甘蔗栽培种单倍体基因组SSR位点的发掘与应用

Development and application of SSR loci in monoploid reference genome of sugarcane cultivar

甘蔗是世界上最重要的糖料作物之一,由于尚未完全破译栽培种基因组,导致SSR标记匮乏,难以覆盖全基因组,限制了甘蔗遗传研究的进展。本研究以栽培种R570的4660个BAC文库片段序列(累计总长为382 Mb,预测到25,316个编码蛋白基因)组装成的一套甘蔗单倍体基因组的模板,利用MISA (Microsatellite identification tool)软件,发掘SSR位点;并综合分析其与4种禾本科植物(高粱、玉米、水稻和二岁短柄草)SSR位点的分布特征;选取50对以TG和AG重复基序的SSR引物,分别利用4个甘蔗属材料(R570、ROC1、LA purple和SES208)和24个重要甘蔗亲本,对SSR引物进行扩增效率验证和多态性分析。共发掘到27,241个SSR位点,平均每个BAC片段有6.29个SSR位点,平均密度为71.33个SSR Mb?1,远低于高粱的平均密度(350.00个SSR Mb?1)。在重复基序中,占比前2位的分别为单核苷酸基序(11,079个)和三核苷酸重复基序(6447个),合计占总SSR位点数的64.33%。与甘蔗不同的是, 4种禾本科植物中的三核苷酸基序类型数量最多、占比最大。此外,在单核苷酸重复基序中, A/T所占比例最高,为84.8%, C/G所占比例最低,为15.2%;在三核苷酸重复基序中, TGT/ACA所占比例最高,为16.04%。总之,禾本科植物基因组富含A/T的基序。在50对SSR引物(TG基序41对和AG基序9对)的多态性验证中,共有45对(90%)能够扩增出清晰的条带,其中35对(70%)在4个甘蔗材料上呈现多态性。进一步利用20对多态性较高的SSR引物对24个甘蔗重要亲本材料进行分析,共扩增到95个等位基因,平均每对引物扩增4.75个,验证了这些引物应用于甘蔗遗传多样性研究的可行性。本研究鉴定的甘蔗栽培种单倍体基因组SSR标记,有效增加了甘蔗遗传研究中可用的分子标记数量,可直接用于甘蔗群体遗传多样性分析和重要性状遗传机制的解析,为甘蔗分子育种的深入研究奠定了基础。

Sugarcane is one of the most important sugar crops in the world. However, it is difficult to develop SSR on a large scale since the genome of cultivar has not been sequenced, which limits the genetic improvement of sugarcane. In this study, a template of monoploid sugarcane genome was assembled using a set of 4660 BAC library sequences(with a cumulative length of 382 Mb,predicting 25,316 genes) from cultivar ’R570’. SSR loci were identified by using MISA(Microsatellite identification tool)software. The distribution characteristics of the monoploid genome ’R570’ was comprehensively analyzed by comparing with the SSR loci of four Gramineae plants(Sorghum bicolor, Zea mays, Oyrza sativa, and Brachypodium distachyon). Fifty pairs of primers with TG and AG repeat motifs were designed to verify the amplification efficiency and polymorphism by PCR amplification in four Saccharum clones(R570, ROC1, LA purple, and SES208) and twenty four core parents of sugarcane. A total of 27,241 SSR loci were identified, with an average of 6.29 SSR loci per BAC clone and an average density of 71.33 SSR Mb–1 which was much lower than that of sorghum(350.00 SSR Mb–1). The mono-nucleotide(11,079) and tri-nucleotide repeat motifs(6447) accounted for 64.33% of the total SSR loci. The number and proportion of tri-nucleotide repeat motifs were the largest in the four Gramineae plants. In addition, A/T(accounting for 84.8%) motif had the highest proportion and C/G(accounting for15.2%) motif the lowest proportion in the mono-nucleotide repeat motifs and TGT/ACA(accounting for 16.04%) motif had the highest proportion in the trinucleotide repeat motifs. In general, the genomes in Gramineae plants are rich in A/T repeat motifs. In the polymorphism validation of 50 pairs of primers(41 pairs of TG motif and 9 pairs of AG motif), 45 pairs of primers(90%)were found to be able to amplify successfully, of which 35(70%) were polymorphic in 4 sugarcane clones. Furthermore, 20 pairs of polymorphic SSR primers were used to detect 24 core parents of sugarcane, a total of 95 alleles were amplified with an average of 4.75 alleles per primer, verifying the application feasibility of these primers for the genetic diversity analysis in sugarcane. The development of SSR markers from the monoploid genome of cultivars ’R570’ not only enriches the number of SSR markers available in sugarcane genetic analysis, but also facilitates the genetic diversity analysis of sugarcane population and the genetic mechanism dissection of important agronomic traits, which provides a foundation for the in-depth research of molecular breeding in sugarcane.