基于重测序的‘金兰柚’基因组InDel标记的开发及应用

doi:10.16420/j.issn.0513-353x.2021-0935

摘要/Abstract

摘要：

基于重测序进行金兰柚全基因组InDel标记开发。利用二代测序技术对‘金兰柚’进行全基因组重测序，运用BWA软件将测序得到的片段与参考基因组序列进行比对，利用严格的参数筛选杂合InDel，结合Primer 3.0设计扩增引物，进行PCR扩增并利用琼脂糖凝胶电泳验证，以重测序材料‘金兰柚’和47份柚品种检测其有效性。在全基因组范围内共筛选出81对扩增条带清晰的引物。筛选出的多态性标记涵盖了整个‘金兰柚’基因组，平均每条染色体上9个。选取的24对 InDel 标记可以将48份柚品种有效区分，最少使用2对引物就可将‘金兰柚’与其他47份柚品种完全区分。利用UPGMA构建48份柚品种的聚类树状图，在遗传相似系数为0.611的阈值处可将48个柚品种分为3大类群。本研究中的InDel标记带型简单易读，适宜柚DNA指纹图谱构建和品种鉴定，可为柚名优品种保护、原产地溯源管理等提供科学依据。

关键词: 柚, 重测序, InDel标记

Abstract:

This study developed a genome-wide InDel marker map of Jinlan pummelo（Citrus maxima）genome. The next generation sequencing（NGS）technology was applied to produce the deep resequencing data of the individual，and was mapped to the reference genome using BWA-MEM. The pipeline with strict parameters improved accuracy and effectiveness of heterozygous InDel markers. The primers designment was performed by using Primer 3.0. The agarose gel electrophoresis of polymerase chain reaction（PCR）product was used to validate the effectiveness of primers. A total of 81 effective paired primers were identified. The corresponding markers covered nearly the whole genome of Jinlan pummelo，with an average number of nine InDel markers per chromosome. Of these markers，24 markerswere succussed in distinguishing the 48 pummelo individuals，which showed that two paired markers could distinguish Jinlan pummelo and other 47 pummelo individuals. Finally，UPGMA was used to construct the phylogeny of 48 pummelo，which was divided into three groups at threshold 0.611 of genetic similarity coefficient. This study suggested that the genome-wide InDel markers developed using deep resequencing was suitable for the identification of the different germplasms of pummelo，and provides a basis in variety protection and origin traceability management.

Key words: pummelo, re-sequencing, InDel marker

中图分类号:

S666.3

汤雨晴, 杨惠栋, 闫承璞, 王斯妤, 王雨亭, 胡钟东, 朱方红. 基于重测序的‘金兰柚’基因组InDel标记的开发及应用[J]. 园艺学报, 2023, 50(1): 15-26.

TANG Yuqing, YANG Huidong, YAN Chengpu, WANG Siyu, WANG Yuting, HU Zhongdong, ZHU Fanghong. Development and Application of Jinlan Pummelo（Citrus maxima）InDel Markers Based on Genome Re-sequencing[J]. Acta Horticulturae Sinica, 2023, 50(1): 15-26.

图/表 7

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序号 No.	品种名 Name	采集地 Place	序号 No.	品种名 Name	采集地 Place
1	金堂绿柚 Jintang Lüyou	重庆Chongqing	25	桃溪蜜柚Taoxi Miyou	江西Jiangxi
2	北碚柚Beibeiyou	重庆Chongqing	26	金沙柚Jinshayou	江西Jiangxi
3	四季抛Sijipao	重庆Chongqing	27	金兰柚Jinlanyou	江西Jiangxi
4	梁沙柚Liangshayou	重庆Chongqing	28	凤凰柚Fenghuangyou	江西Jiangxi
5	暹罗柚Siamese Pummelo	重庆Chongqing	29	永嘉早香柚Yongjia Zaoxiangyou	江西Jiangxi
6	橘红彩柚Juhong Caiyou	重庆Chongqing	30	奥兰柚Oroblanco Grapefruit	广西Guangxi
7	彭县漳州柚Pengxian Zhangzhouyou	重庆Chongqing	31	橙柚Chengyou	广西Guangxi
8	纳西樱桃柚Naxi Yingtaoyou	重庆Chongqing	32	西施柚Xishiyou	广西Guangxi
9	安江石榴柚Anjiang Shiliuyou	重庆Chongqing	33	沙田柚Shatianyou	广西Guangxi
10	暹沙柚Xianshayou	重庆Chongqing	34	泰国晚金柚Thailand Wanjinyou	广西Guangxi
11	永安柚Yonganyou	湖南Hunan	35	云南脆香甜Yunnan Cuixiangtian	广西Guangxi
12	金香柚Jinxiangyou	湖南Hunan	36	泰国早2号Taiguozao 2	广西Guangxi
13	文旦柚Wendanyou	湖南Hunan	37	西双版纳1号Xishuangbanna 1	广西Guangxi
14	安江香柚Anjiang Xiangyou	湖南Hunan	38	鸡尾葡萄柚Cocktail Grapefruit	福建Fujian
15	冰糖柚Bingtangyou	湖南Hunan	39	星露比葡萄柚Star Ruby Grapefruit	福建Fujian
16	黔阳红心柚Qianyang Hongxinyou	湖南Hunan	40	麻步文旦柚Mabu Wendanyou	福建Fujian
17	左氏柚Zuoshiyou	湖南Hunan	41	泰国金丝柚Thailand Jinsiyou	福建Fujian
18	安江红心柚Anjiang Hongxinyou	湖南Hunan	42	尤溪橘柚Youxi Juyou	福建Fujian
19	土柚Tuyou	湖南Hunan	43	白肉琯溪蜜柚Bairou Guanximiyou	福建Fujian
20	缅甸柚Myanmar Pummelo	湖南Hunan	44	红肉琯溪蜜柚 Hongrou Guanximiyuo	福建Fujian
21	安江冰糖柚10号 Anjiang Bingtangyou 10	湖南Hunan	45	可口柚Kekouyou	广东Guangdong
22	华农红柚Huanong Hongyou	湖北Hubei	46	翡翠柚Feicuiyou	广东Guangdong
23	秭归蜜柚1号Zigui Miyou1	湖北Hubei	47	坪山柚Pingshanyou	广东Guangdong
24	秭归蜜柚2号Zigui Miyou2	湖北Hubei	48	火焰葡萄柚Flame Grapefruit	广东Guangdong

序号 No.	品种名 Name	采集地 Place	序号 No.	品种名 Name	采集地 Place
1	金堂绿柚 Jintang Lüyou	重庆Chongqing	25	桃溪蜜柚Taoxi Miyou	江西Jiangxi
2	北碚柚Beibeiyou	重庆Chongqing	26	金沙柚Jinshayou	江西Jiangxi
3	四季抛Sijipao	重庆Chongqing	27	金兰柚Jinlanyou	江西Jiangxi
4	梁沙柚Liangshayou	重庆Chongqing	28	凤凰柚Fenghuangyou	江西Jiangxi
5	暹罗柚Siamese Pummelo	重庆Chongqing	29	永嘉早香柚Yongjia Zaoxiangyou	江西Jiangxi
6	橘红彩柚Juhong Caiyou	重庆Chongqing	30	奥兰柚Oroblanco Grapefruit	广西Guangxi
7	彭县漳州柚Pengxian Zhangzhouyou	重庆Chongqing	31	橙柚Chengyou	广西Guangxi
8	纳西樱桃柚Naxi Yingtaoyou	重庆Chongqing	32	西施柚Xishiyou	广西Guangxi
9	安江石榴柚Anjiang Shiliuyou	重庆Chongqing	33	沙田柚Shatianyou	广西Guangxi
10	暹沙柚Xianshayou	重庆Chongqing	34	泰国晚金柚Thailand Wanjinyou	广西Guangxi
11	永安柚Yonganyou	湖南Hunan	35	云南脆香甜Yunnan Cuixiangtian	广西Guangxi
12	金香柚Jinxiangyou	湖南Hunan	36	泰国早2号Taiguozao 2	广西Guangxi
13	文旦柚Wendanyou	湖南Hunan	37	西双版纳1号Xishuangbanna 1	广西Guangxi
14	安江香柚Anjiang Xiangyou	湖南Hunan	38	鸡尾葡萄柚Cocktail Grapefruit	福建Fujian
15	冰糖柚Bingtangyou	湖南Hunan	39	星露比葡萄柚Star Ruby Grapefruit	福建Fujian
16	黔阳红心柚Qianyang Hongxinyou	湖南Hunan	40	麻步文旦柚Mabu Wendanyou	福建Fujian
17	左氏柚Zuoshiyou	湖南Hunan	41	泰国金丝柚Thailand Jinsiyou	福建Fujian
18	安江红心柚Anjiang Hongxinyou	湖南Hunan	42	尤溪橘柚Youxi Juyou	福建Fujian
19	土柚Tuyou	湖南Hunan	43	白肉琯溪蜜柚Bairou Guanximiyou	福建Fujian
20	缅甸柚Myanmar Pummelo	湖南Hunan	44	红肉琯溪蜜柚 Hongrou Guanximiyuo	福建Fujian
21	安江冰糖柚10号 Anjiang Bingtangyou 10	湖南Hunan	45	可口柚Kekouyou	广东Guangdong
22	华农红柚Huanong Hongyou	湖北Hubei	46	翡翠柚Feicuiyou	广东Guangdong
23	秭归蜜柚1号Zigui Miyou1	湖北Hubei	47	坪山柚Pingshanyou	广东Guangdong
24	秭归蜜柚2号Zigui Miyou2	湖北Hubei	48	火焰葡萄柚Flame Grapefruit	广东Guangdong

区域 Region	标记数量（比例） Number of markers（Percentage）	区域 Region	标记数量（比例） Number of markers（Percentage）
外显子 Exonic	7 311（1.67%）	内含子 Intronic	74 432（16.96%）
基因上游1 kb区域 Upstream	97 700（22.27%）	基因下游1 kb区域 Downstream	68 129（15.53%）
3'UTR	7 825（1.78%）	5'UTR	5 695（1.30%）
基因上游1 kb区域，同时也在另一基因下游1 kb区域 Up-and downstream	86 720（19.77%）	基因间区 Intergenic	90 938（20.73%）

区域 Region	标记数量（比例） Number of markers（Percentage）	区域 Region	标记数量（比例） Number of markers（Percentage）
外显子 Exonic	7 311（1.67%）	内含子 Intronic	74 432（16.96%）
基因上游1 kb区域 Upstream	97 700（22.27%）	基因下游1 kb区域 Downstream	68 129（15.53%）
3'UTR	7 825（1.78%）	5'UTR	5 695（1.30%）
基因上游1 kb区域，同时也在另一基因下游1 kb区域 Up-and downstream	86 720（19.77%）	基因间区 Intergenic	90 938（20.73%）

引物 Primer	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
M4	TAGTGGCAACCGTGTGACTG	GAAGTTCCTTTTGTCTTTGTGGA
M6	TGCATGCATGTAGACAGATGTAG	CATCTTGTCGATGATCTGGTG
M8	AAATCTTGTTGTTAGTCTAATGTGTTG	AACTAAATCTTCTCTTTGACCATCC
M12	GGCCACTCGAGTTGGAATAA	AGGAGTGGCCATGTGGATAG
M19	GCCAAACGGGTCTTTAATTG	CATGAACTTTTGTTGGCGACT
M26	TGGAACCCAATTGATATGTGTC	TTATTCCCTTTTGCCGTCAG
M29	TTTTTCGTCTTCTGCAAGTATTGA	TAAGTGATAATGGGCACAGTTCTT
M32	GTGTTCATTGTCAGCGTTATACA	TTTTCGAGGGACAATGTCGT
M38	GTGGGATTATTGAGACCCACT	GGACTGCTGGGAATTTGGTA
M40	ACTGGTGCTCCCACATTAGG	ATTTGCAACCGTATGGGAAC
M47	CCACATGGCGATATAACATGA	CATCGCCCTTCTGATTCCT
M51	TGTTCTGACCATATTTGACCAAC	TGGGAGGGCATAAGAGAGTG
M62	TCCGTCGTCAATGACTTCAA	TGCTGCAGTTTTCTTTTTCAA
M63	AATCTGAGTTTTCCGAAACTTTTA	TTCGAATGTAGATGGAGGTCAA
M65	TGTCCGCAACTTTTAAGACG	TGAATAGGGTGGCTTGCTTT
M67	CACAGCGACATGCTTTCATT	GCTCCTAGTGACGTGATGCTT
M69	ATCCGAGCATTCAATTTTGTTT	CGGAGGGCAATTTGAATTTT
M72	CGGCATAAACTCTTGCAACTT	TCACAGGAGCAATTATGTCGTC
M73	TTACCGAGAGCCCTCAAATG	CCATCCTTATCAAGGACATGAA
M74	GGTCAAAAGTACCCCTTCTCG	TGGCCAAACGAACTTGTATTC
M84	AGCTGCAGGTCGCAATATCT	GGTAAGGCGGTGTGTGAAGT
M86	GGCCACCATGTTTAAGGAAA	GCCATATTATTGTTGAGATACAACTTT
M90	GCGTGAGCTTGTATCCTTGC	CTCGGTCCTCTGGATTTTCA
M93	CAGAGTTGCGAATTTATCAGAAAG	GCGTTACAACACAAAGCTTGA