中国农业科学 ›› 2025, Vol. 58 ›› Issue (5): 851-863.doi: 10.3864/j.issn.0578-1752.2025.05.003

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

蔗茅全基因组SSR标记特征分析与开发

罗正英1(), 胡鑫2,3(), 吴转娣2,3, 钱禛锋1, 田春艳2,3, 刘新龙2,3(), 李富生1()   

  1. 1 云南农业大学农学与生物技术学院/云南省作物生产与智慧农业重点实验室,昆明 650201
    2 云南省农业科学院甘蔗研究所/云南甘蔗遗传改良重点实验室/农业农村部甘蔗生物学与遗传育种重点实验室(云南),云南开远 661699
    3 云南省农业科学院热带作物生物育种全国重点实验室,昆明 650205
  • 收稿日期:2024-08-04 接受日期:2024-09-27 出版日期:2025-03-07 发布日期:2025-03-07
  • 通信作者:
    刘新龙,E-mail:
    李富生,E-mail:
  • 联系方式: 罗正英,E-mail:zhengyluo@163.com。胡鑫,E-mail:sugarhuxin@163.com。罗正英和胡鑫为同等贡献作者。
  • 基金资助:
    云南省农业科学院科研项目(2024KYZX-03); 云南省种子种业联合实验室项目(202205AR070001-09)

Genome-Wide Survey and Development of Novel SSR Markers in Erianthus fulvus

LUO ZhengYing1(), HU Xin2,3(), WU ZhuanDi2,3, QIAN ZhenFeng1, TIAN ChunYan2,3, LIU XinLong2,3(), LI FuSheng1()   

  1. 1 College of Agronomy and Biotechnology, Yunnan Agricultural University/The Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Kunming 650201
    2 Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences/Yunnan Key Laboratory of Sugarcane Genetic Improvement/Key Laboratory of Sugarcane Biology and Genetic Breeding (Yunnan), Ministry of Agriculture and Rural Affairs, Kaiyuan 661699, Yunnan
    3 National Key Laboratory for Biological Breeding of Tropical Crops, Yunnan Academy of Agricultural Sciences, Kunming 650205
  • Received:2024-08-04 Accepted:2024-09-27 Published:2025-03-07 Online:2025-03-07

摘要:

【目的】蔗茅(Erianthus fulvus)是甘蔗重要的野生资源,可用于改良品种的抗逆性和产量。系统鉴定和开发蔗茅基因组中的简单重复序列(simple sequence repeat,SSR)位点,筛选可利用的多态性SSR标记,解析蔗茅资源的遗传多样性特征,开发重要性状关联分子标记,对于利用蔗茅基因资源改良品种性状具有重要意义。【方法】采用生物信息学软件TBtools中的SSRminer模块,对二倍体蔗茅全基因组序列进行系统性SSR位点挖掘,并对所得数据进行统计分析,揭示其在基因组中的分布模式及规律。利用Batch Target Region Primer Design功能批量设计SSR引物,并通过Primer check工具评估引物的特异性。在6个蔗茅种质中,通过对随机合成的50对SSR引物和14对来自甘蔗的SSR引物进行扩增效率和多态性比较分析,验证引物多态性。【结果】共鉴定出152 707个蔗茅SSR位点,平均检测频率为5.64 kb/个,大部分分布在基因间区。SSR类型分布以单碱基、二碱基、三碱基为主。二核苷酸SSR类型中的基序重复次数变化最大,而五核苷酸的基序重复次数变异最低。共检测出883种不同的SSR基序重复类型,其中,A/T和AT/TA是最为丰富。共设计144 692对SSR引物,其中85 025对显示高特异性。这些特异性引物在基因组上呈现出两端密集、中间稀疏的分布特点。扩增试验显示,50对随机合成的SSR引物中,有42对在蔗茅中扩增出稳定清晰的条带,其中32对显示出多态性,多态率为64.0%。与甘蔗SSR引物相比,蔗茅SSR引物表现出更高的扩增效率和更好的多态性水平。经过筛选,从32对有效的蔗茅SSR引物中确定了16对多态性好且扩增条带清晰的引物。这16对引物共扩增出72条条带,多态性信息量(polymorphism information content,PIC)范围为0.63—0.83,平均PIC值为0.74,表明它们在蔗茅种质资源的多态性分析和分子标记研究中具有有效性和实用性。【结论】揭示了蔗茅基因组中高丰度和多样性的SSR分布特征。获得16对扩增效率高、多态性良好的SSR引物。

关键词: 蔗茅, 基因组, 简单序列重复, 特异扩增, 多态性分析

Abstract:

【Objective】 Erianthus fulvus, serving as a crucial wild resource for sugarcane, is capable of enhancing the stress tolerance and yield of varieties. In order to utilize E. fulvus for sugarcane breeding, it is important to systematically identify and develop simple sequence repeat (SSR) loci in the E. fulvus genome, screen for polymorphic SSR markers, analyse the genetic diversity characteristics of E. fulvus resources and then develop SSR markers associated with important traits. 【Method】Using the SSRminer module in the software TBtools, a comprehensive exploration of SSR loci was conducted on the diploid E. fulvus whole genome sequence. The obtained data were statistically analyzed to reveal their distribution patterns and regularities within the genome. The Batch Target Region Primer Design function was employed for batch designing SSR primers, and the specificity of the primers was evaluated using the Primer check tool. To comparethe SSR polymorphism betweenE. fulvus and sugarcane, amplification experiments were performed on 50 pairs of randomly synthesized SSR primers and 14 pairs of SSR primers sourced from sugarcane across 6 E. fulvus germplasms. 【Result】A total of 152 707 SSR loci, which were distributed on E. fulvus genome with an average density of 5.64 kb/locus, were identified. The majority were located in intergenic regions. In terms of SSR type distribution, mononucleotide, dinucleotide, and trinucleotide had the highest density. Dinucleotide SSR types exhibited the greatest variation in motif repeat numbers, while pentanucleotide motif repeat variations were the least. Across the entire genome, 883 distinct SSR motif repeat types were identified, with A/T and AT/TA being the most abundant. A total of 144 692 pairs of SSR primers, of which 85 025 pairs exhibited high specificity, were designed. These specific primers displayed a distribution characteristic of dense ends and sparse middles on the genome. Amplification experiments showed that 42 out of the 50 randomly synthesized SSR primer pairs yielded stable and clear bands in E. fulvus, with 32 exhibiting polymorphisms, yielding a polymorphism rate of 64.0%. In contrast to the 14 sugarcane SSR primers, the E. fulvus SSR primers demonstrated superior amplification efficacy and greater polymorphism. After screening, 16 pairs of SSR primers with good polymorphism and clear amplification bands were determined from the 32 effective SSR primer pairs. These 16 pairs of primers amplified a total of 72 bands, with polymorphism information content (PIC) ranging from 0.63 to 0.83, and an average PIC value of 0.74, indicating their effectiveness and practicality in polymorphism analysis and molecular marker research of E. fulvus germplasm resources. 【Conclusion】This study comprehensively identified SSR loci in the E. fulvus genome, revealing the high abundance and diversity of SSR distribution features. Sixteen pairs of highly specific and polymorphic SSR primers were successfully screened.

Key words: Erianthus fulvus, genome, simple sequence repeat, specific amplification, polymorphic analysis