中国农业科学 ›› 2019, Vol. 52 ›› Issue (15): 2567-2580.doi: 10.3864/j.issn.0578-1752.2019.15.002
王玉龙1,2,黄冰艳2,王思雨2,3,杜培2,齐飞艳2,房元瑾2,孙子淇2,郑峥2,董文召2,张新友1,2()
收稿日期:
2019-03-17
接受日期:
2019-05-15
出版日期:
2019-08-01
发布日期:
2019-08-06
通讯作者:
张新友
作者简介:
王玉龙,E-mail:基金资助:
WANG YuLong1,2,HUANG BingYan2,WANG SiYu2,3,DU Pei2,QI FeiYan2,FANG YuanJin2,SUN ZiQi2,ZHENG Zheng2,DONG WenZhao2,ZHANG XinYou1,2()
Received:
2019-03-17
Accepted:
2019-05-15
Online:
2019-08-01
Published:
2019-08-06
Contact:
XinYou ZHANG
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。
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. 四倍体野生种花生A.monticola全基因组SSR的开发与特征分析[J]. 中国农业科学, 2019, 52(15): 2567-2580.
WANG YuLong, HUANG BingYan, WANG SiYu, DU Pei, QI FeiYan, FANG YuanJin, SUN ZiQi, ZHENG Zheng, DONG WenZhao, ZHANG XinYou. Development and Characterization of Whole Genome SSR in Tetraploid Wild Peanut (Arachis monticola)[J]. Scientia Agricultura Sinica, 2019, 52(15): 2567-2580.
表1
供试材料的基本信息"
材料名称 Materials | 区组 Section | 种别 Species | 倍性 Ploidy | 基因组 Genomes |
---|---|---|---|---|
PI 219823 | 花生区组Section arachis | A.duranesis | 二倍体Diploid (2n=2x=20) | A |
PI 468322 | 花生区组Section arachis | A.ipaensis | 二倍体Diploid (2n=2x=20) | B |
PI 219824 | 花生区组Section arachis | A.moticola | 四倍体Tetraploid (2n=2x=40) | AB |
Tifrunner | 花生区组Section arachis | A.hypogaea | 四倍体Tetraploid (2n=2x=40) | AB |
表2
A. monticola、A. duranensis和A.ipaensis基因组SSR位点信息"
材料 Materials | 统计量 Statistic | 单核苷酸Mono-nucleotide | 2核苷酸Di-nucleotide | 3核苷酸Tri-nucleotide | 4核苷酸Tetra-nucleotide | 5核苷酸Penta-nucleotide | 6核苷酸Hexa-nucleotide | 总计 Total |
---|---|---|---|---|---|---|---|---|
A.duranesis (AA) | 数目Amount | 29689 | 47805 | 42529 | 4988 | 7658 | 2860 | 135529 |
类型比例 Type ratio (%) | 21.91 | 35.27 | 31.38 | 3.68 | 5.65 | 2.11 | 100.00 | |
平均距离 Average distance (kb) | 36.5 | 22.7 | 25.5 | 217.4 | 141.6 | 379.1 | 8.0 | |
A.ipaensis (BB) | 数目Amount | 57589 | 75334 | 45717 | 6736 | 11092 | 3489 | 199957 |
类型比例 Type ratio (%) | 28.80 | 37.68 | 22.86 | 3.37 | 5.55 | 1.74 | 100 | |
平均距离 Average distance (kb) | 23.5 | 18.0 | 29.6 | 201.0 | 122.1 | 388.0 | 6.7 | |
A.moticola (AABB) | 数目Amount | 316197 | 208181 | 113794 | 12170 | 19552 | 6984 | 676878 |
类型比例 Type ratio (%) | 46.71 | 30.76 | 16.81 | 1.80 | 2.89 | 1.03 | 100.00 | |
平均距离 Average distance (kb) | 8.3 | 12.6 | 23.0 | 215.2 | 133.9 | 375.0 | 3.8 | |
A.moticola (A-subgenome) | 数目Amount | 114570 | 81364 | 46531 | 4664 | 7445 | 2816 | 257390 |
类型比例 Type ratio (%) | 44.51 | 31.61 | 18.08 | 1.81 | 2.89 | 1.09 | 100.00 | |
平均距离 Average distance (kb) | 9.0 | 12.7 | 22.2 | 221.2 | 138.5 | 366.3 | 4.0 | |
A.moticola (B-subgenome) | 数目Amount | 174453 | 118620 | 62638 | 7032 | 11358 | 3912 | 378013 |
类型比例 Type ratio (%) | 46.15 | 31.38 | 16.57 | 1.86 | 3.00 | 1.03 | 100.00 | |
平均距离 Average distance (kb) | 8.5 | 12.5 | 23.7 | 210.7 | 130.5 | 378.8 | 3.9 |
表4
A.monticola A、B亚基因组特异性重复基元类型"
重复基元类型 Motif types | A亚基因组特异重复基元 Specific motifs in A-subgenome | B亚基因组特异重复基元 Specific motifs in B-subgenome |
---|---|---|
四核苷酸 Tetra-nucleotide | AACG/CGTT AGCC/CTGG AGCG/CGCT | AAGC/CTTG AGGC/CCTG |
五核苷酸 Penta-nucleotide | ACCTG/AGGTC AGCCT/AGGCT ACGGG/CCCGT AGCCC/CTGGG ACTGG/AGTCC ACGTC/ACGTG | AACGC/CGTTG AACTT/AAGTT AAGGC/CCTTG ACAGT/ACTGT ACATG/ATGTC ACCGT/ACGGT ACTAG/AGTCT AGCGC/CGCTG AGCGG/CCGCT ATGCC/ATGGC |
六核苷酸 Hexa-nucleotide | AAACCC/GGGTTT AAAGCT/AGCTTT AACAGG/CCTGTT AACCGT/ACGGTT AACGGT/ACCGTT AACGTC/ACGTTG AACGTT/AACGTT AACTCC/AGTTGG AAGTGT/ACACTT AATACC/ATTGGT AATTCG/AATTCG ACACCG/CGGTGT ACATCG/ATGTCG ACCCAG/CTGGGT ACCCAT/ATGGGT ACCTCG/AGGTCG ACGCCG/CGGCGT ACGGAG/CCGTCT ACTCAT/AGTATG ACTCGG/AGTCCG ACTGCC/AGTGGC AGCATG/ATGCTC AGCCCG/CGGGCT AGCCCT/AGGGCT AGCTAT/AGCTAT | AAACGC/CGTTTG AAAGGT/ACCTTT AACGAC/CGTTGT AACGAT/ATCGTT AACTCT/AGAGTT AACTGG/AGTTCC AAGACC/CTTGGT AAGCAT/ATGCTT AAGGCG/CCTTCG AAGGGC/CCCTTG AAGTAC/ACTTGT AATCCG/ATTCGG AATCGC/ATTGCG AATGAC/ATTGTC AATGCG/ATTCGC AATGGT/ACCATT ACAGAT/ATCTGT ACAGGC/CCTGTG ACAGGG/CCCTGT ACATAG/ATGTCT ACATGC/ATGTGC ACCATG/ATGGTC ACCCTG/AGGGTC ACCGAG/CGGTCT ACCTAG/AGGTCT ACGCGG/CCGCGT ACGCTC/AGCGTG ACGTCC/ACGTGG ACTAGG/AGTCCT ACTCTG/AGAGTC ACTGGC/AGTGCC ACTGGG/AGTCCC AGCCAT/ATGGCT AGGCCG/CCTCGG AGGGAT/ATCCCT ATCGGC/ATGCCG |
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