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四倍体野生种花生A.monticola全基因组SSR的开发与特征分析
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  • 英文篇名:Development and Characterization of Whole Genome SSR in Tetraploid Wild Peanut(Arachis monticola)
  • 作者:王玉龙 ; 黄冰艳 ; 王思雨 ; 杜培 ; 齐飞艳 ; 房元瑾 ; 孙子淇 ; 郑峥 ; 董文召 ; 张新友
  • 英文作者:WANG YuLong;HUANG BingYan;WANG SiYu;DU Pei;QI FeiYan;FANG YuanJin;SUN ZiQi;ZHENG Zheng;DONG WenZhao;ZHANG XinYou;College of Agriculture, Henan University of Science and Technology;Industrial Crops Research Institute, Henan Academy of Agricultural Sciences/Key Laboratory of Oil Crops in Huanghuaihai Plain/Henan Provincial Key Laboratory for Oil Crops Improvement;School of Life Sciences, Zhengzhou University;
  • 关键词:花生 ; Arachis ; monticola ; 全基因组序列 ; SSR位点 ; 基序
  • 英文关键词:peanut;;Arachis monticola;;whole genome sequence;;SSR locus;;motif
  • 中文刊名:ZNYK
  • 英文刊名:Scientia Agricultura Sinica
  • 机构:河南科技大学农学院;河南省农业科学院经济作物研究所/农业部黄淮海油料作物重点实验室/河南省油料作物遗传改良重点实验室;郑州大学生命科学学院;
  • 出版日期:2019-08-01
  • 出版单位:中国农业科学
  • 年:2019
  • 期:v.52
  • 基金:国家花生产业技术体系(CARS-13);; 河南省重大科技专项(16110011100);; 河南省花生产业技术体系(2012-5)
  • 语种:中文;
  • 页:ZNYK201915002
  • 页数:19
  • CN:15
  • ISSN:11-1328/S
  • 分类号:15-33
摘要
【目的】通过对四倍体野生种花生Arachis monticola(AABB,2n=4x=40)全基因组SSR位点搜索,研究其全基因组SSR分布特征及规律,开发并验证其全基因组SSR引物,为花生属植物遗传进化分析及重要性状分子标记开发提供依据。【方法】在华大基因GigaScience数据库下载A.monticola全基因组序列,并利用生物信息学软件MISA进行SSR位点搜索,Primer 3进行引物设计,通过电子PCR进行单位点SSR分析,并随机合成100对SSR引物验证通用性。【结果】SSR在四倍体野生种花生A.monticola基因组上共搜索到SSR位点676 878个,平均每3.8 kb就会出现一个SSR,分布于5 127条scaffold中,单核苷酸至六核苷酸均有分布,且数量上差异较大,以单碱基、二碱基、三碱基为主,三者占SSR总数的94.28%,其中单碱基重复数量最多,占46.71%,密度最高;六核苷酸重复数目最少,分布最稀疏。大多数SSR分布在基因间区,基因区SSR多分布于内含子区域;全基因组共鉴定出395个不同的重复基元,其中A亚基因组342种,B亚基因组356种;A/T是最丰富的重复基元;在1—6个核苷酸的重复基元中,数量最多的依次是A/T、AT/AT、AAT/ATT,AAAT/ATTT、AAAAT/ATTTT、AAAAAT/ATTTTT;整体来看,重复基元的重复次数多集中在50次以内,不同类型的motif的重复次数差异很大;同一种类型重复基元的SSR位点,随着motif重复次数增加,SSR的数量逐渐降低;B03染色体上SSR数量最多,A08染色体中SSR密度最高。A.monticola全基因组SSR比A.duranensis、A.ipaensis基因组SSR数量多,密度也更高,A.monticola单核苷酸重复最丰富,2个野生种二核苷酸数量最多。共设计出SSR引物192 303对,单位点SSR标记检出率50.35%,单点SSR标记在基因组上的分布呈现两端密集,中间稀疏的特点;随机合成的100对引物中,90对能在A. monticola中扩增出稳定清晰的条带,且在4份不同的花生基因组DNA中扩增目的条带表现出不同的特点。【结论】A.monticola基因组内SSR种类和数量丰富,单核苷酸至六核苷酸均有分布,单核苷酸重复基元数量最多,且最密,六核苷酸重复基元数量最少,出现频率最低,不同重复基元频数高低与核苷酸数量没有严格相关性,SSR多分布在基因间区,基因区内含子区域SSR数量最多;A.monticola A、B亚基因组具有其各自特异的重复基元类型;单个类型重复基元数量最多的均为AT富集的重复基元,而GC富集的重复基元相对较少;同一种类型重复基元的SSR位点,随着motif重复次数增加,SSR的数量逐渐降低;A.monticola全基因组SSR较2个二倍体野生种数量更多,密度也更高且重复基元分布规律不同;经过初步验证,开发的SSR引物在4份花生材料中表现出部分通用性。
        【Objective】We aimed to identify simple sequence repeat(SSR) loci throughout the genome of tetraploid wild peanut Arachis monticola(AABB, 2 n = 4 x = 40), to identify their distribution characteristics, and to develop and validate SSR primers. These markers have potential uses in genetic evolution analyses and in the development of molecular markers for important traits in peanut.【Method】Using the bioinformatics software MISA, we searched for SSR loci in the whole genome sequence of A.monticola, which was downloaded from the GigaScience database of the BGI. One hundred SSR loci were randomly selected and primers were designed and synthesized. The primers were used to amplify products from four different Arachis genomes, and the products were analyzed by polyacrylamide gel electrophoresis(PAGE).【Result】 A total of 676 878 SSRs were found in the genome of tetraploid wild peanut A. monticola in 5 127 scaffolds(average, one SSR per 3.8 kb). The SSRs ranged from single nucleotides to hexanucleotides. Single nucleotide SSRs were significantly more abundant than hexanucleotide SSRs. Single, double, and triple nucleotide SSRs were predominant, accounting for 94.28% of all the SSRs. Single nucleotide SSRs accounted for the largest proportion of total SSRs(46.71%) and showed the highest density. Hexanucleotide SSRs accounted for the smallest proportion and showed the sparsest density. Most SSRs were located in intergenic regions, and most of the SSRs in gene sequences were located in introns. A total of 395 different repeat motifs were identified in the whole genome, of which 342 were in the A-subgenome and 356 were in the B-subgenome. The most abundant repeat motif was A/T. The most abundant repeat motifs for SSRs with 1–6 nucleotides were A/T, AT/AT, AAT/ATT, AAAT/ATTT, AAAAT/ATTTT, and AAAAAT/ATTTTT, respectively. There were less than 50 of each type of SSR repeat motif, but the number of each type of SSR motif varied greatly. The number of each type of SSRs repeat motif decreased with increasing number of nucleotides in the motif. Chromosome B03 had the most SSRs, and chromosome A08 showed the highest density of SSRs. We designed 192 303 pairs of SSR primers, and the detection rate of single-locus SSR markers was50.35%. The distribution of SSR markers in the genome was dense at both ends and sparse in the middle. Among the 100 synthesized primer pairs, 90 pairs amplified stable and clear bands from A. monticola genomic DNA. The bands amplified from four different peanut genomic DNAs showed different characteristics. 【Conclusion】The A. monticola genome was rich in SSRs ranging from single nucleotides to hexanucleotides. Single nucleotide repeats were the most abundant and densely distributed, and hexanucleotides showed the lowest frequency and the sparsest distribution. There was no strict correlation between the frequency of different repeats and the repeat type. The A-subgenome and B-subgenome had their own specific SSRs. The AT-enriched repeat motifs were the most abundant, while GC-enriched repeat motifs showed much lower frequencies. The number of SSRs with the same type of repeat motif decreased with increasing numbers of nucleotides in the motif. Compared with the genomes of the two diploid wild species, the tetraploid genome of A. monticola had more SSRs, a higher density of SSRs, and a different SSR distribution pattern. Preliminary validation analyses showed that the SSR primers designed in this study shared certain universal properties among four Arachis genomes.
引文
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