核桃重要种质遗传分析及主栽品种指纹鉴定
发布时间：2018-08-21 09:47
【摘要】：核桃重要种质资源的遗传分析是核桃育种中亲本选择的基础工作,可为育种中种质的创新、资源合理利用和遗传改良提供科学支持。核桃种质鉴定则是遗传改良和知识产权保护的重要依据。传统的形态学鉴定方法简单、直观,但由于核桃育种亲本利用的集中化,使选育的新品种在形态上愈加趋于相似,形态学方法已无法满足众多品种的鉴定和纯度分析。分子标记可直接反映个体间DNA水平的差异,能稳定遗传,信息量大,与形态学鉴定结合有效弥补了形态学鉴定的不足,可快捷、准确、稳定的进行资源评价。 本研究从核桃BAC文库末端序列(BES)中开发了SSR引物,将其用于核桃种质遗传分析与品种鉴定中。以自交的亲本和子代为对象开展了形态和分子方面的研究,探讨了核桃自交形成的遗传学机制及其对表型的影响。同时,以坚果表型为基础,SSR标记辅助建立了部分品种的指纹图谱。获得了以下主要结果： 1.从NCBI数据库下载了核桃BAC末端序列22740条。应用MISA软件搜索获得SSR位点4732个,SSR出现频率为1/2.8kb。其中,A/T、AT/AT、ATT/AAT分别为最丰富的单核苷酸、二核苷酸和三核苷酸重复。选择具不同类型和重复次数核心基元的序列设计合成310对引物,筛选后获得特异性较高的多态性引物116对。 选取其中19对SSR引物对核桃属中6个种及1个种间杂交种的20个基因型进行SSR分析,每对引物检测到2-9个多态性位点,平均多态性位点为5.4个。其中17个位点的多态信息含量高于0.5,总平均值为0.662,多态性较高。聚类分析显示不同基因型依存种属分类及地理起源分组。它们在核桃(J. regia L.)、泡核桃(J. sigillata Dode)、麻核桃(J. hopeiensis Hu)和核桃楸(J. mandshurica Maxim)中多态性的比例分别为92%(107对)、71%(82对)、49%(57对)和41%(47对)。 2.以晚实品种‘大泡核桃’1979-1982年自交产生的实生后代9个单株(SP1～SP9)为研究对象,观察其植物表型并对亲本和9个自交后代进行了遗传分析。自交后代的表型出现明显差异,主要表现在童期长短：SP1～SP4播种后2年开花结果,SP5～SP8为7-8年开花结果,而SP9自1980年播种至今未开花；所结的种子包括泡核桃(坚果壳厚0.1-0.9mm)、夹棉核桃(壳厚1.0-1.5mm)和铁核桃(壳厚1.6-2.0mm或超过2.0mm)三种类型；部分植株生长量小,抗性差,树龄超过30年树高仅4.5-5.5m。用58个SSR标记分析自交后代的遗传组成,结果显示在‘大泡核桃’后代表现一定程度的近交衰退。所有子代的基因组纯合度增加,并且在SP9中表现最为明显。SP5表型和遗传组成都与亲本最相似,相同SSR位点的比例为82.8%。 3.用28对SSR引物分析了我国62份核桃(J. regia)种质的遗传关系。每个引物扩增的等位基因数为2-7个,期望杂合度(He)范围从0.382-0.821,平均为0.609；香农信息指数从0.637(WJR291)到1.750(WJR265)不等,平均为1.122。以19对BES-SSR引物计算的62个品种间的遗传相似系数从0.47-0.97,遗传多样性水平较高。聚类分析显示62份种质可分为趋向于地理起源分类的三组。本研究的结果可对品种分类和杂交亲本选择提供参考,杂交育种中用亲缘关系较远的不同组的材料为亲本进行杂交,有利于培育出变异类型丰富的后代群体。 4.建立了坚果表型与SSR标记结合进行核桃(J. reiga)品种鉴指纹鉴定的体系。用12个BES-SSR标记辅助核桃属新品种DUS测试指南中22个坚果形状特征,对我国35个主栽核桃品种进行区分,最终选定4个果形特征[坚果纵切面形状——圆形、(阔)卵圆形、(阔)椭圆形和果顶形状——尖]和4对引物(WJR031、WJR069、WJR265和WJR281)建立了35个品种的指纹图谱。首先以果形特征将35个品种分为4组,每组中用最多2对引物就可区分组内品种,每一品种均具唯一的果形特征和SSR标记等位基因的组合。如果不考虑果形特征,用上述4对SSR引物也可将所分析的35个品种有效区分。
[Abstract]:Genetic analysis of important walnut germplasm resources is the basic work of parental selection in walnut breeding. It can provide scientific support for germplasm innovation, rational utilization and genetic improvement in walnut breeding. The centralized utilization of parents in peach breeding has made the morphological similarity of new peach varieties more and more similar. Morphological methods have been unable to satisfy the identification and purity analysis of many varieties. Foot can evaluate resources quickly, accurately and steadily.
In this study, SSR primers were developed from the BAC terminal sequence (BES) of walnut and used in genetic analysis of walnut germplasm and variety identification. SSR fingerprinting assisted the establishment of fingerprints of some cultivars. The following results were obtained:
1. 22740 BAC terminal sequences of walnut were downloaded from NCBI database. 4732 SSR loci and 1/2.8 KB SSR frequency were obtained by MISA software. Among them, A/T, AT/AT and ATT/AAT were the most abundant single nucleotide, dinucleotide and trinucleotide repeats, respectively. 310 pairs of primers with different types and repeats were designed and synthesized. After screening, 116 pairs of polymorphic primers with high specificity were obtained.
Nineteen pairs of SSR primers were selected to analyze 20 genotypes of six species and one interspecific hybrid of Walnut. Each pair of primers detected 2-9 polymorphic loci, with an average polymorphic locus of 5.4. The polymorphic information content of 17 loci was higher than 0.5, with a total average value of 0.662 and high polymorphism. Taxonomy and geographic origin of the genus Juglans. The percentage of polymorphism in walnut (J. regia L.), walnut (J. sigillata Dode), Juglans (J. hopeiensis Hu) and Juglans (J. mandshurica Maxim) was 92% (107), 71% (82), 49% (57) and 41% (47) respectively.
2. Nine seedling progenies (SP1-SP9) of late-bearing cultivar'Dahuo Walnut'from 1979 to 1982 were selected as the research objects. The phenotypes of the progenies were observed and their parents and 9 progenies were analyzed. It is 7-8 years flowering and fruiting, but SP9 has not flowered since 1980; the seeds include walnut (nut shell thickness 0.1-0.9 mm), cotton walnut (shell thickness 1.0-1.5 mm) and iron walnut (shell thickness 1.6-2.0 mm or more than 2.0 mm); some plants have small growth, poor resistance, tree height over 30 years old is only 4.5-5.5 mm. The results showed that the progenies of'Bubble Walnut'showed a certain degree of inbreeding decline. Genome homozygosity of all progenies increased and was most obvious in SP9. The phenotype and genetic composition of SP5 were most similar to those of parents, and the proportion of the same SSR locus was 82.8%.
3. The genetic relationship of 62 walnut (J. regia) Germplasms in China was analyzed with 28 pairs of SSR primers. The number of alleles amplified by each primer was 2-7, and the expected heterozygosity (He) ranged from 0.382-0.821, averaging 0.609; Shannon's information index ranged from 0.637 (WJR291) to 1.750 (WJR265), averaging 1.122. The genetic similarity coefficient ranged from 0.47 to 0.97, and the level of genetic diversity was high. Cluster analysis showed that 62 germplasms could be divided into three groups tending to geographical origin classification. The offspring group with abundant variation types.
4. A system of fingerprint identification of walnut (J. reiga) cultivars was established by combining the phenotype of walnut with SSR markers. Twelve BES-SSR markers were used to assist the identification of 22 nuts in the DUS test guidelines for new walnut cultivars. Thirty-five main Walnut Cultivars in China were distinguished and four fruit-shaped characteristics were selected. The fingerprints of 35 cultivars were established by using round, (broad) oval and apical shape-tip primers and four pairs of primers (WJR031, WJR069, WJR265 and WJR281). If the fruit shape characteristics are not considered, the 4 pairs of SSR primers can be used to distinguish the 35 varieties.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S664.1
本文编号：2195326
[Abstract]:Genetic analysis of important walnut germplasm resources is the basic work of parental selection in walnut breeding. It can provide scientific support for germplasm innovation, rational utilization and genetic improvement in walnut breeding. The centralized utilization of parents in peach breeding has made the morphological similarity of new peach varieties more and more similar. Morphological methods have been unable to satisfy the identification and purity analysis of many varieties. Foot can evaluate resources quickly, accurately and steadily.
In this study, SSR primers were developed from the BAC terminal sequence (BES) of walnut and used in genetic analysis of walnut germplasm and variety identification. SSR fingerprinting assisted the establishment of fingerprints of some cultivars. The following results were obtained:
1. 22740 BAC terminal sequences of walnut were downloaded from NCBI database. 4732 SSR loci and 1/2.8 KB SSR frequency were obtained by MISA software. Among them, A/T, AT/AT and ATT/AAT were the most abundant single nucleotide, dinucleotide and trinucleotide repeats, respectively. 310 pairs of primers with different types and repeats were designed and synthesized. After screening, 116 pairs of polymorphic primers with high specificity were obtained.
Nineteen pairs of SSR primers were selected to analyze 20 genotypes of six species and one interspecific hybrid of Walnut. Each pair of primers detected 2-9 polymorphic loci, with an average polymorphic locus of 5.4. The polymorphic information content of 17 loci was higher than 0.5, with a total average value of 0.662 and high polymorphism. Taxonomy and geographic origin of the genus Juglans. The percentage of polymorphism in walnut (J. regia L.), walnut (J. sigillata Dode), Juglans (J. hopeiensis Hu) and Juglans (J. mandshurica Maxim) was 92% (107), 71% (82), 49% (57) and 41% (47) respectively.
2. Nine seedling progenies (SP1-SP9) of late-bearing cultivar'Dahuo Walnut'from 1979 to 1982 were selected as the research objects. The phenotypes of the progenies were observed and their parents and 9 progenies were analyzed. It is 7-8 years flowering and fruiting, but SP9 has not flowered since 1980; the seeds include walnut (nut shell thickness 0.1-0.9 mm), cotton walnut (shell thickness 1.0-1.5 mm) and iron walnut (shell thickness 1.6-2.0 mm or more than 2.0 mm); some plants have small growth, poor resistance, tree height over 30 years old is only 4.5-5.5 mm. The results showed that the progenies of'Bubble Walnut'showed a certain degree of inbreeding decline. Genome homozygosity of all progenies increased and was most obvious in SP9. The phenotype and genetic composition of SP5 were most similar to those of parents, and the proportion of the same SSR locus was 82.8%.
3. The genetic relationship of 62 walnut (J. regia) Germplasms in China was analyzed with 28 pairs of SSR primers. The number of alleles amplified by each primer was 2-7, and the expected heterozygosity (He) ranged from 0.382-0.821, averaging 0.609; Shannon's information index ranged from 0.637 (WJR291) to 1.750 (WJR265), averaging 1.122. The genetic similarity coefficient ranged from 0.47 to 0.97, and the level of genetic diversity was high. Cluster analysis showed that 62 germplasms could be divided into three groups tending to geographical origin classification. The offspring group with abundant variation types.
4. A system of fingerprint identification of walnut (J. reiga) cultivars was established by combining the phenotype of walnut with SSR markers. Twelve BES-SSR markers were used to assist the identification of 22 nuts in the DUS test guidelines for new walnut cultivars. Thirty-five main Walnut Cultivars in China were distinguished and four fruit-shaped characteristics were selected. The fingerprints of 35 cultivars were established by using round, (broad) oval and apical shape-tip primers and four pairs of primers (WJR031, WJR069, WJR265 and WJR281). If the fruit shape characteristics are not considered, the 4 pairs of SSR primers can be used to distinguish the 35 varieties.
【学位授予单位】：北京林业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：S664.1
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