基于SSR标记的四倍体鸭茅遗传图谱加密

【目的】利用转录组测序开发的EST-SSR标记和鸭茅基因组调研测序开发的基因组SSR(genomic-SSR)标记,对已构建的四倍体鸭茅遗传图谱加密,为定位控制鸭茅重要农艺性状的QTL位点奠定基础。【方法】基于拟测交策略,以"楷模"(高杆、多分蘖、宽叶、早熟)和"01436"(矮秆、少分蘖、细叶、晚熟)作为亲本材料进行杂交,得到一个含有214株鸭茅材料的作图群体,利用亲本和随机选取的5个单株对574对EST-SSR标记和150对Genomic-SSR进行引物筛选,PCR产物经8%非变性聚丙烯酰胺凝胶电泳检测后,将扩增条带清晰、在亲本之间存在差异且子代间存在分离的多态性引物用于亲本及群体扩增。将扩增结果按标记类型统计分析,对于亲本间存在差异的条带,按条带有无(有带计1,无带记0)对DNA扩增产物按进行统计,经卡方检验,将分离比例符合1﹕1(亲本基因型为Aaaa×aaaa或aaaa×Aaaa)和3﹕1(亲本基因型为Aaaa×Aaaa)的标记,用于遗传连锁图谱构建。符合作图要求的标记采用High Map软件进行遗传图谱构建。【结果】最终筛选出符合要求的EST-SSR引物31对和Genomic-SSR引物17对,引物多态性分别为5.4%和11.3%,总的多态性为6.6%。对鸭茅214个作图群体单株及亲本DNA进行扩增,共得到169个多态性位点,其中EST-SSR101个,Genomic-SSR68个位点。169个标记位点经卡方检验分析表明,有89个标记符合孟德尔分离规律,标记可用率为52.7%,其中呈Aaaa×aaaa或aaaa×Aaaa分离类型的标记有79个,呈Aaaa×Aaaa的有10个,其余80个为偏分离标记。将SSR标记整合以前的标记信息,重新构建了一张包含2 551个标记,覆盖7个连锁群,总长度为758.4 cM的鸭茅高密度遗传图谱。加密后的图谱包含SNP标记4 187个,SSR标记84个,各连锁群标记数在166—709个,每个连锁群的平均标记数为364个,LG1包含最多标记数有709个,LG7标记数最少166个,各连锁群长度在60.28—147.09 cM,标记平均密度为0.19—0.76 cM,总的平均图距由原来的0.37 cM缩至0.3 cM,且由于标记密度的改变,各连锁群上标记分布的位置也发生较大变动。【结论】增加了部分SSR标记后,新构建了一张包含2 551个标记,覆盖7个连锁群总长度为758.4 cM的四倍体鸭茅遗传图谱,总长度增加42.63 cM,平均图距由0.37 cM缩至为0.3 cM。

Enhancement of the Genetic Linkage Map Density of Tetraploid Based on SSR Markers

【Objective】In order to obtain a high density genetic map of tetraploid orchardgrass previously established, we used EST-SSR and genomics-SSR markers to enhance the density of the genetic map. These results will be beneficial and helpful to orchardgrass selection and QTL analysis，especially QTL analysis of recessive genes.【Method】Based on the proposed test-hybridization strategy, an F1 population of 214 individuals derived from the cross between two Chinese orchardgrass cultivars–Kaimo (tall height plant, more tillers, broad leaves and early-maturing) and 01436 (dwarf, less tillers, narrower leaves and late-maturing) was used for map construction. 574 pairs of EST-SSR markers and 150 pairs of Genomic-SSR markers were selected as the screening primers. Five of the 214 progenies were randomly selected and amplified together with their parents. Amplified fragments were separated on 8% denatured polyacrylamide gels. The primers which could amplify clear bands and the presence of separated polymorphic were used for population and parental DNA amplification. The amplified results were statistically analyzed according to the marker type. According to the presence or absence of bands (with band count 1, or count 0), the amplification products of the DNA were statistically analyzed. According to theχ2 test, the marker which separation ratio was in accordance with 1﹕1 (Aaaa × aaaa or aaaa × Aaaa) and 3﹕1 (Aaaa × Aaaa) for genetic linkage map construction by using HighMap software.【Result】Finally, 31 pairs of EST-SSR primers and 17 pairs of Genomic-SSR primers were used for population and parental DNA amplification, The primer polymorphism percentage were 5.4%, 11.3% and 6.6%.A total of 169 clear bands were obtained, and 89 were used to construct the genetic linkage map of orchardgrass. There were 79 markers with Aaaa × aaaa or aaaa × Aaaa segregation types, 10 with Aaaa × Aaaa and the remaining 80 with distorted markers. A high-density linkage map of orchardgrass was constructed using 2,551 markers, which were distributed on seven linkage groups spanning 758.4 cM. The encrypted map including 4187 SNP markers, 84 SSR markers, the number of markers in the LGs from166 to709, with average 364. LG1 contains the largest maker with 709, while the LG7 was the least with 166. The sizes of the individual LGs ranged from 60.28 to 147.09 cM, with average inter-marker distances ranging 0.19—0.76 cM. The average distance between adjacent distance markers was reduced from 0.37 cM to 0.3 cM. Due to the change of marker density, the position of the markers distribution on each linkage group also changed greatly.【Conclusion】A high-density genetic linkage map of tetraploid Dactylis glomerata was reconstructed using 2551 markers, which were distributed on seven linkage groups spanning 758.4 cM. The new map added a number of SSR markers, which total length increased by 42.63 cM and average distance between adjacent distance markers was reduced from 0.37 cM to 0.3 cM.