巨桉和细叶桉群体适应性和枝瘿姬小蜂抗性的关联分析

发布时间：2018-01-05 09:17

  本文关键词：巨桉和细叶桉群体适应性和枝瘿姬小蜂抗性的关联分析　出处：《中国林业科学研究院》2016年博士论文　论文类型：学位论文

  更多相关文章： 巨桉 细叶桉 关联分析 转录组分析 分子标记辅助选择

【摘要】：森林是陆地生态系统的主体,在人类的生态文明建设、经济发展和环境保护等方面发挥着重要作用。我国森林覆盖率低,国家木材战略储备工作和林木育种工程面临挑战。传统的杂交育种虽取得了显著成效,但是林木遗传杂合度高、生命周期长且经济性状多为数量性状等原因使得传统林木遗传改良进展缓慢。并且,早期的林木遗传改良主要关注木材产量而忽略了林木适应性和抗性。因此,深入研究林木适应性和抗性的生物学基础,解析其遗传基础,有助于有效管理和利用森林资源、加快林木遗传改良的进程。本论文以巨桉(Eucalyptus grandis)和细叶桉(E.tereticornis)天然群体子代苗建立的大田试验林为材料,基于全基因组低密度的简单序列重复(Simple sequence repeat,SSR)标记,检测了巨桉和细叶桉适应性和抗枝瘿姬小蜂(Leptocybe invasa)关联的基因组位点;通过转录组分析,发现了一批与枝瘿姬小蜂抗性相关的候选基因;对其中1个候选基因与枝瘿姬小蜂抗性的关联分析表明,存在显著影响枝瘿姬小蜂抗性的等位变异;探索了适应性和抗性的标记辅助选择的可行性。主要研究内容如下:(1)巨桉天然群体遗传多样性和受选择位点。参试材料为巨桉16个群体的159株样品(1株子代苗/自由授粉家系),参试标记为覆盖巨桉全基因组的110个SSR位点(包括45个基因组SSRs和65个EST-SSRs)。31个中性的基因组SSRs揭示了较高水平的群体多样性,HE=0.744,AR=4.929;群体间分化水平较低,平均FST=0.037,分子方差分析中群体间方差分量仅占3.7%、遗传变异主要存在于群体内。所有110个位点中,共检测到58个FST值异常位点;线性回归分析验证了3个等位片段与气候因子(Embra180-120 bp与年均温、EUCeSSR0755-276 bp与年均温和等温性)的显著线性回归(P0.05)。这对理解多年生林木适应气候的遗传基础和巨桉种质资源的保存和利用具有重要作用。(2)细叶桉天然群体遗传多样性和受选择位点。参试材料为细叶桉9个群体的77株样品(1株子代苗/自由授粉家系),参试标记为覆盖巨桉全基因组的108个SSR位点(包括44个基因组SSRs和64个EST-SSRs)。25个中性的基因组SSRs揭示了较高的群体多态性,HE=0.800,AR=3.246;群体间分化水平较低,平均FST=0.012,分子方差分析中群体间方差分量仅占1.2%、遗传变异主要存在于群体内。所有位点中,检测到78个fst值异常位点;线性回归分析验证了1个等位片段(eucessr485-140bp)与季节性降水变异系数的回归显著性(p0.05)。(3)巨桉枝瘿姬小蜂抗性关联的ssr标记及其在细叶桉中的验证。利用已经发表的、覆盖巨桉全基因组的816个ssr标记,通过pcr优化以及对8株高抗和8株高感基因池的筛选,得到在两类基因池中等位频率差异较大的87个ssr标记用于16个群体470株巨桉的关联分析。得到了与抗性关联的7个ssr位点,对表型变异的解释率为3.3%~37.8%;7个关联位点对细叶桉9个群体303株样品的验证表明,4个位点在细叶桉中与抗性关联,对表型变异的解释率为24.3%~48.5%。这为桉树抗枝瘿姬小蜂的分子育种提供了有潜力的标记资源。(4)巨桉感染枝瘿姬小蜂的转录组分析。通过转录组测序比较巨桉4个种源在自然条件下感蜂(有虫瘿s-2和无虫瘿s-1)和抗蜂表型(r)的基因表达差异,结果表明s-2vss-1的差异表达基因为19个,显著富集的keggpathway有10条;s-1vsr的差异表达基因为0;s-2vsr的差异表达基因有7个,分别富集在7个不同的keggpathway。其中大部分差异表达基因与呼吸作用、et途径、ga途径以及多条植物逆境胁迫应答代谢途径有关。基因eucgr.g02880可能是巨桉抗枝瘿姬小蜂的重要候选基因。(5)候选基因与巨桉抗枝瘿姬小蜂的关联分析。利用转录组分析发现的候选基因eucgr.g02880,再对巨桉16个群体470株进行基因测序和关联分析,扩增的767bp片段中有38个单核苷酸多态性(singlenucleotidepolymorphism,snp)和2个插入/缺失(insert/deletion,indel)标记,其中9个snp的等位基因频率≥5%,基于混合线性模型的关联分析表明其中3个snp与枝瘿姬小蜂抗性显著关联,对抗性变异的解释率为0.876~1.959%。研究也表明转录组分析与关联分析相结合是解析重要性状的基因组位点的有效手段。(6)巨桉和细叶桉适应性和枝瘿姬小蜂抗性的标记辅助选择。基于适应性和抗枝瘿姬小蜂的关联分析结果,确定了巨桉和细叶桉两类性状的最大增效效应优势位点联合体,并预测了优势位点的组合。巨桉种源/家系试验林中没有个体能够聚合所有优势位点,建议将聚合较多优势位点的单株进行一次或者多次杂交,再利用关联标记对子代中聚合了所有优势位点的单株进行选择。细叶桉中编号为111的单株聚合了所有优势位点,具有较好的应用潜力。
[Abstract]:Forests are the main terrestrial ecosystems, in the construction of ecological civilization of human, plays an important role in economic development and environmental protection. China's forest coverage rate is low, the national strategic reserve of timber and forest tree breeding engineering challenges. Traditional breeding has made remarkable achievements, but the genetic heterozygosity is high, life long period and economic traits for number of characters makes progress of traditional tree breeding is slow. And the early genetic improvement of trees mainly focus on timber production while ignoring the forest adaptability and resistance. Therefore, the biological basis for further research of forest adaptability and resistance, analysis of its genetic basis, contribute to the effective management and utilization of forest resources to speed up the process, genetic improvement of trees. In this paper, Eucalyptus grandis (Eucalyptus grandis) and e.tereticornis (E.tereticornis) in natural populations of progeny seedling establishment of the Field testing of forest materials, genome-wide simple sequence repeat (low density based on Simple sequence repeat, SSR) markers, the detection of Eucalyptus grandis and Eucalyptus tereticornis adaptability and resistance of Leptocybe invasa (Leptocybe invasa) genomic loci associated; through transcriptome analysis, found a number of Leptocybe invasa candidate gene for resistance related; the association of 1 candidate genes and Leptocybe invasa resistance analysis showed that there was significant effect of Leptocybe invasa resistance alleles; to explore the adaptability and resistance of marker assisted selection is feasible. The main research contents are as follows: (1) the genetic diversity of natural populations of Eucalyptus grandis and selection site. The materials for Eucalyptus grandis in 16 groups of 159 strains (1 strains of progeny seedling / open pollinated families), tested markers for 110 SSR loci covering the whole genome of Eucalyptus grandis (including 45 genomic SSRs and 65 EST-SSRs).31 neutral The genomic SSRs revealed a higher level of population diversity, HE=0.744, AR=4.929; the low level of differentiation among populations, the average FST=0.037, the molecular analysis of variance between groups variance components accounted for only 3.7% of the genetic variation mainly exists within the population. All the 110 loci, 58 FST were detected abnormal sites; linear regression the analysis confirms the 3 allele and climatic factors (Embra180-120 BP and EUCeSSR0755-276 BP and the annual mean temperature, annual mean temperature and isothermal) significant linear regression (P0.05). And use this to play an important role in understanding the genetic basis of perennial trees adapt to climate and eucalypt germplasm preservation. (2) natural populations. The genetic diversity of Eucalyptus and selected sites. The materials for e.tereticornis 9 groups of 77 strains (1 strains of progeny seedling / open pollinated families), tested markers for 108 SSR loci covering the whole genome of Eucalyptus grandis (including 44 The genome of SSRs and 64 EST-SSRs).25 neutral genomic SSRs revealed high polymorphism group, HE=0.800, AR=3.246; the low level of differentiation among populations, the average FST=0.012, the molecular analysis of variance between groups variance components accounted for only 1.2% of the genetic variation mainly exists within the population. All loci detected, 78 FST the value of abnormal loci; linear regression analysis verified the 1 allele (eucessr485-140bp) and regression of seasonal precipitation variation coefficient (P0.05). (3) SSR markers in Eucalyptus grandis Leptocybe invasa resistance connection and its verification in e.tereticornis. The use of already published, 816 SSR markers covering the giant the Eucalyptus genome, the PCR optimization and screening of high resistant strains and 8 highly susceptible gene pool of 8 strains, in two kinds of medium frequency differences in the gene pool of 87 SSR markers for larger correlation analysis of Eucalyptus grandis in 16 populations of 470 strains obtained with anti. 7 SSR loci associated, explain phenotypic variation rate of 3.3%~37.8%; verification 7 related sites for the 9 groups of 303 strains of e.tereticornis samples showed that 4 loci in e.tereticornis and resistance Association, provides a potential marker resource to explain phenotypic variation rate of 24.3%~48.5%. for the molecular breeding anti Eucalyptus Leptocybe invasa. (4) the transcriptome analysis of Leptocybe invasa Eucalyptus grandis infection. Through comparative transcriptome sequencing of 4 provenances of Eucalyptus grandis sense bee under natural conditions (with and without S-1 S-2 gall gall) and anti bee phenotype (R) differentially expressed genes, the results showed that the expression the difference of s-2vss-1 gene was 19, significantly enriched keggpathway 10; the expression of s-1vsr gene was 0 s-2vsr; there were 7 differentially expressed genes, were enriched in 7 different keggpathway. most of the differentially expressed genes and respiration, et pathway, GA pathway and The plant stress response pathway. Eucgr.g02880 gene may be an important candidate gene of Eucalyptus grandis anti Leptocybe invasa. (5) analysis of candidate genes and associated resistance of Eucalyptus grandis Leptocybe invasa. Using transcriptome analysis of candidate gene eucgr.g02880, 16 populations of Eucalyptus grandis 470 strains were analyzed sequencing and association, there are 38 single nucleotide polymorphisms amplified 767bp fragments (singlenucleotidepolymorphism, SNP) and 2 insertion / deletion (insert/deletion, indel) markers, including 9 SNP allele frequency is more than 5%, associated with mixed linear model analysis showed that 3 SNP and Leptocybe invasa resistance significantly based on the association of mutation rate against 0.876~1.959%. studies also showed that the combination of transcriptome analysis and association analysis is an effective means to analysis the genomic loci of important traits. (6) of Eucalyptus grandis and eucalyptus leaves fine adaptability And marker assisted selection of Leptocybe invasa resistance. Correlation adaptability and anti Leptocybe invasa. Based on the results of the analysis, determine the maximum synergistic effect of combination of advantages of site Eucalyptus grandis and Eucalyptus tereticornis two kinds of traits, and forecasts the combination advantage sites. Eucalyptus grandis provenances / families in the experimental forest, no individual can all the advantages of single site polymerization, polymerization should be more advantage of one or more sites of hybridization, using markers associated with offspring in polymerization plant all the advantages of site selection. The number 111 of the plant all advantages in site polymerization of Eucalyptus tereticornis, has good potential.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S792.39
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本文编号：1382474