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作物学报 ›› 2018, Vol. 44 ›› Issue (12): 1809-1817.doi: 10.3724/SP.J.1006.2018.01809

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

高丹草杂种及其亲本转录组SNP及等位基因特异性表达分析

董婧1,逯晓萍1,*(),张坤明1,薛春雷1,张瑞霞2   

  1. 1内蒙古农业大学农学院, 内蒙古呼和浩特010019
    2呼和浩特市种子管理站, 内蒙古呼和浩特 010020
  • 收稿日期:2017-12-21 接受日期:2018-07-20 出版日期:2018-12-12 网络出版日期:2018-07-25
  • 通讯作者: 逯晓萍
  • 基金资助:
    本研究由国家自然科学基金项目(31160302);本研究由国家自然科学基金项目(31460375);呼和浩特市科技计划项目(2012-重-计-8-2)资助

Analysis of SNP and Allele-specific Expression in Transcriptome of Sorghum bicolor × Sorghum sudanense and Their Parents

Jing DONG1,Xiao-Ping LU1,*(),Kun-Ming ZHANG1,Chun-Lei XUE1,Rui-Xia ZHANG2   

  1. 1 Agronomy College, Inner Mongolia Agricultural University, Huhhot 010019, Inner Mongolia, China
    2 Huhhot Seed Management Station, Huhhot 010020, Inner Mongolia, China
  • Received:2017-12-21 Accepted:2018-07-20 Published:2018-12-12 Published online:2018-07-25
  • Contact: Xiao-Ping LU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31160302);This study was supported by the National Natural Science Foundation of China(31460375);the Science and Technology Plan Projects of Hohhot (2012-major-plans-8-2)

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摘要：

为探究高丹草杂种及其亲本间单核苷酸变异与其杂种优势形成的关系, 以高丹草杂种及其亲本的根、茎和叶组织为试验材料, 采用Illumina Hiseq 2000进行转录组测序。对平均长度达58 122 160 bp的各测序样品序列信息进行检测后, 均检测到不少于58 000个SNP位点, 位于基因内的SNP个数显著多于位于基因间的SNP个数, SNP发生频率为1/741 bp, 平均转换颠换比为1.00∶1.53。在所有变异类型中, C/T和G/A发生频率最高。经过筛选得到198 (21%)个极显著偏向性等位基因表达偏向性SNP, 其中65%偏向父本白壳苏丹草, 并且很多在白壳苏丹草中具有高水平基因表达的转录本, 在高丹草杂种中的等位基因表达也偏向白壳苏丹草。在3种组织中, 分别有79%、78%和82%转录本的2个亲本等位基因表现出相对平衡的表达水平, 说明与顺式作用相比, 反式作用可能更多地影响了等位基因的特异性表达。选择6个极显著偏向性等位基因表达偏向性SNP-unigene进行qRT-PCR验证, 这些基因的差异基因表达模式与RNA-Seq分析结果一致。本研究采用Illumina 测序技术研究等位基因表达, 为高丹草杂种优势分析提供了依据, 也为其他饲草作物的相关研究提供了理论参考。

关键词: 高丹草, 杂种优势, 转录组, 单核苷酸多态性, 功能注释

Abstract:

Taking root, stem and leaf tissues of S. sudanense hybrids and their parents as test materials, Illumina Hiseq 2000 was used to analyze the transcriptome to explore the relationship between single nucleotide variation and heterosis in the hybrids of Sorghum bicolor × S. sudanense and their parents. About 58 000 SNP loci were detected from the sequencing samples with an average length of 58 122 160 bp. The number of genic SNP was significantly more than that of intergenic SNP. The frequency of SNP was 1/741 bp, and the conversion ratio of the average conversion was 1.00:1.53. Among all the types of variation, C/T and G/A had the highest frequency. After screening, 198 (21%) extremely significant biased alleles were expressed in bias SNP, and 65% of them were biased towards paternal white shell S. sudanense, and many of the transcriptional copies with high level gene expression in the white shell S. sudanense were also expressed in the Sorghum bicolorss × S. sudanense hybrid. The two parental alleles with 79%, 78%, and 82% transcripts showed a stable level of expression in the three tissues. It is suggested that the trans-acting may affect the specific expression of the allele more than the cis-acting. Six highly-biased SNP-unigene alleles were selected for qRT-PCR validation. The differential gene expression pattern of these genes was consistent with that of RNA-Seq analysis. Illumina sequencing technology was used to study allelic expression in this study, provides a basis for heterosis analysis of Sorghum bicolor × S. sudanense and also a theoretical reference for related studies of other forage crops.

Key words: Sorghum bicolor × S. sudanense;, heterosis, transcriptomics, single nucleotide polymorphism, functional annotion

表 1

材料编号及名称"

编号
No.		 材料名称(组织)
Material name (tissue)		 编号
No.		 材料名称(组织)
Material name (tissue)
1 白壳苏丹草(根I) White shell Sudan grass (root I) 15 高粱11A (叶II) Sorghum 11A (leaf II)
2 白壳苏丹草(茎I) White shell Sudan grass (stem I) 16 F1 (根II) F1 (root II)
3 白壳苏丹草(叶I) White shell Sudan grass (leaf I) 17 F1 (茎II) F1 (stem II)
4 高粱11A (根I) Sorghum 11A (root I) 18 F1 (叶II) F1 (leaf II)
5 高粱11A (茎I) Sorghum 11A (stem I) 19 白壳苏丹草(根 III) White shell Sudan grass (root III)
6 高粱11A (叶I) Sorghum 11A (leaf I) 20 白壳苏丹草(茎 III) White shell Sudan grass (stem III)
7 F1 (根I) F1 (root I) 21 白壳苏丹草(叶 III) White shell Sudan grass (leaf III)
8 F1 (茎I) F1 (stem I) 22 高粱11A (根 III) Sorghum 11A (root III)
9 F1 (叶I) F1 (leaf I) 23 高粱11A (茎 III) Sorghum 11A (stem III)
10 白壳苏丹草(根II) White shell Sudan grass (root II) 24 高粱11A (叶 III) Sorghum 11A (leaf III)
11 白壳苏丹草(茎II) White shell Sudan grass (stem II) 25 F1 (根III) F1 (root III)
12 白壳苏丹草(叶II) White shell Sudan grass (leaf II) 26 F1 (茎III) F1 (stem III)
13 高粱11A (根II) Sorghum 11A (root II) 27 F1 (叶III) F1 (leaf III)
14 高粱11A (茎II) Sorghum 11A (stem II)

表2

SNP位点统计表"

材料编号
Material number		 总读长
Total reads		 SNP数
SNP number		 基因内SNP
Genic SNP		 基因间SNP
Intergenic SNP		 转换
Transition (%)		 颠换
Transversion (%)		 杂合型
Heterozygosity (%)
1 63 420 052 64 836 59 809 5027 60.19 39.81 10.11
2 59 951 742 71 331 66 793 4538 60.20 39.80 8.32
3 57 812 158 61 330 57 316 4014 60.69 39.31 9.28
4 50 219 934 62 583 57 875 4708 60.56 39.44 23.85
5 50 930 220 70 397 66 138 4259 60.25 39.75 21.43
6 47 779 664 58 033 54 415 3618 60.54 39.46 23.60
7 58 868 406 68 886 64 489 4397 60.09 39.91 22.36
8 64 250 174 65 248 61 364 3884 60.25 39.75 16.52
9 72 335 474 64 670 60 166 4504 60.60 39.40 16.67
10 53 344 576 99 040 92 057 6983 60.30 39.70 31.46
11 58 821 334 107 700 100 064 7636 60.35 39.65 31.87
12 61 991 178 84 379 78 540 5839 60.27 39.73 28.42
13 49 206 246 99 539 92 695 6844 60.46 39.54 34.03
14 54 672 342 98 391 91 657 6734 60.39 39.61 33.68
15 61 319 270 81 486 75 946 5540 60.36 39.64 30.89
16 56 744 190 109 658 102 075 7583 60.26 39.74 31.77
材料编号
Material number		 总读长
Total reads		 SNP数
SNP number		 基因内SNP
Genic SNP		 基因间SNP
Intergenic SNP		 转换
Transition (%)		 颠换
Transversion (%)		 杂合型
Heterozygosity (%)
17 59 733 160 95 723 89 741 5982 60.17 39.83 33.54
18 72 158 580 81 267 75 641 5626 60.34 39.66 29.79
19 78 016 508 95 205 87 506 7699 60.50 39.50 36.17
20 53 568 218 82 079 77 412 4667 60.47 39.53 30.25
21 49 779 342 73 905 69 659 4246 60.75 39.25 30.17
22 59 151 356 89 068 82 884 6184 60.36 39.64 38.21
23 50 348 066 75 276 70 970 4306 60.23 39.77 36.31
24 51 025 424 72 978 68 695 4283 60.48 39.52 36.28
25 55 095 668 81 945 76 448 5497 60.75 39.25 31.16
26 57 676 612 87 673 82 696 4977 60.37 39.63 31.16
27 61 078 424 78 864 74 174 4690 60.74 39.26 30.43

图1

SNP类型统计"

图2

SNP-unigene Nr比对结果 括号内为该类型unigene数及其在所有unigene中所占比例。"

图3

SNP-unigene COG比对结果"

图4

等位基因表达偏向性SNP"

图5

高丹草杂种中等位基因表达偏向性"

表3

10个等位基因表达偏向性SNPs"

编号
No.		 基因名称
Gene ID		 位置
Position		 ♂ F1 ♀ 深度
Depth		 染色体
Chr.
1 Sobic.001G191200 16936477 A A R 477 1
2 Sobic.001G293800 50043262 G R R 394 1
3 Sobic.002G215700 60749644 C C S 518 2
4 Sobic.002G215700 60750650 T T Y 476 2
5 Sobic.003G085700 7390162 T T W 353 3
6 Sobic.003G206800 53754265 T Y Y 361 3
7 Sobic.003G314500 64282541 T T K 492 3
8 Sobic.003G314500 64279654 C C Y 419 3
9 Sobic.004G225100 56832538 C C Y 476 4
10 Sobic.004G253000 59205755 T T Y 335 4

图6

高丹草杂种及其亲本3个组织中各基因表达量"

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