【作者】 马明；

【导师】 杨克强； 侯立群；

【作者基本信息】 山东农业大学 ， 林木遗传育种， 2007， 硕士

【摘要】 核桃的早实基因是一份珍贵的自然资源,研究核桃早实基因的遗传规律,寻找与其相连锁的DNA分子标记,不仅对于核桃育种和栽培实践,而且对于揭示木本植物童期发育的机理都有重要意义。近年来的研究表明,核桃的早实特性的遗传是数量性状遗传,有分离现象,是由多基因控制的性状。本研究以早实核桃优系绿园和晚实优系绿丰的F1代杂种后代以及部分核桃品种为试材,以RAPD标记OPB-08958定位核桃早实基因,计算该标记与核桃早实基因间的遗传距离,对该标记进行克隆测序及序列分析,并将该特异标记转化为SCAR标记;还初步确立了适于核桃的SRAP标记的反应体系。主要结果如下:①核桃基因组DNA的提取本研究证明改良的CTAB法是适合核桃基因组DNA提取的快速、简便的方法。该方法提取的核桃基因组DNA条带清晰,亮度均一,纯度较高,没有明显拖尾,无RNA,酶切反应均能被完全切开,酶切产物均一,酶切反应彻底,说明用本法提取的基因组DNA从质量和数量上都可以满足进一步研究的需要。②RAPD特异标记在亲本与杂交F1后代中的分离状况分析RAPD特异标记OPB-08900在早实亲本绿园上扩增出一条约900bp的特异性条带,而在晚实亲本绿丰上则无此条带;对于70株早实后代,有69株出现该条带,1株未出现(5号);84株晚实后代中,82株未出现该条带,2株出现(18号、108号)。χ2检测显示,供试杂交群体早实和晚实性状的分离比例符合1:1的分离比例,标记OP-B08900与早实性状共分离。在154株杂交F1后代中,共有3株表现为重组类型,则其重组率为1.95%。利用Haldane函数M=–㏑(1-2r)/2,计算得标记与核桃早实基因间的遗传距离为1.99cM,表明RAPD标记OPB-08900与核桃早实基因相连锁。③RAPD特异标记的回收、克隆、测序及序列分析本研究对RAPD特异标记片断OPB-08900进行了回收,并将回收产物与质粒PMD18-T连接,转化大肠杆菌JM109菌株感受态细胞,涂在预先涂布X-Gal/IPTG的含Amp的LB固体培养基上进行蓝白斑筛选,克隆了该片断。对该片断进行测序,结果显示序列大小为958bp,提交GenBank,登录号为DQ673614。运用序列分析软件DNAMAN进行序列分析,表明该片断共有36个酶切位点,不具有基因的结构,应属于与核桃早实基因紧密连锁的DNA片断。在NCBI上特异标记DNA序列的同源性进行了比较,结果发现在GenBank中有26条来自于其它生物的DNA序列和DQ673614序列有部分相似性,但相似性位点片段大小大都在27-30bp之间,E值为0.002-7.0﹥﹥1×10-5,同源性较小说明特异标记OPB-08958的DNA序列为核桃基因组所独有。④SCAR标记的转化分析根据测序结果,在原有的OPB08随机引物及其互补序列基础上,设计了SEA、SEB两对SCAR引物,经检验,引物SEA在退火温度56℃条件下,对反应条件、反应体系如Taq酶、Mg2+、模板和引物的浓度等不敏感,重复性好,可以用于分子辅助育种的鉴定与选择。SEA在早实亲本及品种上扩增出和OPB-08958大小相似的条带,在晚实亲本及品种上则无此条带。检测其在杂交F1后代中的分离状况,发现与RAPD标记相一致。对该SCAR标记进行回收、克隆以及测序,与RAPD标记OPB-08958大小一致,表明本研究成功的将与核桃早实基因相连锁的RAPD标记OPB-08958成功转化为SCAR标记。⑤适于核桃的SRAP标记反应体系的确立分析本研究初步确立了适于核桃的SRAP标记反应体系,并利用不同引物组合对核桃早实和晚实亲本进行了SRAP-PCR扩增,挑选出一个多态性比较高的引物组合Em10-Me4对20株杂交后代上进行了检测,引物组合Em10-Me4在这20株杂交后代个体上共产生196条DNA条带,其中多态性条带为107条,多态性比率达54.6%,这说明SRAP标记适用于核桃遗传多样性的分析。更多还原

【Abstract】 Precocious gene of walnut is rare natural resource. Studying on inheritance of walnut precocious gene and finding DNA molecular markers linked to it are not only significative to walnut breeding and cultivation, but also to revealing flowering regulating genes and their relations with juvenility in woodyplant. Studies of recent years indicated that walnut pecocious characteristic is quantitative characters, and it is controled by multiple genes. In this research, RAPD technique was used to study segregation of a specific DNA marker OPB-08900 in walnut F1 hybrids of cross between precocious parent Lüyuan and later mature parent Lüfeng, and genetic distance to this marker is counted; we cloned and sequencinged this marker, and transformed it to SCAR marker; SRAP marker reaction system in walnut were also established in this research. Main results are followed.①Extraction of walnut genomic DNAThis research indicated that improved CTAB method is a fast, convenient and appropriate way for genomic DNA extraction in walnut. Both the enzyme digestion and agarose gel electrophoresis test indicated that the quality of genomic DNA was high pure, and they were fit for the further studies.②Segregation of RAPD marker OPB-08900 in walnut parents and F1 hybrids of crossA 900bp DNA fragment was amplified by RAPD primer OPB08 in precocious parent Lüyuan, but this fragment did not appear in late mature parent Lüfeng. In 70 precocious hybrids of cross, this fragment appeared in 69 progenies, and in 84 late mature hybrids of cross, this fragment did not appear in 82 progenies.χ2 test indicated that precocious and late mature hybrids of cross accorded with 1:1 segregation proportion, and the marker OP-B08900 segregated with precocious character. In 154 hybrids of cross, there were 3 recombinant types, and the recombinant rate was 1.95%. It indicated that RAPD marker OP-B08900 linked with walnut precocious gene. ③Retrieval, cloning and sequencing of RAPD marker OPB-08900RAPD marker OPB-08900 was retrievaled and cloned into PMD18-T vector in this research, and then transferred into E.coli JM109. White screen was selected from the LB solid culture medium and cultivated in fluid culture medium for 12 hours. Plasmid was extracted from bacteria liquid with MiniBest Plasmid Purification Kit. Sequencing final showed that this DNA fragment was actually 958bp in length. It was submitted to GenBank and the accession number was DQ673614. Sequence analysis carried by software of DNAMAN indicated that there are 36 enzyme digestion sites and it has no gene structure. So it should belong to the DNA fragments which linked to precocious gene of walnut. GeneBank accession number of this marker is DQ673614. Comparability of this fragment was analysed in NCBI, and 26 sequences of other species were found to be homologous to this fragment, but comparability sites are mostly between 27 to 30bp, and E value=0.002-7.0﹥﹥1×10-5. So the homology are very small, and the DQ673614 are unique to the walnut genome.④Transformation of SCAR markerTwo pairs of SCAR primers SEA and SEB were designed according to sequence of OPB08 and its complementary sequence. After identification, pimer pairs SEA are not insensitive to reaction factors at annealing temperature 56℃, and can be used on molecular assistance breeding. SEA amplified a fragment which similar with OPB-08958 on precocious parent, and can not amplify this fragment on late mature parent. Segregation of the SCAR marker in walnut parents and F1 hybrids of cross was in agreement with RAPD marker OPB-08958. This SCAR marker was reclaimed and cloned, and then results of DNA sequencing indicated that it is in agreement with RAPD marker OPB-08958. So this research successfully transformed RAPD marker to SCAR marker.⑤Establishment of SRAP marker reaction system of walnutSRAP marker reaction system of walnut were established in this research. Different primer pairs were used at SRAP-PCR amplification in walnut parents, and a higher polymorphism primer pair, Em10-Me4, was selected and identified in 20 F1 hybrids of across. This primer pair amplified 196 DNA fragments, in which 107 were polymorphism, and diversity rate was 54.6%. So SRAP marker can be used for analyse at genetic diversity of walnut.更多还原