【作者】 张晓明；

【导师】 王国英； 张开春；

【作者基本信息】 中国农业大学 ， 遗传学， 2005， 博士

【摘要】 自交不亲和是高等植物在进化过程中为保持遗传变异而发展起来的一种重要机制。甜樱桃与蔷薇科诸多果树(苹果、梨、杏等)一样具有配子体自交不亲和现象。生产中为获得最佳产量,往往需要配置大量授粉树保证授粉受精,因此自交不亲和基因型的鉴定对樱桃生产具有重要的意义。常规的杂交鉴定方法需要在结果后才能进行,周期长,占地面积大,并且结果往往不够准确。本研究以甜樱桃主栽品种为试材,建立了AS-PCR技术体系,对不同甜樱桃品种的花柱S基因型进行鉴定,用于指导生产中授粉树的选择选配。 最近发现的SFB(S-单元型特异F-box蛋白基因)是配子体自交不亲和花粉决定基因。本研究鉴别了自交亲和的S4’单元型的SFB4’基因,并根据该基因的特点,发展了一种简单实用的分子标记用于樱桃育种中自交亲和基因型的早期鉴定。 具体结果如下: 1.建立了基于PCR技术的甜樱桃品种S基因型鉴定技术。所设计的两对引物组合BFP73/74,BFP93/94扩增产物分别包含了甜樱桃S-RNase基因的第二和第一个内含子,结合S1,S5基因的特异扩增引物,通过扩增片段长度的不同,就可以对大部分樱桃品种的S基因型进行鉴定,其可靠性用已知基因型品种进行了验证。该技术体系的建立可以从DNA水平鉴定甜樱桃S基因型,不受取材时间的限制,比传统的杂交授粉方法和S蛋白鉴定方法简便实用,准确可靠。 2.对61个国内外的甜樱桃品种进行了自交不亲和基因型的鉴定,首次鉴定了我国主栽的一些甜樱桃品种的S基因型,所获得的甜樱桃品种S基因型列表对于指导生产中品种配置和授粉树的选择具有很大的作用。 3.依据甜樱桃自交不亲和的花粉SFB基因设计引物,鉴别出了自交亲和突变体SFB4’基因中的缺失突变,发现S4’单元型中花粉部分自交不亲和功能的丧失与SFB4’基因中存在缺失突变有关。基于SFB4’基因中的缺失突变,获得了SFB4’基因特异分子标记,从而区别开了利用S-RNase基因不能区分的S4和S4’单元型。该分子标记可以在自交亲和品种选育工作中用于自交亲和品种的早期选择。 4.培育自交亲和甜樱桃品种对樱桃生产具有重要意义,为获得自交亲和突变体S4纯合的种质,以自交亲和品种斯坦拉(Stella)为亲本构建自交亲和群体,根据S基因分离的特点可获得基因型为S3S4’和S4’S4’自交亲和后代,利用AS-PCR技术体系对自交后代的基因型进行鉴定,在获得的337棵植株中S3S4’基因型178株,S4’S4’基因型140株。这些种质的获得对于深入开展甜樱桃自交亲和育种工作具有重要的意义。更多还原

【Abstract】 In order to assure genetic variability, many flowering plants have evolved strategies to prevent inbreeding and promote out-crosses. The predominant form of self-incompatibility (SI) is the Gametophytic self-incompatibility (GSI) system , in which the compatibility of a cross is determined by the haploid genome of the pollen and the diploid genome of the pistil. Sweet cherry exhibits the monofactorial gametophytic self-incompatibility system similar to other self-incompatible fruit tree species of the Rosaceae. Therefore, they will not set fruit unless pollinated with cultivars with different S-genotypes. Determination of correct pollen incompatibility groups and assignment of cultivars to the groups are essential for good crop production. Pollen incompatibility groups in sweet cherry have been identified by controlled pollination tests and/or pollen tube growth tests, but these tests is time-consuming and prone to be affected by environmental factors. The allele-specific PCR system was established to identify the S-genotype of 61 sweet cherry cultivars in this study.Recently SFBs (S-haplotype specific F-box protein gene) were found to associate with pollen determinant of GSI. This report identified the SFB4 ’of the self-compatible S4’ haplotype, and the SFB4’ gene-specific molecular marker was established.All results were summarized as follows:1. The PCR based identification technology of 7 known self-incompatibility alleles of sweet cherry was established .Two pairs of consensus primers BFP73/74 and BFP93/94 were designed from published cDNA sequences of 57 to S6 S-RNase to reveal length variation of the first and the second introns.With the consensus and allele-Specific primers of S1 and 55, most sweet cherry varities S-genotype can be visualized directly on an agarose gel. The use of the consensus and allele-specific primers was demonstrated by detecting genotypes that have been published recently. This method is a rapid and convenient technique that can be used on vegetative material.2. The allele-specific PCR system was used to identify the S-genotype of 61 sweet cherry cultivars. We genotyped some main Chinese sweet cherry cultivars for the first time using the alleles specific PCR, The new listing of cultivars S-allele assignments was presented and proved to be an effective reference for selecting pollinator and pre-selection the new selections for breeders and growers.3. This report identified the SFB4’ of the self-compatible S4’ haplotype. The alignment of the sequences of SFB4’ and SFB4 by the BLAST program revealed that there is a 4 bp deletion in SFB4’ — — TTTA. The sequence polymorphism generated by the ’TTTA’ deletion in the SFB4’ was exploited to develop the SFB4 ’gene-specific molecular marker specific for the detection of the S4’ haplotype but not S4 haplotype. The SFB4’ gene-specific molecular marker can be visualized directly on an agarose gel, so it can be immediately applied to a marker-assistant cherry breeding program.4. It was known that the self-compatible sweet cherry cultivar ’Stella’ has the genotype S3S4’. Using the AS-PCR, we analysed progeny from selfing ’Stella’ and detected three genotypes: S3S4’, S4’S4’, S3S3, respectively. S4’S4’ and S3S4’ segregated approximately 1:1.27 in the seedlings, and the genotype of S3S3 is 2.08%. The S4 ’S4’ homozygous germplasm is very valuable in the breeding of SC sweet cherry cultivars.更多还原