【作者】 赵鹏；

【导师】 张硕新； Keith E. Woeste；

【作者基本信息】 西北农林科技大学 ， 生态学， 2011， 博士

【摘要】 核桃属植物栽培历史久远,种质资源十分丰富,约有21个种,且具有重要的生物学和经济价值。美国白核桃(Juglans cinerea L.),温带落叶阔叶乔木,生长于美国东部和加拿大南部,是一种价值很高的坚果和用材树种。上世纪70年代,该树种受到一种外来病菌Sirococcus clavigignenti-juglandacearum Nair(Scj)的危害已濒临灭绝。日本核桃(Juglans ailantifolia Carr.)源于日本和库页岛,于1870年引入美国,该树种也受到病菌(Scj)的侵害。美国白核桃(J. cinerea)和日本核桃(J. ailantifolia)杂交的杂合核桃(buartnuts or buarts, J. ailantifolia×J. cinerea)很难从形态学上与其父母亲本区分开来。美国黑核桃(Juglans nigra L.)源于美国东部从新英格兰州到新泽西州,坚果可以榨油,木材质量高,是一个非常有价值的经济和生态树种。本文利用40对随机扩增多态性DNA(RAPD)引物组合对美国白核桃、日本核桃、杂种核桃和美国黑核桃进行了DNA分群鉴定分析。共筛选了530对RAPD引物组合,找出了32个核桃属植物中的差异DNA片段,纯化这些DNA后将其连接到pGEM-T载体上,然后转到大肠杆菌JM109细胞中,并用“蓝白筛选法”将DNA产物进行克隆、质粒DNA提取和测序等遗传分析研究。经测序后,将这些RAPD分子标记成功转化为可以鉴定美国白核桃、日本核桃、美国黑核桃和杂合核桃的SCAR和CAPS等分子标记,并且用这些分子标记对190多个美国、日本及其杂合核桃DNA样品材料进行了检测试验,同时采用两种Scj病菌对美国白核桃和杂合核桃进行了抗病试验;采用12对微卫星(SSR)分子遗传标记对美国印第安纳州9年生来自20个母本家系的美国黑核桃(J. nigra L.)328个子代群体进行了基因型测定、家系分析和群体遗传分析。结合表现型和基因型的遗传数据,对该自由授粉核桃子代群体的四个表现型指标:树高、胸径、干型和综合质量进行了数量群体遗传分析,并对不同性状进行了相关性分析;采用10对微卫星分子标记技术对密苏里大学美国黑核桃(J. nigra L.)育种基地22个优良栽培品种家系的285个子代无性系个体进行了基因型测定、指纹鉴定、家系分析等遗传分析研究。对所有优良栽培品种个体无性系植株的七个表现型包括形态学特征进行了测定,并结合表现型和基因型指标鉴定每个无性系的栽培品种家系名称,同时进行了典型相关性分析和双复式分析。主要研究结果如下:1.“Buartnut”的杂合核桃植株[日本核桃(J. ailantifolia)×美国白核桃(J. cinerea)]相比父母本对病菌(Scj)更具有抗病性。杂合核桃结合了亲本的遗传表现特征,其中,大多杂合核桃为F1代,其余杂合核桃为杂合F2代、F3代、F4代,甚至为多个核桃种的杂交后代,被检测的杂合核桃具有复杂的遗传历史背景。2.克隆筛选了32个RAPD分子标记,成功获得24个重要DNA片段序列,并且发现了9个DNA分子标记,分别是:1个核糖体内转录间隔区(ITS)分子标记;1个叶绿体(TrnT (UAA)–trnF (GAA) region)分子标记;1个线粒体分子标记(3-9);6个核DNA分子标记(16R-2、22-5、14R-1、39-6、40-1、15R-8)。其中有5个为共显性分子标记(22-5、15R-8、14R-1、39-6、40-1),4个为显性分子标记(ITS、TrnT–trnF、3-9、16R-2)。上述2个细胞质DNA分子标记(TrnT–trnF、Mitochondrial)和7个核DNA分子标记(ITS、16R-2、22-5、14R-1、39-6、40-1、15R-8)在鉴定和区分美国白核桃、日本核桃、美国黑核桃和杂合核桃品种中,全面实用、简单灵活,且在实验室容易操作,是一个新的核桃种资源管理工具。美国农业部林务局等合作单位已经开始将这9个分子标记应用到濒危植物美国白核桃种子园建立、抗病育种、基因保护和分子系统进化等研究中。3.采用上述9个分子标记和1个叶绿体CPS05分子标记对24个日本核桃、51个杂合核桃、111个美国白核桃、1个胡桃楸和4个美国黑核桃样品进行了分子水平的种质资源鉴定检测。其中用所有分子标记测试杂种核桃J. mandshurica均显示日本核桃表现型特征;接近100 %的日本核桃DNA样品显示日本核桃本身的表现型特征,在被检测的236个日本核桃样品中,只有两个样品(008和014)在用核DNA分子标记15R-8测试时,显示美国白核桃表现型特征;9.8 %美国白核桃经测试之后确定其为杂合核桃;11.7 %的样品用细胞质分子标记检测时,显示美国白核桃表现型特征,实质上这些样品基因型为杂合核桃;若只用细胞质分子标记检测时,90.2 %的杂合核桃仅显示日本核桃表现型特征。4.采用微卫星(SSR)分子标记技术和数量遗传分析可鉴定核桃的优良基因型和提高核桃的遗传特性,同时父系分析取决于子代个体基因型和表现型的无性系之间的差异和父系分析的排斥原则。该核桃子代群体遗传多样性水平高(Ho=0.802, He=0.778),12对微卫星平均每个位点等位基因数为18个,其中最多的基因座WGA82等位基因数为35个,最少的AAG001等位基因数为7个。5.接近89 %的子代个体可以根据基因型鉴定出母本家系,6 %的子代个体可以从20个候选的母本家系中根据其等位基因大小准确找到其父本家系个体。采用12对微卫星分子标记技术对该328个子代个体进行基因型测定,99.99 %可以鉴定出子代个体正确的父母本家系和个体亲缘之间的关系。根据基因型数据可以找出自由授粉子代群体的半同胞和全同胞家系。美国黑核桃用材群体家系的胸径、树高、质量和综合等性状指标之间存在相关性。树高和胸径的相关系数为0.88,相关性极显著(P≤0.0001)。综合质量与树高和胸径显著相关(r值分别为0.57和0.46, P≤0.0001),而干型只与树高相关性极显著(r = 0.18, P≤0.001),干型与其他表现型特征无显著相关性。6.结合基因型数据,22个美国黑核桃栽培品种家系的七个表现型和形态学特征的数量遗传分析表明,相比其他表现型和形态学特征,BBDAD(Bud break date (dyasafter‘Davidson’),发芽期在栽培品种‘Davidson’之后的天数)和FPLD(First pistillate date,雌花期)两个表现型/形态学特征可以非常有效地对美国黑核桃生产优良果实的栽培品种进行家系和个体亲缘关系鉴定。基因型数据显示结果表明,该黑核桃285个子代无性系群体遗传多样性水平也很高(Ho=0.834, He=0.837),10对微卫星平均每个位点等位基因数为13个。其中最多的基因座WGA82等位基因数为20个,最少的WGA69等位基因数为7个。7.结合基因型和表现型数据显示,一些优良的核桃栽培品种无性系家系名称相同,但表现型、基因型不相同。同时,一些基因型相同的无性系栽培品种个体有多个不同栽培品种家系名称。多种因素造成的误差,导致在优良核桃栽培品种无性系中很难找出合适的栽培名称和基因型。8.所使用的微卫星(SSR)基因位点分子标记技术为核桃群体遗传育种提供了有效工具,具有较大的应用价值。如将基因型测定数据用于遗传育种、子代群体型测定、数量遗传、鉴定重要栽培品种家系等遗传分析研究中,有利于减少遗传分析误差和错误,并会对下一个育种项目和实践产生重大影响。更多还原

【Abstract】 The genus Juglans (Juglandaceae) consists of about 21 species, many of which are of biologically and economically important because of their high quality timer and nutritious nuts. There are rich germplasm resources for Juglans. Butternut (J. cinerea L.) is a temperate deciduous hardwood native to the eastern United States and southern Canada valued for its nuts and wood. Butternut’s survival is threatened by butternut canker; a disease caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Japanese walnut (J. ailantifolia Carr.) native to Japan and Sakhalin, which was introduced into North America from Japan about 1870 by nurserymen can also serve as a host to S. clavigignenti-juglandacearum (Scj). Hybrids between butternut and Japanese walnut (J.×bixbyi but commonly called buartnuts or just buarts) are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. Black walnut (J. nigra L.) is a large tree native throughout the eastern United States from New England to Texas. It is a valuable hardwood species that also produces a high quality, edible nut.We used pair-wise combinations of forty random primers to screen bulked DNA pools of butternut, Japanese walnut and buartnuts to identify genomic regions unique to Japanese walnut. About 530 randomly amplified polymorphic DNA (RAPD) panels were examined. We found about thiry-two DNA amplicons present in Japanese walnut and buartnut hybrids but absent in butternut. We cloned and sequenced twenty four amplicons in order to convert them into useful genomic markers. Nine CAPS or SCAR markers were developed successfully from these RPAD markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. A battery of 12 microsatellite markers was used to carry out parentage analysis of 328 9-year-old black walnut progeny and 20 parent trees associated with a black walnut progeny test in Indiana, U. S. A. We obtained the phenotypic data for each open-pollinated progeny family and compared it with data for the same families after each seedling’s membership in the family was either confirmed or disproved based on genotypic data. The four parameters height, diameter at breast height (DBH), leader (LDR), and quality were measured for each progeny. The genotypic data also permitted the assignment of some progeny into paternal half-sib and full-sib families. The black walnut nut breeding orchard at the University of Missouri contains about 70 accessions used for breeding. The 22 cultivars were characterized for seven phenological descriptors: bud break date (dyas after‘Davidson’(BBDAD), first pistillate date (FPLD), first pollen date (FPD), season length (SL), harvest date after‘Davidson’(HDAD), mean pistillate date after‘Davidson’(MPLDAD), and mean pollen date after‘Davidson’(MPDAD). A battery of 10 microsatellite markers was used to carry out cultivar identification and genetic analysis of 285 samples and 22 cultivars (clones). We combined morphological, phenological, and genotype data from ten microsatellites to group individual accessions by cultivar (clone). The main results were as follows:1. Field observations indicate that trees commonly called buartnut [a hybrid of butternut and its close congener Japanese walnut (J. ailantifolia×J. cinerea)] may be more resistant to butternut canker disease fungus (Scj)than either parental species. We confirmed that hybrids currently growing in the landscape typically have a complex genetic history and present features of the parental species in all possible combinations.2. We cloned and sequenced 32 RAPD amplicons apparently present in JA but absent from JC samples. Of these, primers were designed to amplify 24 loci for re-sequencing. We ultimately identified one internal transcribed spacer region (ITS) marker, one chloroplast (TrnT (UAA)–trnF (GAA) region) marker, one mitochondrial marker (3-9), and six nuclear markers (16R-2, 22-5, 14R-1, 39-6, 40-1, and 15R-8). Five markers were co-dominant (22-5, 14R-1, 39-6, 40-1, and 15R-8), and four markers were dominant (ITS, trnT–trnF, 3-9, and 16R-2).3. The molecular markers described here for identifying buarts and other hybrids were robust, simple to use, easy to score in the lab, and they provide a new resource-management tool. The markers are also flexible, and they have already been used to identify non-hybrid trees in National Forests of United States, for establishing seed orchards, and to further butternut resistant breeding and conservation efforts by the USDA Forest Service and cooperating institutions. The utility of the markers for identifying hybrids was tested and verified using 24 Japanes walnuts, 51 hybrids, 111 butternuts, one J. mandshurica, and four black walnuts. J. mandshurica had a phenotype identical to JA at all the loci we tested, and that all JA samples (100 %) had a JA phenotype at all the loci except for one sample (008) at one locus (15R-8). It was 9.8 % of butternuts we tested turned out to be hybrids, 11.7 % of the butternuts had JA cytoplasm (and so they turned out to be hybrids), 90.2 % of the hybrids had JA cytoplasm.4. Using microsatellite markers and quantitative genetic analyses we identified elite genotypes for further genetic improvement or the establishment of production populations. A sequential exclusion procedure was used in an attempt to determine paternity for all progeny. A high level of genetic diversity was revealed among the seedling in the progenty test (Ho=0.802, He=0.778), and the mean number of alleles per locus was 18, locus WGA82 has 35 alleles, and locus AAG001 has 7 alleles.5. Approximately 89% of the offspring were assigned by parentage analysis to their putative female. Based on the female parents’genotype, paternity tests revealed that about 6% of the offsprings could be assigned a male parent. The probability of error in first parent assignment, second parent assignment, parent pair assignment, individual identity and sib identity was < 0.01 % using 12 microsatellite loci. The genotypic data was also could found the assignment of some progeny into paternal half-sib and full-sib families.6. In black walnut we observed high correlations among height, DBH, LDR, and quality. The correlation between height and DBH was 0.88 and highly significant (P≤0.0001). The quality ratings at age nine were strongly and significantly correlated with both height and diameter (r=0.57 and 0.46, respectively, P≤0.0001), but the presence of a central leader (LDR, an important factor in determining if a young hardwood will develop into a crop tree) was only weakly associated with height (r=0.18, P≤0.001) and not significantly associated with diameter. Analyses based on six morphological descriptors of black walnut showed that BBDAD and FPLD were better than other evaluated traits for determing clone identify. The genotypic data showed that the clones used for black walnut nut breeding possessed a highly level of genetic diversity (Ho=0.834, He=0.837), and the mean number alleles per locus was 13, including the locus WAG82 has 20 alleles, locus WGA69 has 7 alleles.7. We found that many black walnut ramets that shared cultivar names did not have identical phenotypes or genotypes, and conversely, some genotypes were given multiple cultivar names. Our results indicate that multiple errors were committed during the propagation of these important cultivars, and that it may be difficult or even impossible to properly assign a name to a genotype.8. Microsatellites provide a powerful and valuable tool for the management of black walnut nut and timber breeding populations. The results showed that genotypic data can be an important supplement to breeding, and that progeny tests and breeding programs are subject to potentially costly errors without genotypic data.更多还原