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作物学报 ›› 2018, Vol. 44 ›› Issue (9): 1400-1410.doi: 10.3724/SP.J.1006.2018.01400

• 研究论文 • 上一篇    下一篇

基于甘蔗热带种(LA-purple)全基因组序列的SSR开发及特征分析

王恒波(),肖乃衍,朱专为,刘翠翠,IntikhabALAM,陈平华,卢运海()   

  1. 福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室 / 作物遗传育种与综合利用教育部重点实验室 / 作物科学学院, 福建福州 350002
  • 收稿日期:2017-11-20 接受日期:2018-03-26 出版日期:2018-09-10 网络出版日期:2018-04-09
  • 通讯作者: 卢运海
  • 基金资助:
    本研究由国家现代农业产业技术体系建设专项(CARS-20-1);国家甘蔗工程技术研究中心2017年主任课题基金项目(ptjh1500117) 资助

Development and Characterization of SSR Markers from the Whole Genome Sequences of Saccharum officinarum (LA-purple)

Heng-Bo WANG(),Nai-Yan XIAO,Zhuan-Wei ZHU,Cui-Cui LIU,ALAM Intikhab,Ping-Hua CHEN,Yun-Hai LU()   

  1. Key Laboratory of Ministry of Agriculture for Sugarcane Biology and Genetic Breeding (Fujian), Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2017-11-20 Accepted:2018-03-26 Published:2018-09-10 Published online:2018-04-09
  • Contact: Yun-Hai LU
  • Supported by:
    This study was supported by the China Agriculture Research System(CARS-20-1);National Sugarcane Engineering Research Center of Director Project Fund in 2017 (ptjh1500117)

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摘要：

现代甘蔗栽培品种(2n = 100~130)是由甘蔗热带种(2n = 80)与割手密(2n = 40~128)种间杂交而来, 形成异源多倍体、非整倍体作物, 使得甘蔗栽培品种中80%~90%的染色体来源于热带种。开发热带种基因组SSR分子标记, 有助于甘蔗遗传多样性分析、分子标记辅助选择、遗传图谱的构建等。本研究基于热带种LA-purple的全基因组测序数据的255 398个预测基因序列(累计总长为1 029 222 285 bp), 利用Perl程序与生物信息学软件结合, 发掘SSR位点, 获得了153 150个SSR位点, 平均每1.67个基因有1个SSR位点, 其中二、三核苷酸重复基序分别为39 556个和50 072个, 占总SSR位点数的58.5%。在二核苷酸重复基序中, TA/AT所占比例最高, 占41.4%, CG/GC所占比例最低, 占4.6%; 在三核苷酸碱基重复基序中, TGT/ACA所占比例最高, 为15.6%。在TA/AT重复类型中选取100个基序重复次数在60~90之间的SSR位点, 进行引物设计与合成, 在12个甘蔗属材料中进行PCR扩增分析, 从中筛选出52对具有多态性SSR引物, 其中有27对引物在研究的2个甘蔗栽培品种间表现为多态。这些基因组SSR标记的开发, 不仅可以用于甘蔗栽培品种DNA指纹图谱分析, 而且为甘蔗属不同种的遗传图谱构建、遗传多样性分析和重要性状的遗传机制解析奠定基础, 为甘蔗分子育种研究提供重要支撑。

关键词: 甘蔗, 基因组, SSR, 标记开发, 多态性

Abstract:

Modern sugarcane cultivars (2n = 100-130) are derived from interspecific hybridization and backcross breeding between Saccharum officinarum (2n = 80) and Saccharum spontaneum (2n = 40-128), forming polyploid and aneuploid crops. The main components (80%-90%) of the sugarcane cultivars’ genome are originated from S. officinarum. The development and mining of genomic SSR molecular marker of S. officinarum, will benefit sugarcane genetic diversity analysis, molecular marker assisted selection, and construction of genetic maps. In this study, we explored the SSR loci from 255 398 predicted gene sequences (with a cumulative length of 1 029 222 285 bp) derived from the whole genome sequencing project of a S. officinarum clone LA-purple, by combining Perl program with bioinformatics software. A total of 153 150 SSR loci, with an average of 1.67 genes per SSR locus, were identified, of which 39 556 (25.8%) were dinucleotide repeat motifs and 50 072 (32.7%) were tri-nucleotide repeat motifs. Among the dinucleotide repeat motifs, TA/AT had the highest proportion, accounting for 41.4%, while CG/GC had the lowest proportion, accounting for only 4.6%. Among the trinucleotide repeat motifs, TGT/ACA had the highest proportion, accounting for 15.6%. One hundred SSR loci with 60-90 repeats of TA/AT motifs were selected and analyzed by PCR amplification in 12 representative Saccharum clones, of which 52 were polymorphic among the 12 clones and 27 were polymorphic between the tested two modern sugarcane cultivars. The genome-wide development of these gene-based SSR markers will not only facilitate the DNA fingerprinting analysis of sugarcane cultivars, but also help to construct the genetic maps, analyze the genetic diversity, study the genetic mechanism of important traits in Saccharum species, and provide important support to the molecular breeding in sugarcane.

Key words: sugarcane, genome sequence, SSR, marker development, polymorphism

表1

供试甘蔗种质资源名称及来源"

序号
Code		 名称
Name		 类型
Type		 倍性
Ploidy
1 57NG208 大茎野生种 S. robustum
2 NG77-004 大茎野生种 S. robustum
3 云南75-2-11 YN75-2-11 割手密 S. spontaneum 八倍体 Octoploid
4 福建89-1-1 FJ89-1-1 割手密 S. spontaneum 九倍体 Nonaploid
5 广东21 GD21 割手密 S. spontaneum 十倍体 Decaploid
6 贵州78-2-28 GZ78-2-28 割手密 S. spontaneum 十二倍体 Dodecaploid
7 黑车里本 Black cheribon 热带种 S. officinarum 八倍体 Octoploid
8 路达士 Loethers 热带种 S. officinarum 八倍体 Octoploid
9 克里斯塔林娜 Crystalina 热带种 S. officinarum 八倍体 Octoploid
10 拔地拉 Badila 热带种 S. officinarum 八倍体 Octoploid
11 桂糖35 GT35 栽培种 Saccharun hybrid
12 科5 K5 栽培种 Saccharun hybrid

Table 2

Frequency distribtiom of various types of simple sequence repent（SSR）motifs with different numbers of repeats among the genes of S.officinarum LA-purple genome"

图1

二核苷酸重复基序的次数分布"

图2

三核苷酸重复基序的次数分布"

图3

4对不同SSR引物在12个供试甘蔗属材料上的代表性PCR扩增图谱1: 57NG208; 2: NG77-004; 3: 云南75-2-11; 4: 福建89-1-1; 5: 广东21; 6: 贵州78-2-28; 7: 黑车里本; 8: 路达士; 9: 克里斯塔林娜; 10: 拔地拉; 11: 桂糖35; 12: 科5; M: 100 bp DNA ladder。"

Table 3

SSR primers information of sugarcane with amplified polymorphism"

图4

基于SSR分子标记的12个甘蔗属材料的UPGMA聚类分析"

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