基于SNP标记的桃矮化基因精细定位

发布时间：2018-01-25 13:22

本文关键词： 桃矮化基因 SNP 精细定位　出处：《中国农业科学》2017年18期 　论文类型：期刊论文

【摘要】：【目的】矮化型桃树体矮小、节间短,是盆栽观赏和砧木育种的重要遗传资源。明确矮化性状形成的遗传机制并对桃矮化基因进行精细定位,是建立目标性状分子辅助选种体系和遗传改良的前提,可为有目标的选育矮化观赏桃和砧木品种奠定基础。【方法】以‘05-2-144’(‘97矮’×‘鸳鸯垂枝’桃)套袋自交获得的395个后代单株构建的分离群体为材料。参考桃基因组信息并基于Sanger技术开发的SNP标记对亲本和后代单株进行分析,在扩大群体单株中进行连锁关系分析,确定连锁的SNP标记,初步定位目标基因。在定位区域内基于二代测序技术开发更多的基因型和表型一致的SNP标记,对后代单株进行基因分型,完成目标性状的精细定位。然后在精细定位区域内开发SNP标记,即杂交群体双亲均为Aa杂合基因型,对‘10-7’×‘96-5-1’杂交后代89个单株进行分子鉴定,以验证基因定位结果的准确性。【结果】通过对桃单株‘05-2-144’自交后代实生苗表型鉴定表明,普通型和矮化型单株数分别为300株和95株,性状分离比例接近3㑳1(P值为0.67;χ2为0.19),符合孟德尔遗传规律,桃矮化性状受隐性单基因控制。用于分子鉴定的单株来源于‘10-7’(普通型)×‘96-5-1’(普通型)杂交组合,共获得89个后代单株,其中普通型66株;矮化型23株(P值为0.854;χ2为0.034)。基于Sanger测序技术开发了SNP标记,在桃基因组数据库Pp06上25 230 425 bp和27 191 090 bp处获得了连锁的SNP标记,且目标基因位于这两个标记的右侧,初步获得了连锁的SNP分子标记。在此基础上,对亲本进行66.89X深度测序,继续开发符合Aa杂合基因型的SNP标记位点。根据参考基因组和物理距离区间共设计了15对SNP引物,其中12对引物与重测序结果中SNP的类型一致,3对引物与重测序结果中SNP的类型不一致,连锁标记的基因分型成功率为80.0%。通过基于SNP基因分型分析,最终完成了目标性状的精细定位,位点位于Pp06的28 712165 bp(引物为JXSNP-5)和28 899 661 bp(引物为JXHRM-SNP-3)之间,遗传距离分别为0.38 c M和0.13 c M,物理距离约为277 kb,精细定位区域内有54个已知转录本。在定位区域内桃基因组Pp06的28 108 436 bp处和29 247 763 bp处开发SNP标记用于杂交后代表型的鉴定,结果表明所有后代单株基因型和表型鉴定结果完全一致,鉴定准确率为100%。【结论】本研究精细定位了桃矮化基因,物理距离约为277 kb,为基因克隆、亲本早期筛选以选育矮化观赏桃和砧木品种等奠定了基础。
[Abstract]:[objective] Dwarf peach is an important genetic resource for potted ornamental and rootstock breeding because of its short body and short internodes. It is the premise of establishing molecular aided seed selection system and genetic improvement for target traits. It can lay a foundation for the selection of dwarf ornamental peach and rootstock varieties. The isolated populations of 395 progenies constructed by bagging self-crossing were used as materials. Referring to the genomic information of peach and based on the SNP markers developed by Sanger technology, the parents and progenies were analyzed. The linkage relationship analysis was carried out in the expanded population, and the linkage SNP markers were determined. Based on the second generation sequencing technology, more genotypic and phenotypic SNP markers were developed for genotyping of single progeny. Then SNP markers were developed in the fine mapping area, that is, both parents of the hybrid population were AA heterozygote genotypes. The molecular identification of 89 individual plants in the progenies of 10 ~ (-7'脳 10 ~ (-7)'脳 10 ~ (-7)'脳 10 ~ (-7)'脳. In order to verify the accuracy of gene mapping results. [results] the phenotypic identification of self-bred seedlings of Peach per plant 05-2-144'showed that the number of common type and dwarf type per plant were 300 and 95, respectively. The segregation ratio of traits is close to 3? 1P value was 0.67; 蠂 2 was 0.19, which was consistent with Mendelian inheritance. The dwarf traits of peach were controlled by recessive single gene. The single plant for molecular identification was derived from the cross combination of 10-7 (common type) 脳 96-5-1 (common type), and 89 progenies were obtained. Among them, 66 were common type; P value of 23 dwarf strains was 0.854; 蠂 2 was 0.034. SNP markers were developed based on Sanger sequencing technique. Linkage SNP markers were obtained at 25 230,425 BP and 27 191 090 BP in peach genome database Pp06, and the target gene was located to the right of the two markers. The linkage SNP markers were obtained, and the parents were further sequenced by 66.89X. Further development of SNP marker sites consistent with AA heterozygote genotype was carried out. A total of 15 pairs of SNP primers were designed according to reference genomes and physical distances. The type of SNP in 12 pairs of primers was the same as that in the result of resequencing. 3 pairs of primers were not consistent with the type of SNP in the result of resequencing. The success rate of genotyping of linkage markers was 80.0%. Based on SNP genotyping analysis, the precise mapping of target traits was finally completed. The loci were between 28 712165 BP (primer JXSNP-5) and 28 899,661bp (primer JXHRM-SNP-3) of Pp06. The genetic distance was 0.38 cm and 0.13 cm, and the physical distance was about 277 kb. There are 54 known transcripts in the fine location region. In the localized region of peach genome Pp06, 28 108 436 BP and 29 247 763. SNP markers were developed to identify the representative type after hybridization. The results showed that the results of genotypic and phenotypic identification of all progenies were identical, and the accuracy of identification was 100. [conclusion] in this study, the dwarf gene of peach was located with a physical distance of 277kb. For gene cloning, early screening of parents for breeding dwarf ornamental peach and rootstock varieties laid the foundation.
【作者单位】：中国农业科学院郑州果树研究所/国家桃葡萄品种改良中心/农业部果树育种技术重点实验室;新西兰植物与食品研究所;
【基金】：国家自然科学基金(31500558) 中国农业科学院创新工程(CAAS-ASTIP-2017-ZFRI) 中央级科研院所基本科研业务费专项(1610192017702)
【分类号】：S662.1
【正文快照】： 0引言【研究意义】桃[Prunus persica(L.)Batsch]是中国栽培面积较大的落叶果树之一。2014年中国桃栽培面积约799 500 hm2,居世界桃栽培第一位(FAO)。其中,矮化型桃因其节间短、成花好是观赏桃和砧木育种的发展方向之一。通过对桃矮化基因进行精细定位是建立目标性状分子辅助

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