中国农业科学 ›› 2020, Vol. 53 ›› Issue (4): 695-706.doi: 10.3864/j.issn.0578-1752.2020.04.003
收稿日期:
2019-07-28
接受日期:
2019-10-21
出版日期:
2020-02-16
发布日期:
2020-03-09
联系方式:
徐志军,E-mail:zhijunxu1990@163.com。
基金资助:
ZhiJun XU1,Sheng ZHAO2,Lei XU1,XiaoWen HU1,DongSheng AN1,Yang LIU1()
Received:
2019-07-28
Accepted:
2019-10-21
Published:
2020-02-16
Online:
2020-03-09
摘要:
【目的】鉴定花生RNA-seq数据中的SSR位点,明确转录组中SSR位点的分布和结构特点,开发与花生基因相关联的SSR标记,为花生重要功能基因的挖掘、等位变异研究和分子标记辅助育种奠定基础。【方法】根据栽培种花生全生育期中22种不同类型的组织RNA-Seq数据,使用MISA软件分析SSR位点分布及特征,采用Primer 3设计基因关联的SSR引物,并利用电子PCR软件对引物的质量进行检测,随机合成38对引物,进行多态性检测。【结果】从52 280条转录本中共鉴定19 143个SSR位点,分布于14 084条转录本,发生频率为26.94%。重复单元类型为单核苷酸—五核苷酸,以单核苷酸和三核苷酸为重复单元的SSR位点数最多,分别占位点总数的39.24%和38.40%。各重复单元优势基序类型分别为A/T、AG/CT、AAG/CTT、AAAG/CTTT和AACAC/GTGTT,占所在重复单元中的比例分别为97.62%、72.01%、30.96%、24.59%和16.67%。重复单元的重复次数为5—47次,单个SSR位点的长度的分布范围为10—47 bp,基序长度主要集中在10—14 bp;复合SSR位点的长度范围为21—249 bp,以31—40 bp为主。鉴定的SSR位点中共有13 477个SSR位点可以进行引物设计,其中5 020条转录本序列对应到特定的基因,共包含5 859个可进行引物设计的SSR位点,这些SSR位点在A基因组和B基因组共20条染色体上不均匀分布,其中B03染色体上SSR位点最多,为484个。对特定基因SSR引物进行电子PCR检测,在A.duranensis、A.ipaensis、A.hypogaea基因组中有效扩增位点分别为4 468、4 929和10 188个,有效引物数分别为3 968(67.74%)、4 232(72.25%)和5 174(88.33%)对,在A. hypogaea基因组中,SSR引物扩增位点主要以2个位点为主,其中,有1 477对引物单位点扩增。根据SSR引物扩增位点在栽培种花生基因组中的位置信息绘制了SSR位点的物理图谱。在38对SSR引物中,共有35对(92.1%)SSR引物可以扩增出清晰的条带,其中,有11对(28.9%)SSR引物在2个品种间扩增出差异条带。【结论】鉴定了13 477个可进行标记开发的SSR位点,开发、检测了5 859个基因相关SSR标记,在栽培种花生基因组中具有较高的扩增效率,并构建了基因相关SSR位点的物理图谱。
徐志军, 赵胜, 徐磊, 胡小文, 安东升, 刘洋. 基于RNA-seq数据的栽培种花生SSR位点鉴定和标记开发[J]. 中国农业科学, 2020, 53(4): 695-706.
ZhiJun XU, Sheng ZHAO, Lei XU, XiaoWen HU, DongSheng AN, Yang LIU. Discovery of Microsatellite Markers from RNA-seq Data in Cultivated Peanut (Arachis hypogaea)[J]. Scientia Agricultura Sinica, 2020, 53(4): 695-706.
表1
栽培种花生RNA-seq SSR位点分布特征"
重复单元类型 Repeat unit type | 基序种类 Motif | SSR位点数量 SSR number | 比例 Ratio (%) | 分布频率 Distribution frequency (%) | 优势基序 Dominating motif | ||
---|---|---|---|---|---|---|---|
基序类型 Motif type | 数量 Number | 比例 Ratio (%) | |||||
单核苷酸 Mononucleotide | 4 | 7513 | 39.24 | 14.37 | A/T | 7334 | 97.62 |
二核苷酸 Dinucleotide | 12 | 3926 | 20.51 | 7.51 | AG/CT | 2827 | 72.01 |
三核苷酸 Trinucleotide | 60 | 7351 | 38.40 | 14.06 | AAG/CTT | 2276 | 30.96 |
四核苷酸 Tetranucleotide | 87 | 305 | 1.59 | 0.58 | AAAG/CTTT | 75 | 24.59 |
五核苷酸 Pentanucleotide | 39 | 48 | 0.25 | 0.09 | AACAC/GTGTT | 8 | 16.67 |
总计 Total | 202 | 19143 | 36.62 |
表2
栽培种花生SSR各重复单元重复次数及分布频率"
重复单元类型 Repeat unit type | 重复次数Repetition times | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | ≥15 | |
单核苷酸Mononucleotide | 2985 | 1519 | 943 | 657 | 468 | 941 | |||||
二核苷酸 Dinucleotide | 1446 | 896 | 640 | 475 | 340 | 115 | 14 | ||||
三核苷酸 Trinucleotide | 4352 | 2108 | 761 | 130 | |||||||
四核苷酸 Tetranucleotide | 246 | 59 | |||||||||
五核苷酸 Pentanucleotide | 48 | ||||||||||
总计 Total | 4646 | 3613 | 1657 | 770 | 475 | 3325 | 1634 | 957 | 657 | 468 | 941 |
分布频率 Distribution frequency(%) | 24.2 | 18.87 | 8.66 | 4.02 | 2.48 | 17.37 | 8.54 | 5.00 | 3.43 | 2.44 | 4.92 |
表3
可进行引物设计的特异基因SSR位点统计"
染色体 Chromosome | SSR位点数Number of SSR loci | 对应基因数Number of genes | 平均SSR位点密度 Average density of SSR loci | ||||
---|---|---|---|---|---|---|---|
单一位点 Single loci | 复合位点 Compound loci | 总计 Total | 单位点基因 Gene contain single loci | 多位点基因 Gene contain multiple loci | 总计 Total | ||
A01 | 252 | 9 | 261 | 136 | 37 | 217 | 1.21 |
A02 | 170 | 15 | 185 | 145 | 13 | 172 | 1.08 |
A03 | 380 | 17 | 397 | 220 | 53 | 335 | 1.19 |
A04 | 222 | 18 | 240 | 160 | 24 | 212 | 1.13 |
A05 | 314 | 13 | 327 | 179 | 48 | 277 | 1.18 |
A06 | 229 | 11 | 240 | 160 | 26 | 213 | 1.13 |
A07 | 175 | 11 | 186 | 110 | 24 | 160 | 1.16 |
A08 | 255 | 13 | 268 | 146 | 36 | 225 | 1.19 |
A09 | 265 | 16 | 281 | 168 | 35 | 242 | 1.16 |
A10 | 198 | 18 | 216 | 148 | 22 | 193 | 1.12 |
B01 | 273 | 16 | 289 | 177 | 34 | 250 | 1.16 |
B02 | 264 | 15 | 279 | 157 | 36 | 236 | 1.18 |
B03 | 451 | 33 | 484 | 328 | 48 | 430 | 1.13 |
B04 | 323 | 21 | 344 | 169 | 49 | 281 | 1.22 |
B05 | 291 | 15 | 306 | 201 | 33 | 270 | 1.13 |
B06 | 293 | 16 | 309 | 152 | 45 | 253 | 1.22 |
B07 | 281 | 20 | 301 | 190 | 31 | 261 | 1.15 |
B08 | 249 | 15 | 264 | 135 | 39 | 219 | 1.21 |
B09 | 339 | 17 | 356 | 188 | 52 | 298 | 1.19 |
B10 | 309 | 17 | 326 | 182 | 44 | 276 | 1.18 |
全基因组 Whole genome | 5533 | 326 | 5859 | 3451 | 729 | 5020 | 1.17 |
表4
基因关联SSR引物e-PCR扩增位点统计"
DNA模板 DNA template | 扩增位点 Amplified loci | 有效扩增位点 Effective amplified loci | 引物扩增位点统计 Primer amplified loci statistics | 有效引物数 Number of effective primer pairs | |||
---|---|---|---|---|---|---|---|
1 | 2 | 3 | >3 | ||||
A.duranensis | 4760 | 4468 (93.86%) | 3721 (93.75%) | 152 (3.83%) | 47 (1.18%) | 49 (1.23%) | 3969 (67.74%) |
A.ipaensis | 5264 | 4929 (93.64%) | 3866 (91.33%) | 249 (5.88%) | 52 (1.23%) | 66 (1.56%) | 4233 (72.25%) |
A.hypogaea | 10818 | 10188 (94.17%) | 1477 (28.54%) | 3221 (62.24%) | 252 (4.87%) | 225 (4.35%) | 5175 (88.33%) |
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