桉树种质资源遗传多样性分析及精准鉴定体系的初步研究
发布时间：2018-08-28 08:51
【摘要】：我国桉树(Eucalyptus)种质资源材料主要来源于桉树原产地澳大利亚,作为世界上生长最快和经济价值极高的树种,桉树已成为我国南方造林的战略性树种。SSR分子标记技术能为桉树种群遗传多样性调查和种质资源鉴定提供重要的技术手段,有效地促进桉树资源的良种选育与种质资源的开发和利用。本研究利用Genbank数据库中桉树基因组序列和EST序列进行桉树SSR标记的开发,以42种159份桉树DNA种质材料为主要研究对象,对桉树种质资源的遗传多样性进行分析,探讨桉树种间亲缘关系,并通过选用种内扩增序列完全保守的SSR位点,初步构建桉树种间种质资源的精准鉴定体系。主要研究结果如下:(1)通过Genbank检索桉树28,691条基因组序列和16,566条EST序列,经序列分析,确认1,785条有效序列,并发现2,292个SSR位点,含SSR位点的序列占序列总数的12.64%。通过分析桉树SSR位点信息,发现桉树SSR位点的重复单元长度与SSR位点的丰度呈负相关关系。桉树EST序列中以三碱基重复单元最为丰富,而桉树基因组序列中则是二碱基重复单元出现频率最高。对所有SSR位点进行引物设计及评估,共合成了395对SSR引物,经过优化PCR体系初步筛选出340对SSR引物,为桉树SSR分子标记在遗传多样性分析和系统发育分析等方面研究提供了引物资源和理论基础。(2)本研究以6个桉树DNA样品为对照组,1个基因组DNA混合池为试验组进行基因组DNA混合池在SSR引物筛选中的适用性分析。通过PCR扩增产物的重测序分析,6个桉树单一DNA样品的扩增结果与基因组DNA混合池的扩增结果差异不显著。利用基因组DNA混合池为模板进行SSR-PCR扩增序列重测序,经序列同源性和SSR位点的比对分析,得到可应用于桉树遗传多样性分析和桉树种质资源鉴定研究的283个SSR有效标记。并利用283对SSR引物在39种桉树中进行SSR-PCR扩增分析,得到268对扩增结果稳定,重复性较好的引物,且在桉属双蒴盖亚属内具有良好的通用性。(3)利用扩增稳定、条带特异的110对SSR有效引物对42种159份桉树种质材料进行PCR扩增结果统计,159份桉树种质材料中共检测到SSR位点的等位基因共200个,平均每个位点等位基因数为1.818,变化范围为1~7个。纯合基因型数162个(81%)远远多于杂合基因型数38(19%)。遗传多样性分析的结果表明,47个多态SSR位点的平均有效等位基因数为1.172,平均Shannon’s信息指数为0.181,平均观察杂合度Ho为0.068,平均多态信息含量PIC为0.182。综合各指标分析得到,位点eSSR-GR018、位点eSSR-GR083和位点eSSR-GR109的多态性程度最高,反映的信息量更大,能够在桉树种质资源的遗传多样性分析和种质鉴定等方面发挥更大的作用。主坐标法分类结果与形态学分类状态基本一致,同时也证实了伞房属与桉属存在明显的遗传差异。非加权类平均法(UPGMA)表明,昆士兰桉和少花桉间具有更近的遗传距离,很有可能产生杂交种,这为桉树种质资源的有效利用和桉树杂交育种工作提供了理论基础和选种依据。(4)分析20对在同一桉树种内扩增片段大小基本一致的特异SSR引物在159份桉树种质材料中的扩增序列,Blast结果显示不同SSR位点对应不同的功能蛋白。通过对20个SSR位点微卫星序列在不同桉树种间和种内不同个体间的变化特征表明,不同种间的变异主要是由于SSR位点的重复次数和侧翼序列的碱基变异引起;种内不同个体间的变异主要是由于SSR位点的重复次数的变化引起。对24种103份桉树种质材料进行亲缘关系分析,提出几种可能产生杂交的桉树组合,为桉树杂交育种提供了理论资料和选种依据。(5)根据4个在种内扩增序列完全保守的SSR位点的扩增序列特征分析发现,4个SSR位点的微卫星序列在不同种间存在重复单元和重复次数的变化,侧翼序列在不同种间存在碱基的变异,证实了SSR等位基因长度变化机制的复杂性。利用4个位点的微卫星重复次数和侧翼序列特异碱基组合构建的桉树种间种质资源的鉴定条码,能够精准鉴定的桉树12种,为系统全面地建立桉树种质资源精准鉴定体系奠定了基础并得到了初步成果。分析共享同一鉴定条码的桉树种间存在较近的亲缘关系,为桉树杂交育种工作提供了生物学依据。
[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.
【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】：2017
【分类号】：S792.39
，
本文编号：2208902
[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.
【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】：2017
【分类号】：S792.39
，
本文编号：2208902
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