位

发布时间：2018-03-14 10:18

本文选题：柑橘　切入点：分子标记　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：柑橘是世界第一大果树,栽培历史悠久,其鲜果和果汁具有重要的经济价值。然而,柑橘育种面临着极大的挑战,如大多数重要性状为复杂的数量性状、世代周期长、遗传背景复杂、无融合生殖和配子体不育现象普遍等。利用分子标记技术进行遗传图谱的构建和QTL定位研究,能找到与育种目标性状紧密连锁的或共分离的标记,利用分子标记辅助选择育种技术可极大的缩短育种年限、提高育种定向性和效率。本研究首先基于克里曼丁橘的全基因组序列开发了大量的共显性SSR标记,并且基于枳基因组重测序数据大规模的开发了可靠的Indel标记,为柑橘的进一步遗传研究提供了宝贵的资源;然后以克里曼丁橘和枳为亲本进行杂交,构建了F1分离群体,利用SSR标记、Indel标记和结合简化基因组测序技术开发的SNP标记,构建了柑橘的高密度遗传连锁图谱;最后对后代落叶分离表型进行QTL定位分析,首次分析了控制枳落叶性状的QTL位点。主要研究结果如下:1.从数据库中获得克里曼丁橘全基因组信息,通过MISA软件共找到80,708个SSR位点,平均密度为268个/Mb,二核苷酸和三核苷酸基序重复类型最多。对33,929个转录组序列进行分析,发现6,834个转录本序列共包含8,989个SSR位点,然后对这些转录本进行了功能注释和不同基序重复类型进行了统计。2.利用获得的SSR位点进行标记开发,设计了105对引物(27个SSR位于genomic和78个位于CDs区域)。对柑橘三大属的18个不同种进行鉴定,获得95个稳定性好、多态性高的SSR标记(成功的标记占比为90.5%)。进一步聚类分析表明,18个柑橘种聚为三大类,各种间聚类结果与前期的研究报道相一致。3.对克里曼丁橘和枳的杂交后代(CP)和枳(PT)进行了基因组深度重测序(测序深度分别为22.86 X和25.98 X)。通过比对,CP中得到了2,591,103个SNP位点,PT中获得了3,105,640个SNP位点,共同的SNP位点有503,132个。此外,CP中得到了722,298个Indel位点,PT中获得了895,643个Indel位点,CP与PT共有的Indel位点有157,471个。4.随机筛选了82个Indel位点并在其两端设计引物,对柑橘三属的32份不同种进行鉴定,最终获得了74个稳定性好、有多态性的Indel标记(去掉无效扩增和无多态性的标记)。进一步聚类分析表明,各种间聚类分析结果与前人的研究报道相一致。结果表明,基于枳基因组深度重测序开发的Indel标记效率(90.24%)和可靠性均较高。5.利用SSR和Indel标记并结合简化基因组测序技术开发的SNP标记,分别构建了双亲的高密度遗传连锁图谱,以及双亲整合的遗传图谱。其中,母本遗传图谱共包含1,491个标记,总图距为1773.53 c M,平均图距为1.19 c M;父本遗传图谱共包含2,980个标记,总图距为1530.88 c M,平均图距为0.51 c M;而双亲整合后的遗传图谱共包含4,179个标记,总图距为1770.37 c M,平均图距为0.42c M。SNP标记统计表明,偏分离标记所占总标记的比例相比于前期的研究稍微偏高;对物理图谱和遗传图谱共线性分析表明构建图谱质量较高,并发现了几个明显的重组热点区域。6.对后代落叶分离表型进行了观察,获得了两年的表型数据,结合构建的遗传连锁图谱,进行落叶性状的QTL定位。再次,利用Mutmap方法定位了染色体1和8上两个区间,区间大小分别为1.37 Mb和0.67 Mb,并且分别包含36个和107个基因。其中,染色体8上定位区间更加显著,具有更大的基因密度。7.对2个定位区间的共143个候选基因组进行了功能注释,并在贡献值较大的染色体8上定位区间验证了几个与表型连锁的Indel标记,并进一步缩小了染色体8上的关联区间范围。由于枳的落叶性与冬季低温关系密切,本研究进一步对枳幼苗进行低温处理,根据功能注释和前期的转录组数据,在染色体1和8定位区间内分别筛选了7和23个候选基因,并利用q RT-PCR验证了候选基因在低温诱导下的表达模式。结果表明,12个基因受低温胁迫诱导后表达显著升高,2个基因下调表达显著,而剩余的16个基因表达变化不明显,这些结果为进一步获得落叶关联基因奠定了基础。
[Abstract]:Is the world's largest citrus fruit, has a long history of cultivation, its fruit and fruit juice has important economic value. However, citrus breeding is facing great challenges, such as the most important traits for complex quantitative traits, the generation of a long cycle, complex genetic background, apomixis and gametophyte sterility phenomenon. Research on construction and QTL the positioning of genetic map by using molecular marker technology, can be found closely linked with breeding target traits or co segregating marker assisted selection, breeding technology can greatly shorten the breeding period using molecular markers, improve breeding orientation and efficiency. Based on the complete genome sequence of the orange Clementina developed codominant SSR marker number the orange, and genome sequencing data based on the large-scale development of Indel markers and reliable, and provide a valuable resource for further genetic studies of citrus; Then to Clementina orange and trifoliate orange parent for hybridization, construct the F1 segregating populations using SSR markers, Indel markers and SNP markers with simplified genome sequencing technology development of the citrus high density genetic linkage map was constructed; finally the offspring phenotype was QTL leaves separation analysis, is analyzed for the first time locus QTL control trifoliate leaf traits the main results are as follows: 1. from the database to obtain information through the whole genome of orange Clementina, MISA found a total of 80708 SSR loci, the average density is 268 /Mb, dinucleotide and trinucleotide repeat motifs. On most types of sequences of 33929 transcriptome analysis, found that 6834 transcripts sequence consists of 8989 SSR site, then these transcripts were annotated and different motifs repeat type statistics.2. marker development using SSR gene was designed. 105 pairs of primers (27 SSR in genomic and 78 in CDs region). Identification of 18 species of Citrus three genera and 95 SSR markers with good stability, high polymorphism (successful markers accounted for 90.5%). Further cluster analysis showed that 18 species were clustered into three categories, research reports and previous clustering results between consistent progeny.3. of trifoliate orange and Clementina (CP) and orange (PT) of genome sequencing (depth of sequencing depth were 22.86 X and 25.98 X). By comparison, the CP received 2591103 SNP loci, obtained 3105640 SNP loci PT, SNP loci in the 503132. In addition, CP received 722298 Indel loci, 895643 Indel loci were obtained in PT, CP and PT shared Indel loci with 157471.4. randomly selected 82 Indel sites and at two ends of the primers of Citrus three genera 32 A different species were identified, got 74 good stability, there are polymorphic Indel markers (remove invalid amplified polymorphism and no further). Cluster analysis showed that consistent between cluster analysis results and previous reports. The results showed that the orange gene Indel labeling efficiency re sequencing depth development group based on (90.24%) and higher reliability.5. using SSR and Indel markers and SNP markers to simplify the genome sequencing technology development, were constructed by the parents of high density genetic linkage map, genetic mapping and integration. The parents, maternal genetic map contains 1491 markers, a total distance of 1773.53 C M, average the map is 1.19 C M; paternal genetic map contains 2980 markers, a total distance of 1530.88 C M, the average map distance of 0.51 C M; and the parents after the integration of the genetic map contains 4179 markers, a total distance of 1770.37 C M, the average map distance of M.SNP markers showed that 0.42c statistics, skewed markers of total markers compared with previous studies on the physical map and slightly higher; genetic map linear analysis showed that the mapping of higher quality, and found several obvious recombination hotspots of.6. were observed on the offspring leaves separation phenotype. The phenotypic data of two years, with the construction of the genetic linkage map and localization of QTL were deciduous traits. Thirdly, using Mutmap method to locate the chromosome 1 and 8 on the two interval, interval sizes were 1.37 Mb and 0.67 Mb, and contains 36 and 107 genes, respectively. Among them, the positioning interval on chromosome 8 more significantly, with greater density of.7. gene were 143 candidate genome of 2 positioning interval of functional annotation, and larger values of chromosome 8 was verified with several positioning interval in phenotypic contribution The Indel markers, and further reduced on chromosome 8 associated range. Due to low temperature in winter and deciduous Poncirus closely, the further study of Poncirus trifoliata seedlings were treated with low temperature, according to the transcriptome data and functional annotation of the 7 and 23 candidate genes were screened in chromosome 1 and 8 positioning area in between, and the use of Q RT-PCR to verify the expression patterns of candidate genes induced by low temperature stress. The results showed that 12 genes induced by low temperature stress was significantly higher, 2 genes downregulated significantly, while the remaining 16 genes expression did not change significantly, these results laid the foundation for further defoliation associated genes.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S666

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