發(fā)布時(shí)間：2018-08-28 08:51

【摘要】：我國(guó)桉樹(Eucalyptus)種質(zhì)資源材料主要來源于桉樹原產(chǎn)地澳大利亞,作為世界上生長(zhǎng)最快和經(jīng)濟(jì)價(jià)值極高的樹種,桉樹已成為我國(guó)南方造林的戰(zhàn)略性樹種。SSR分子標(biāo)記技術(shù)能為桉樹種群遺傳多樣性調(diào)查和種質(zhì)資源鑒定提供重要的技術(shù)手段,有效地促進(jìn)桉樹資源的良種選育與種質(zhì)資源的開發(fā)和利用。本研究利用Genbank數(shù)據(jù)庫(kù)中桉樹基因組序列和EST序列進(jìn)行桉樹SSR標(biāo)記的開發(fā),以42種159份桉樹DNA種質(zhì)材料為主要研究對(duì)象,對(duì)桉樹種質(zhì)資源的遺傳多樣性進(jìn)行分析,探討桉樹種間親緣關(guān)系,并通過選用種內(nèi)擴(kuò)增序列完全保守的SSR位點(diǎn),初步構(gòu)建桉樹種間種質(zhì)資源的精準(zhǔn)鑒定體系。主要研究結(jié)果如下:(1)通過Genbank檢索桉樹28,691條基因組序列和16,566條EST序列,經(jīng)序列分析,確認(rèn)1,785條有效序列,并發(fā)現(xiàn)2,292個(gè)SSR位點(diǎn),含SSR位點(diǎn)的序列占序列總數(shù)的12.64%。通過分析桉樹SSR位點(diǎn)信息,發(fā)現(xiàn)桉樹SSR位點(diǎn)的重復(fù)單元長(zhǎng)度與SSR位點(diǎn)的豐度呈負(fù)相關(guān)關(guān)系。桉樹EST序列中以三堿基重復(fù)單元最為豐富,而桉樹基因組序列中則是二堿基重復(fù)單元出現(xiàn)頻率最高。對(duì)所有SSR位點(diǎn)進(jìn)行引物設(shè)計(jì)及評(píng)估,共合成了395對(duì)SSR引物,經(jīng)過優(yōu)化PCR體系初步篩選出340對(duì)SSR引物,為桉樹SSR分子標(biāo)記在遺傳多樣性分析和系統(tǒng)發(fā)育分析等方面研究提供了引物資源和理論基礎(chǔ)。(2)本研究以6個(gè)桉樹DNA樣品為對(duì)照組,1個(gè)基因組DNA混合池為試驗(yàn)組進(jìn)行基因組DNA混合池在SSR引物篩選中的適用性分析。通過PCR擴(kuò)增產(chǎn)物的重測(cè)序分析,6個(gè)桉樹單一DNA樣品的擴(kuò)增結(jié)果與基因組DNA混合池的擴(kuò)增結(jié)果差異不顯著。利用基因組DNA混合池為模板進(jìn)行SSR-PCR擴(kuò)增序列重測(cè)序,經(jīng)序列同源性和SSR位點(diǎn)的比對(duì)分析,得到可應(yīng)用于桉樹遺傳多樣性分析和桉樹種質(zhì)資源鑒定研究的283個(gè)SSR有效標(biāo)記。并利用283對(duì)SSR引物在39種桉樹中進(jìn)行SSR-PCR擴(kuò)增分析,得到268對(duì)擴(kuò)增結(jié)果穩(wěn)定,重復(fù)性較好的引物,且在桉屬雙蒴蓋亞屬內(nèi)具有良好的通用性。(3)利用擴(kuò)增穩(wěn)定、條帶特異的110對(duì)SSR有效引物對(duì)42種159份桉樹種質(zhì)材料進(jìn)行PCR擴(kuò)增結(jié)果統(tǒng)計(jì),159份桉樹種質(zhì)材料中共檢測(cè)到SSR位點(diǎn)的等位基因共200個(gè),平均每個(gè)位點(diǎn)等位基因數(shù)為1.818,變化范圍為1~7個(gè)。純合基因型數(shù)162個(gè)(81%)遠(yuǎn)遠(yuǎn)多于雜合基因型數(shù)38(19%)。遺傳多樣性分析的結(jié)果表明,47個(gè)多態(tài)SSR位點(diǎn)的平均有效等位基因數(shù)為1.172,平均Shannon’s信息指數(shù)為0.181,平均觀察雜合度Ho為0.068,平均多態(tài)信息含量PIC為0.182。綜合各指標(biāo)分析得到,位點(diǎn)eSSR-GR018、位點(diǎn)eSSR-GR083和位點(diǎn)eSSR-GR109的多態(tài)性程度最高,反映的信息量更大,能夠在桉樹種質(zhì)資源的遺傳多樣性分析和種質(zhì)鑒定等方面發(fā)揮更大的作用。主坐標(biāo)法分類結(jié)果與形態(tài)學(xué)分類狀態(tài)基本一致,同時(shí)也證實(shí)了傘房屬與桉屬存在明顯的遺傳差異。非加權(quán)類平均法(UPGMA)表明,昆士蘭桉和少花桉間具有更近的遺傳距離,很有可能產(chǎn)生雜交種,這為桉樹種質(zhì)資源的有效利用和桉樹雜交育種工作提供了理論基礎(chǔ)和選種依據(jù)。(4)分析20對(duì)在同一桉樹種內(nèi)擴(kuò)增片段大小基本一致的特異SSR引物在159份桉樹種質(zhì)材料中的擴(kuò)增序列,Blast結(jié)果顯示不同SSR位點(diǎn)對(duì)應(yīng)不同的功能蛋白。通過對(duì)20個(gè)SSR位點(diǎn)微衛(wèi)星序列在不同桉樹種間和種內(nèi)不同個(gè)體間的變化特征表明,不同種間的變異主要是由于SSR位點(diǎn)的重復(fù)次數(shù)和側(cè)翼序列的堿基變異引起;種內(nèi)不同個(gè)體間的變異主要是由于SSR位點(diǎn)的重復(fù)次數(shù)的變化引起。對(duì)24種103份桉樹種質(zhì)材料進(jìn)行親緣關(guān)系分析,提出幾種可能產(chǎn)生雜交的桉樹組合,為桉樹雜交育種提供了理論資料和選種依據(jù)。(5)根據(jù)4個(gè)在種內(nèi)擴(kuò)增序列完全保守的SSR位點(diǎn)的擴(kuò)增序列特征分析發(fā)現(xiàn),4個(gè)SSR位點(diǎn)的微衛(wèi)星序列在不同種間存在重復(fù)單元和重復(fù)次數(shù)的變化,側(cè)翼序列在不同種間存在堿基的變異,證實(shí)了SSR等位基因長(zhǎng)度變化機(jī)制的復(fù)雜性。利用4個(gè)位點(diǎn)的微衛(wèi)星重復(fù)次數(shù)和側(cè)翼序列特異堿基組合構(gòu)建的桉樹種間種質(zhì)資源的鑒定條碼,能夠精準(zhǔn)鑒定的桉樹12種,為系統(tǒng)全面地建立桉樹種質(zhì)資源精準(zhǔn)鑒定體系奠定了基礎(chǔ)并得到了初步成果。分析共享同一鑒定條碼的桉樹種間存在較近的親緣關(guān)系,為桉樹雜交育種工作提供了生物學(xué)依據(jù)。
[Abstract]:Eucalyptus germplasm resources in China mainly come from Australia. As one of the fastest growing and high economic value species in the world, Eucalyptus has become a strategic tree species for afforestation in southern China. SSR molecular marker technology can provide important techniques for Genetic Diversity Investigation and Germplasm Resources Identification of Eucalyptus population. Eucalyptus genome sequence and EST sequence in Genbank database were used to develop SSR markers for eucalyptus. 42 Eucalyptus DNA germplasm materials were used as the main research object to analyze the genetic diversity of Eucalyptus germplasm resources. The main results are as follows: (1) Eucalyptus 28,691 genomic sequences and 16,566 EST sequences were searched by Genbank, and 1,785 valid sequences were identified by sequence analysis, and 2,292 SS sequences were found. By analyzing the information of Eucalyptus SSR loci, it was found that there was a negative correlation between the length of repeating units and the abundance of SSR loci. A total of 395 pairs of SSR primers were synthesized, and 340 pairs of SSR primers were screened by optimized PCR system. These primers provide a theoretical basis for the study of Eucalyptus SSR molecular markers in genetic diversity and phylogenetic analysis. (2) Six Eucalyptus DNA samples were used as control group, one was used as control group. The suitability of genomic DNA mixing pools in SSR primer screening was analyzed. Sequence re-sequencing of PCR products showed that there was no significant difference between the amplification results of six Eucalyptus single DNA samples and that of genomic DNA mixing pools. SSR-PCR amplification using genomic DNA mixing pools as templates was performed. 283 SSR markers were obtained by sequence homology analysis and SSR loci comparison, which could be used to analyze the genetic diversity of Eucalyptus and identify Eucalyptus germplasm resources. (3) A total of 200 alleles of SSR loci were detected in 159 Eucalyptus germplasms from 42 species using 110 pairs of SSR primers. The average number of alleles per locus was 1.818, and the variation range was 1-7. The results of genetic diversity analysis showed that the average effective allele number of 47 polymorphic SSR loci was 1.172, the average Shannon's information index was 0.181, the average observed heterozygosity Ho was 0.068, and the average polymorphic information content PIC was 0.182. SR-GR018, eSSR-GR083 and eSSR-GR109 had the highest degree of polymorphism and reflected more information, which could play a greater role in the analysis of genetic diversity and germplasm identification of Eucalyptus germplasm resources. Unweighted Class Average (UPGMA) showed that there was a close genetic distance between Eucalyptus Queensland and Eucalyptus oligoflora, and it was possible to produce hybrids, which provided theoretical basis and selection basis for effective utilization of Eucalyptus germplasm resources and Eucalyptus cross breeding. (4) Analysis of the size of amplified fragments in the same Eucalyptus species. The results of Blast showed that different SSR loci corresponded to different functional proteins. The variation of microsatellite sequences of 20 SSR loci in Different Eucalyptus species and in different individuals showed that the variation between different species was mainly due to the repetition times of SSR loci and the variation of SSR loci among Different Eucalyptus species. The variation of flanking sequence was mainly caused by the change of repetition times of SSR loci in different individuals. The genetic relationship of 103 Eucalyptus germplasm materials from 24 species was analyzed, and several possible Eucalyptus combinations were put forward, which provided theoretical data and breeding basis for Eucalyptus hybridization. (5) According to four species in existence The analysis of the amplified sequence characteristics of SSR loci with completely conserved internal amplification sequence showed that the microsatellite sequences of four SSR loci had the variation of repeating units and repeating times among different species, and the variation of flanking sequences among different species, which confirmed the complexity of the variation mechanism of SSR allele length. The identification barcodes of Eucalyptus interspecific germplasm resources constructed by complex number and flank sequence specific base combinations can accurately identify 12 species of Eucalyptus. These barcodes lay a foundation for the establishment of an accurate identification system of Eucalyptus germplasm resources systematically and comprehensively, and preliminary results were obtained. It provided biological basis for the cross breeding of Eucalyptus.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S792.39
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本文編號(hào)：2208902