基于转录组的阿拉善地区多枝柽柳多态Genic-SSRs的识别与开发

doi:10.11983/CBB20185

植物学报 ›› 2021, Vol. 56 ›› Issue (4): 433-442.DOI: 10.11983/CBB20185

基于转录组的阿拉善地区多枝柽柳多态Genic-SSRs的识别与开发

张雅楠¹, 黄蕾¹, 李佳彬¹, 张雷²^,³, 党振华¹^,^*()

¹内蒙古大学生态与环境学院, 蒙古高原生态与资源利用教育部重点实验室/内蒙古草地生态学重点实验室, 呼和浩特 010021
²内蒙古自治区林业科学研究院, 呼和浩特 010010
³内蒙古大青山森林生态系统定位观测研究站, 呼和浩特 010010

收稿日期:2020-11-20 接受日期:2021-05-27 出版日期:2021-07-01 发布日期:2021-06-30
通讯作者: 党振华
作者简介:*E-mail: zhdang_1982@aliyun.com
基金资助:
国家林业和草原局林业科技发展项目(KJZXSA2019047);内蒙古自然科学基金(2020MS03005)

Identification and Development of Polymorphic Genic-SSRs in Tamarix ramosissima in Alxa Region Based on Transcriptome

Yanan Zhang¹, Lei Huang¹, Jiabin Li¹, Lei Zhang²^,³, Zhenhua Dang¹^,^*()

¹Ministry of Education Key Laboratory of Ecology and Resource Use of the Mongolian Plateau/Inner Mongolia Key Laboratory of Grassland Ecology, School of Ecology and Environment, Inner Mongolia University, Hohhot 010021, China
²Inner Mongolia Academy of Forestry Sciences, Hohhot 010010, China
³Daqing Mountains Forest Ecosystem Research Station, Hohhot 010010, China

Received:2020-11-20 Accepted:2021-05-27 Online:2021-07-01 Published:2021-06-30
Contact: Zhenhua Dang

摘要/Abstract

摘要： 基因内部的简单重复序列(Genic-SSR)可在植物适应环境变化中发挥重要作用。通过对阿拉善5个样点多枝柽柳(Tamarix ramosissima)的转录组进行测序、组装和比较, 经CandiSSR软件分析, 共鉴定出代表157个基序类型的1 185个多态性Genic-SSR位点, 位于1 123个转录本中。其中, 三核苷酸重复序列(596, 50.30%)最多, 其次是二核苷酸重复序列(486, 41.01%)。定位分析表明, 分别有411、239和163个Genic-SSRs位于相应基因的CDSs、5′UTRs和3′UTRs; 78.47%的三核苷酸重复SSRs位于基因的CDSs, 94.07%的二核苷酸重复SSRs位于基因的UTRs; 在CDS中, AGC/GCT、AGG/CCT、AAG/CTT、CCG/CGG和ATC/GAT重复相对丰富, 占所有Genic-SSRs的64.48%; AG/CT和AT/AT是UTRs中最丰富的重复类型, 占UTR中所有Genic-SSRs的55.22%。功能注释表明, 含有多态Genic-SSRs的基因可注释到多个与植物逆境应答相关的GO条目和KEGG通路中。在随机选取的15个多态性SSR位点中, 14个被成功扩增, 共检测到64个等位基因。遗传多态性估算表明, 它们的期望杂合度(He)、观测杂合度(Ho)和多态性信息含量(PIC)平均值分别为0.553、0.421和0.493, 均属于中、高多态SSR标记, 表明利用RNA-seq技术开发SSR标记可行。

关键词: Genic-SSR, 多态性, 转录组测序, 多枝柽柳

Abstract: Simple sequence repeats located in gene transcribed regions (Genic-SSR) can play important roles in plant adaptation to environmental changes. In this study, the transcriptomes of Tamarix ramosissima from five different locations in Alax were sequenced, assembled, and compared. By using CandiSSR software, a total of 1 185 polymorphic Genic-SSRs representing 157 motif types were identified in 1 123 transcripts. Among them, the trinucleotide repeats (596, 50.30%) were the most abundant, followed by dinucleotide repeats (486, 41.01%). Location analysis showed that 411, 239, and 163 Genic-SSRs were located in CDSs, 5′UTRs, and 3′UTRs of the relevant transcripts, respectively; 78.47% of the trinucleotide SSRs were located in CDSs, and 94.07% of the dinucleotide SSRs were located in UTRs. Among SSRs distributed in CDSs, AGC/GCT, AGG/CCT, AAG/CTT, CCG/CGG, and ATC/GAT were relatively abundant, accounting for 64.48% of all the Genic-SSRs; AG/CT and AT/AT were the most abundant repeat types in UTRs, which together account for 55.22% of all the Genic-SSRs in UTRs. Functional annotation showed that polymorphic Genic-SSRs containing genes enriched in a wide range of GO terms and KEGG pathways that highly related to stress response in T. ramosissima. Of the 15 randomly selected Genic-SSRs, 14 were successfully amplified by using polymerase chain reaction technology and 64 alleles were found in these SSR loci. Genetic polymorphism estimation showed that the mean of expected and observed heterozygosity (He, Ho), polymorphism information content (PIC) of these SSRs were 0.553, 0.421, and 0.493, respectively, demonstrating the feasibility of developing SSR markers by RNA-seq.

Key words: Genic-SSR, polymorphism, RNA-seq, Tamarix ramosissima

张雅楠, 黄蕾, 李佳彬, 张雷, 党振华. 基于转录组的阿拉善地区多枝柽柳多态Genic-SSRs的识别与开发. 植物学报, 2021, 56(4): 433-442.

Yanan Zhang, Lei Huang, Jiabin Li, Lei Zhang, Zhenhua Dang. Identification and Development of Polymorphic Genic-SSRs in Tamarix ramosissima in Alxa Region Based on Transcriptome. Chinese Bulletin of Botany, 2021, 56(4): 433-442.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB20185

https://www.chinbullbotany.com/CN/Y2021/V56/I4/433

图/表 6

表1 采样点具体信息

Table 1 Detailed information of the sampling locations

Population	Longitude (E)	Latitude (N)	Altitude (m)	Habitats
TR1	101°00′06″	41°52′16″	939	Wetland
TR2	101°11′44″	41°57′27″	927	Sandy land
TR3	101°03′36″	42°07′02″	915	Wetland
TR4	101°12′26″	41°58′45″	924	Wetland
TR5	101°16′25″	42°01′55″	921	Wetland

表2 测序与组装结果统计

Table 2 Statistics of sequencing and assembly results

Sample	CR (No.)	Q30 (%)	GC (%)	Ug (No.)	ML (bp)	N50 (bp)
TR1	47893646	93.12	41.95	37790	1053.09	1953
TR2	43861774	93.06	41.96	35818	1083.87	1983
TR3	46194046	93.04	42.33	39252	995.67	1812
TR4	44817976	93.17	42.87	32175	1099.48	1899
TR5	44190698	92.92	42.49	34364	1082.91	1930
All-unigene				81728	823.46	1364

图1 基因内部简单重复序列(Genic-SSRs)的分布 x轴表示多态性Genic-SSRs的分布和motif序列类型; y轴表示Genic-SSRs的数目。

Figure 1 Distribution of the identified Genic-SSRs The x-axis represents the distribution and motif types of polymorphic Genic-SSRs; The y-axis represents the number of Genic- SSRs.

图2 含Genic-SSR序列基因的GO和KEGG富集分析 (A) GO分析, 3列气泡分别代表5′UTRs、CDSs和3′UTRs含有Genic-SSRs基因的GO富集分析结果, GO条目的基因数目由气泡大小表示; 每个基因区域显示基因数目≥10的GO条目; (B) KEGG富集分析, x轴表示5′UTRs、CDSs和3′UTRs含有Genic-SSRs基因富集的代谢通路; y轴表示富集在KEGG通路中的基因数目。

Figure 2 GO and KEGG enrichment of the Genic-SSR-containing sequences (A) GO analysis, the three columns of the bubbles represent GO enrichment analysis of transcripts that containing Genic-SSRs in the 5′UTRs, CDSs and 3′UTRs, respectively, and the number of unigenes assigned to each term is indicated by the size of each bubble; GO terms that contained unigenes more than or equal to ten in one of the gene regions are shown; (B) KEGG enrichment analysis, the x-axis indicates the enriched pathways assigned to the 5°UTRs, CDSs and 3°UTRs Genic-SSR-containing sequences; the y-axis represents the number of unigenes enriched in KEGG pathways.

图3 多态Genic-SSR序列比对及毛细管电泳分析 (A) TR1-TR5转录组中多态Genic-SSR 104关联基因序列的比对结果; (B) 图(A)中多态Genic-SSRs的毛细管电泳结果

Figure 3 Multiple sequence alignment of polymorphic Genic-SSR and capillary electrophoresis analysis (A) Multiple sequence alignment for five transcripts assembled in the TR1-TR5 transcriptomes that correspond to polymorphic Genic-SSR 104; (B) The polymorphism of Genic-SSRs in (A) revealed by capillary electrophoresis

表3 14个多态Genic-SSRs标记的基本信息及遗传学参数

Table 3 Basic information and genetic parameters of the 14 polymorphic Genic-SSRs

SSR ID		PS (5′→3′)	RM	Tm (°C)	AS (bp)	Na	PIC	Ho	He
104		F: CAAGGAGGAGCTGTCGTCTG	AG	59.9	123-139	8	0.516	0.286	0.558
	R: GCAGACACGAAGTTTGCGAT		AG	59.9	123-139	8	0.516	0.286
221		F: TGAAGCAGCTGTGTTGGTGA	AT	53.4	158-162	3	0.562	0.321	0.645
	R: TCCTCCTCGATTCCCTACTGA		AT	53.4	158-162	3	0.562	0.321
300		F: GAAGGGTTTGGGTGTTTTCAGA	AT	59.5	155-161	4	0.641	0.643	0.708
	R: AAAACGCACCCTCTCAGCAG		AT	59.5	155-161	4	0.641	0.643
392		F: CGCAACAAGCACAACATCCA	CAG	57.9	100-109	4	0.407	0.608	0.489
	R: GTTAACCGGTCGCACAACTG		CAG	57.9	100-109	4	0.407	0.608
438		F: GGCACCGATACACAAGGACA	CCTT	55.4	192-204	4	0.512	0.500	0.593
	R: CCCACAGGCTTACACCCATT		CCTT	55.4	192-204	4	0.512	0.500
441		F: GCCACCGCCACATTTATCTT	CGAG	50.8	106-122	5	0.520	0.286	0.570
	R: GGAGCTTGACAGGTACAGCA		CGAG	50.8	106-122	5	0.520	0.286
563		F: TCCTGTGCAACGAACTGAGT	GA	51.7	176-186	6	0.593	0.536	0.666
	R: GGGTTTAGCCATGGGTGACA		GA	51.7	176-186	6	0.593	0.536
668		F: ATGGCGATGATGGAGCAACA	GAG	59.9	146-173	7	0.711	0.429	0.758
	R: AGGGATTGGCGGAAGTGAAG		GAG	59.9	146-173	7	0.711	0.429
670		F: GAAGACACAGCACCAAGGGA	GAG	56.7	186-201	5	0.546	0.714	0.627
	R: CGTCTTCCCCAAGTCCGATC		GAG	56.7	186-201	5	0.546	0.714
947		F: TCCCCACACGTAATCCCTTC	TC	50.8	139-149	5	0.445	0.607	0.488
	R: GCGGATGGAAGGAGAAGAGG		TC	50.8	139-149	5	0.445	0.607
1024		F: TGCGCATTTCTTGATTGCCA	TCT	52.1	168-180	5	0.476	0.464	0.516
	R: GGGTTGATGCGGCTTGATTG		TCT	52.1	168-180	5	0.476	0.464
1053		F: GTCGACACTGCAAGCATCAC	TG	56.7	202-204	2	0.293	0.036	0.363
	R: GCACGTACGAGCACATCTCT		TG	56.7	202-204	2	0.293	0.036
1093		F: GAATAGTGGTGGCGGGAGTC	TGC	54.7	117-126	2	0.195	0.250	0.223
	R: GCTTGGCATCGTACCCCTAT		TGC	54.7	117-126	2	0.195	0.250
1114		F: GGTCCTCAGTGTGGCCATAG	TGG	54.1	156-171	4	0.487	0.214	0.543
	R: GTGTTAGAGATGGCGGGCAA		TGG	54.1	156-171	4	0.487	0.214
Mean						4.5	0.493	0.421	0.553

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基于转录组的阿拉善地区多枝柽柳多态Genic-SSRs的识别与开发

Identification and Development of Polymorphic Genic-SSRs in Tamarix ramosissima in Alxa Region Based on Transcriptome

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