本文關(guān)鍵詞：巨桉和細(xì)葉桉群體適應(yīng)性和枝癭姬小蜂抗性的關(guān)聯(lián)分析　出處：《中國林業(yè)科學(xué)研究院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 巨桉 細(xì)葉桉 關(guān)聯(lián)分析 轉(zhuǎn)錄組分析 分子標(biāo)記輔助選擇

【摘要】：森林是陸地生態(tài)系統(tǒng)的主體,在人類的生態(tài)文明建設(shè)、經(jīng)濟(jì)發(fā)展和環(huán)境保護(hù)等方面發(fā)揮著重要作用。我國森林覆蓋率低,國家木材戰(zhàn)略儲備工作和林木育種工程面臨挑戰(zhàn)。傳統(tǒng)的雜交育種雖取得了顯著成效,但是林木遺傳雜合度高、生命周期長且經(jīng)濟(jì)性狀多為數(shù)量性狀等原因使得傳統(tǒng)林木遺傳改良進(jìn)展緩慢。并且,早期的林木遺傳改良主要關(guān)注木材產(chǎn)量而忽略了林木適應(yīng)性和抗性。因此,深入研究林木適應(yīng)性和抗性的生物學(xué)基礎(chǔ),解析其遺傳基礎(chǔ),有助于有效管理和利用森林資源、加快林木遺傳改良的進(jìn)程。本論文以巨桉(Eucalyptus grandis)和細(xì)葉桉(E.tereticornis)天然群體子代苗建立的大田試驗(yàn)林為材料,基于全基因組低密度的簡單序列重復(fù)(Simple sequence repeat,SSR)標(biāo)記,檢測了巨桉和細(xì)葉桉適應(yīng)性和抗枝癭姬小蜂(Leptocybe invasa)關(guān)聯(lián)的基因組位點(diǎn);通過轉(zhuǎn)錄組分析,發(fā)現(xiàn)了一批與枝癭姬小蜂抗性相關(guān)的候選基因;對其中1個候選基因與枝癭姬小蜂抗性的關(guān)聯(lián)分析表明,存在顯著影響枝癭姬小蜂抗性的等位變異;探索了適應(yīng)性和抗性的標(biāo)記輔助選擇的可行性。主要研究內(nèi)容如下:(1)巨桉天然群體遺傳多樣性和受選擇位點(diǎn)。參試材料為巨桉16個群體的159株樣品(1株子代苗/自由授粉家系),參試標(biāo)記為覆蓋巨桉全基因組的110個SSR位點(diǎn)(包括45個基因組SSRs和65個EST-SSRs)。31個中性的基因組SSRs揭示了較高水平的群體多樣性,HE=0.744,AR=4.929;群體間分化水平較低,平均FST=0.037,分子方差分析中群體間方差分量僅占3.7%、遺傳變異主要存在于群體內(nèi)。所有110個位點(diǎn)中,共檢測到58個FST值異常位點(diǎn);線性回歸分析驗(yàn)證了3個等位片段與氣候因子(Embra180-120 bp與年均溫、EUCeSSR0755-276 bp與年均溫和等溫性)的顯著線性回歸(P0.05)。這對理解多年生林木適應(yīng)氣候的遺傳基礎(chǔ)和巨桉種質(zhì)資源的保存和利用具有重要作用。(2)細(xì)葉桉天然群體遺傳多樣性和受選擇位點(diǎn)。參試材料為細(xì)葉桉9個群體的77株樣品(1株子代苗/自由授粉家系),參試標(biāo)記為覆蓋巨桉全基因組的108個SSR位點(diǎn)(包括44個基因組SSRs和64個EST-SSRs)。25個中性的基因組SSRs揭示了較高的群體多態(tài)性,HE=0.800,AR=3.246;群體間分化水平較低,平均FST=0.012,分子方差分析中群體間方差分量僅占1.2%、遺傳變異主要存在于群體內(nèi)。所有位點(diǎn)中,檢測到78個fst值異常位點(diǎn);線性回歸分析驗(yàn)證了1個等位片段(eucessr485-140bp)與季節(jié)性降水變異系數(shù)的回歸顯著性(p0.05)。(3)巨桉枝癭姬小蜂抗性關(guān)聯(lián)的ssr標(biāo)記及其在細(xì)葉桉中的驗(yàn)證。利用已經(jīng)發(fā)表的、覆蓋巨桉全基因組的816個ssr標(biāo)記,通過pcr優(yōu)化以及對8株高抗和8株高感基因池的篩選,得到在兩類基因池中等位頻率差異較大的87個ssr標(biāo)記用于16個群體470株巨桉的關(guān)聯(lián)分析。得到了與抗性關(guān)聯(lián)的7個ssr位點(diǎn),對表型變異的解釋率為3.3%~37.8%;7個關(guān)聯(lián)位點(diǎn)對細(xì)葉桉9個群體303株樣品的驗(yàn)證表明,4個位點(diǎn)在細(xì)葉桉中與抗性關(guān)聯(lián),對表型變異的解釋率為24.3%~48.5%。這為桉樹抗枝癭姬小蜂的分子育種提供了有潛力的標(biāo)記資源。(4)巨桉感染枝癭姬小蜂的轉(zhuǎn)錄組分析。通過轉(zhuǎn)錄組測序比較巨桉4個種源在自然條件下感蜂(有蟲癭s-2和無蟲癭s-1)和抗蜂表型(r)的基因表達(dá)差異,結(jié)果表明s-2vss-1的差異表達(dá)基因?yàn)?9個,顯著富集的keggpathway有10條;s-1vsr的差異表達(dá)基因?yàn)?;s-2vsr的差異表達(dá)基因有7個,分別富集在7個不同的keggpathway。其中大部分差異表達(dá)基因與呼吸作用、et途徑、ga途徑以及多條植物逆境脅迫應(yīng)答代謝途徑有關(guān)�；騟ucgr.g02880可能是巨桉抗枝癭姬小蜂的重要候選基因。(5)候選基因與巨桉抗枝癭姬小蜂的關(guān)聯(lián)分析。利用轉(zhuǎn)錄組分析發(fā)現(xiàn)的候選基因eucgr.g02880,再對巨桉16個群體470株進(jìn)行基因測序和關(guān)聯(lián)分析,擴(kuò)增的767bp片段中有38個單核苷酸多態(tài)性(singlenucleotidepolymorphism,snp)和2個插入/缺失(insert/deletion,indel)標(biāo)記,其中9個snp的等位基因頻率≥5%,基于混合線性模型的關(guān)聯(lián)分析表明其中3個snp與枝癭姬小蜂抗性顯著關(guān)聯(lián),對抗性變異的解釋率為0.876~1.959%。研究也表明轉(zhuǎn)錄組分析與關(guān)聯(lián)分析相結(jié)合是解析重要性狀的基因組位點(diǎn)的有效手段。(6)巨桉和細(xì)葉桉適應(yīng)性和枝癭姬小蜂抗性的標(biāo)記輔助選擇�；谶m應(yīng)性和抗枝癭姬小蜂的關(guān)聯(lián)分析結(jié)果,確定了巨桉和細(xì)葉桉兩類性狀的最大增效效應(yīng)優(yōu)勢位點(diǎn)聯(lián)合體,并預(yù)測了優(yōu)勢位點(diǎn)的組合。巨桉種源/家系試驗(yàn)林中沒有個體能夠聚合所有優(yōu)勢位點(diǎn),建議將聚合較多優(yōu)勢位點(diǎn)的單株進(jìn)行一次或者多次雜交,再利用關(guān)聯(lián)標(biāo)記對子代中聚合了所有優(yōu)勢位點(diǎn)的單株進(jìn)行選擇。細(xì)葉桉中編號為111的單株聚合了所有優(yōu)勢位點(diǎn),具有較好的應(yīng)用潛力。
[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.

【學(xué)位授予單位】：中國林業(yè)科學(xué)研究院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S792.39
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本文編號：1382474