基于SSR的光敏型饲草高粱分子辅助育种体系

Molecular Aided Breeding System of Photosensitive Forage Sorghum Based on SSR

【目的】利用分子标记技术对光敏型饲草高粱新品种传统选育方法进行改良,构建分子辅助育种体系,从而提高品种选育效率,降低育种成本,为形成新的育种体系奠定理论基础。【方法】以光敏型(不抽穗)饲草高粱品种晋光1R去雄后为母本、非光敏型(抽穗)饲草高粱BMRC-3-2为父本进行杂交,构建F2群体。按照光敏与非光敏性状将群体分为两类,各选30株,取叶片提取DNA,利用微卫星分子标记技术(SSR)和集团分离分析法(BSA)对晋光1R/BMRC-3-2的F2群体进行光敏感基因定位分析,以及特异性SSR引物的设计、筛选;将筛出的特异性引物对F1杂交种(以晋光1R为亲本选育的高代稳定恢复系作为父本与其他不育系测配得到的F1代杂交种)进行光敏性鉴定,通过与田间表型对照,确定最终目标引物,从而构建新的光敏型饲草高粱新品种选育体系。【结果】经过SNP index分析,选取99%阈值线最高点前后约250 kb的区域作为性状相关的候选区域,区域总长度为500 kb,共400个SNP位点,对这些SNP位点注释,其中非同义突变或者stop-gain或者stop-loss的SNP位点有6个,最终确定2个候选基因与高粱光敏感性相关,这两个基因位于高粱第7染色体。利用微卫星标记技术开发引物51对,对光敏和非光敏的亲本及F1杂交种进行毛细管电泳检测,选定1对特异性引物70.2-3,结果表明,以251 bp处出现"单峰"为判断依据,引物70.2-3对50个非光敏型F1杂交种的鉴选准确率达到100%,所有材料均在251 bp附近出现峰值。以214和251 bp两处附近出现"双峰"为判断依据,该引物对另外50个光敏型F1杂交种鉴选的准确率达到90%,其中F1-69、F1-70、F1-71这3个材料在214和262 bp附近出现"双峰";F1-81仅在251 bp处有"单峰",而F1-86则在251和232 bp两处附近出现"双峰"。【结论】发现了控制高粱光敏性的候选基因LOC8068537和LOC8068548,位于高粱第7染色体810 000—1 310 000 bp,获得特异性引物70.2-3,可在一定程度上改良传统育种手段,用实验室验证替代田间测交观察,节省了育种成本,提高了育种效率。

【Objective】 The aim of this study is to use molecular marker to construct a breeding system to improve the traditional breeding method,thereby reducing breeding costs and improving breeding efficiency. At the same time, it lays a theoretical foundation for the formation of a new breeding system. 【Method】 The F2 population was constructed by crossing the photosensitive(non-heading) forage sorghum variety Jinguang 1 R as female and the photo insensitive(heading) sorghum variety BMRC-3-2 as male. According to heading and non-heading phenotypes, F2 population was divided into two groups. Thirty plants were selected from each group and DNA was extracted from leaves, SSR and BSA were used to map photosensitive genes in F2 population of Jinguang 1 R/BMRC-3-2 and screen for specific SSR primers. The specific primers were used to identify the photosensitivity trait in F1 generation(F1 Hybrids breeded from high generation stable restorer line with Jinguang 1 R as parent and matched with other male sterile lines), and the final target primers were determined by comparing with the phenotype in the field, so as to construct a new photosensitive breeding system for forage sorghum. 【Result】 After SNP index analysis, the region of about 250 Kb before and after the highest point of 99% threshold line was selected as the candidate region for trait correlation. The total length of the region was 500 kb, with 400 SNP loci, and six of them were non-synonymous mutations or stop gain or stop loss SNP loci. Finally, two candidate genes were identified to be related to photosensitivity, which were located on chromosome 7. A pair of specific primers 70.2-3 was developed by SSR. The primers can distinguish between heading and non-heading sorghum plants to a great extent. The results showed that according to the "single peak" at 251 bp, the accuracy of primers 70.2-3 in the identification and selection of 50 non-photosensitive F1 hybrids reached 100%, and all the materials showed a peak value near 251 bp. Based on the "double peaks" in the vicinity of 214 bp and 251 bp, the accuracy rate of the primer for the identification and selection of the other 50 photosensitive F1 hybrid species reached 90%. Three materials, No. F1-69, F1-70 and F1-71, showed "double peaks" near 214 bp and 262 bp. Material F1-81 has a "single peak" at 251 bp, while material No. 86 has a "double peak" near 251 bp and 232 bp.【Conclusion】 In this study, we revealed that the candidate genes controlling heading date of sorghum were LOC8068537 and LOC8068548, which were between 810000 and 1310000 bp on chromosome 7 of sorghum. The acquisition of specific primers 70.2-3 improved the traditional breeding methods. The primer validation in laboratory could replace the field crossing observation, saving the breeding cost and improving the breeding efficiency.