Development of novel EST-SSR markers in pineapple (<i>Ananas comosus</i>) by data mining from public EST sequences

CHEN Junhao; LU Yang; NI Yanjie; ZHANG Zhijun; ZHONG Yaping; BAO Jingjing; LUO Shuping

doi:10.11833/j.issn.2095-0756.2017.02.018

Volume 34 Issue 2

Mar. 2017

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CHEN Junhao, LU Yang, NI Yanjie, ZHANG Zhijun, ZHONG Yaping, BAO Jingjing, LUO Shuping. Development of novel EST-SSR markers in pineapple (Ananas comosus) by data mining from public EST sequences[J]. Journal of Zhejiang A&F University, 2017, 34(2): 332-339. doi: 10.11833/j.issn.2095-0756.2017.02.018

Citation:

CHEN Junhao, LU Yang, NI Yanjie, ZHANG Zhijun, ZHONG Yaping, BAO Jingjing, LUO Shuping. Development of novel EST-SSR markers in pineapple (Ananas comosus) by data mining from public EST sequences[J]. Journal of Zhejiang A&F University, 2017, 34(2): 332-339. doi: 10.11833/j.issn.2095-0756.2017.02.018

Development of novel EST-SSR markers in pineapple (Ananas comosus) by data mining from public EST sequences

doi: 10.11833/j.issn.2095-0756.2017.02.018

1.
The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin'an 311300, Zhejiang, China
2.
College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China

Received Date: 2016-03-16
Rev Recd Date: 2016-05-07
Publish Date: 2017-04-20

Abstract

Expressed sequence tags (ESTs) are used for microsatellite development. EST derived from simple sequence repeats (EST-SSRs), one of the most common new microsatellite markers, are more likely to be embedded in functional gene sequences, to be less costly and time effective, and may provide more information. In the present study, analysis of 5 659 publicly available expressed sequence tags of pineapple (Ananas comosus) was conducted with the Webstat online program. Results of 636 (SSRs), identified electronically in 617 ESTs, showed that the average density was one SSR per 7.39 kb of ESTs screened. Dinucleotide (42.61%), trinucleotide (29.25%), and hexanucleotide (20.13%) repeats were the dominant types among the obtained motifs. The least frequent was tetranucleotide repeats (3.46%). TC/AG was most abundant in the dinucleotide motif. Of the 30 functional pairs, 27 primer pairs showed polymorphisms by polymerase chain reaction (PCR) amplifications. Thus, the EST-SSRs developed here with the first large-scale development of SSR markers in pineapple have the potential to be used for development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping, and finger printing cultivars.
- molecular biology,
- Ananas comosus,
- ESTs,
- SSR

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References

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[2]	ZHAO Shengjie, LIU Wenge, YAN Zhihong, et al. Analysis of SSR information in EST resource of watermelon (Citrullus lanatus)[J]. Chin Veget, 2009, 1(24):47-51.
[3]	ZHANG Jun'e. Analysis of microsatellites derived from apple ESTs[J]. J Guangxi Agric Biol Sci, 2008, 27(4):378-380.
[4]	WANG Yanfang, ZHAO Yanhong, WANG Aiyun, et al. Analysis of SSR information in EST resource of Lentinus edodes[J]. Food Sci, 2010, 31(1):137-140.
[5]	CORDEIRO G M, CASU R, MCINTYRE C L, et al. Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum[J]. Plant Sci, 2001, 160(6):1115-1123.
[6]	CHEN Junfang, REN Zhenglong, GAO Lifeng, et al. Developing new SSR markers from EST of wheat[J]. Acta Agron Sin, 2005, 31(2):154-158.
[7]	ZHANG Huiyun, WU Qingsong, SUN Weisheng, et al. Method of modified CTAB for DNA extraction from pineapple[J]. Southwest China J Agric Sci, 2009, 22(2):440-443.
[8]	TEMNYKH S, DECLERCK G, LUKASHOVA A, et al. Computational and experimental analysis of microsatellites in rice (Oryza sativa L.):frequency, length variation, transposon associations, and genetic marker potential[J]. Genome Res, 2001, 11(8):1441-1452.
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沈阳化工大学材料科学与工程学院沈阳 110142

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菠萝Ananas comosus原产巴西，为热带多年生草本植物，16世纪时传入中国，有70多个品种，岭南四大名果之一。菠萝含有大量的果糖，葡萄糖，维生素A，维生素B，维生素C，磷，柠檬酸和蛋白酶等营养物质。其果味甘性温，具有解暑止渴、消食止泻之功，为夏令医食兼优的时令佳果。另外，菠萝皮中富含菠萝酶，有丰富的药用价值，长期食用菠萝皮，心脑血管，糖尿病发病率显著降低，并有一定的抗癌效果。近年来分子生物学的发展以及各种分子标记技术不断出现，使得植物遗传分析研究得以迅速发展，其中以简单重复序列（simple sequence repeat，SSR）标记在植物遗传研究上应用最为广泛。随着测序技术成本的降低，GenBank中大量的植物表达序列标签（expressed sequence tag，EST）数据为SSR分子标记的开发提供了新的途径。EST-SSR除具一般SSR分子标记特点外，还有信息量大，通用性好，开发简单、快捷、成本低等^[1]的特殊优势。目前，许多作物^[2-5]已开发大量的EST-SSR，并应用于遗传作图、遗传多样性等^[6]研究上，但在菠萝栽培种上至今尚未见从EST中开发SSR的相关报道。本研究对现有菠萝EST中SSR信息进行全面分析，以明确菠萝EST-SSR发生频率和特点，为进一步建立EST-SSR标记并探索其在菠萝研究中的遗传作图、育种材料评价、品种鉴定等的应用奠定基础。

1. 材料与方法

1.1. 材料

采集菠萝品种‘巴厘’Ananas comosus ‘Comte de Paris’的幼嫩叶片于-20 ℃保存。

1.2. 菠萝EST的获得及EST-SSR序列查找

从美国生物技术中心（NCBI）的植物基因组数据库（http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList.html）共搜索到5 659条菠萝的EST序列。应用Websat（http://wsmartins.net/websat/）在线程序搜索EST-SSR。搜索的标准为：二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复序列的重复次数分别大于或等于9，6，5，4和3。

1.3. EST-SSR引物设计

利用Primer Premier 3.0在线程序对包含有SSR的EST设计引物，引物设计的原则为EST序列长度大于100 bp，SSR序列的开始和结束位置分别距5′和3′端不少于20 bp。引物设计的主要参数为：引物长18~27 bp，最适为22 bp；引物退火温度T_m值57~60 ℃，上游与下游引物的T_m值相差±1 ℃；PCR预期产物长100~400 bp；尽量避免引物二聚体，发夹结构和错配等。按重复类型的比例挑选30对引物由上海生物工程技术服务有限公司合成。

1.4. EST数据冗余分析

利用VecScreen（http://www.ncbi.nlm.nih.gov/VecScreen）及RepeatMasker（http://www.repeatmasker.org）去除载体污染和重复序列，对于那些能设计出引物的EST序列，最后通过T_m值和引物序列比对进一步删除冗余序列。

1.5. DNA提取

采用改良十六烷基三甲基溴化铵法（CTAB）^[7]提取菠萝的基因组DNA。

1.6. PCR扩增、电泳检测

PCR反应体系（25 μL）：10×缓冲液2.5 μL，镁离子（Mg²⁺）（25.0 mmol·L^-1）1.5 μL，dNTP（10.0 mmol·L^-1）0.5 μL，引物（100.0 μmol·L^-1）1.0 μL，Taq酶（5.0×16.67 nkat）0.2 μL，模板DNA（20.0 mg·L^-1）3.0 μL。

反应程序为：94 ℃预变性5 min，94 ℃变性30 s，51 ℃退火40 s，72 ℃延伸50 s，38个循环，72 ℃终延伸7 min，8 ℃保存。

PCR产物用质量分数为6%非变性聚丙烯酰胺凝胶电泳进行分离检测，120 V电压电泳1.5 h后，采用银染法染色，BIO-RAD Gel Doc2000凝胶成像系统中成像。

1.7. SSR位点的功能分析

对筛选出的SSR提取其所在的基因序列，翻译成蛋白质序列后，利用Blastp比对，提取相似性最高的序列注释信息，作为SSR靶向基因的功能注释，并对SSR位点的注释信息进行分类。

3. 讨论

利用扩增片段长度多态性（AFLP），限制性片段长度多态性（RFLP），随机扩增多态性DNA标记（RAPD）和简单重复间序列（ISSR）分子标记技术研究菠萝遗传多样性已有文献报道^[10-14]。在菠萝栽培种上至今尚未见从EST中开发SSR的相关报道。随着近年来NCBI中EST数据库的完善，EST-SSR作为新一代分子标记展现了广阔的前景。本实验通过对菠萝5 659条EST进行SSR筛选，共发现636个SSR，分布于617条EST中，SSR占整个EST的11.24%，高于水稻Oryza sativa（4.70%）^[15]，甘蔗Saccharum（2.90%）^[5]，普通小麦Triticum aestivum（5.40%）^[16]，辣椒Capsicum annuum（2.97%）^[17]，白桦Betula platyphylla（10.20%）^[18]，亚麻Linum usitatissimum（2.73%）^[19]，花生Arachis hypogaea（7.80%）^[20]，香蕉Musa nana（5.30%）^[21]，西瓜Citrullus vulgaris（6.89%）^[2]，甜瓜Cucumis melo（8.36%）^[22]，香菇Lentinus edodes（2.96%）^[4]，略低于橡胶Hevea brasiliensis（11.42%）^[1]，核桃Juglans regia（10.75%）^[23]，猕猴桃Actinidia（14.08%）^[24]。上述差异的原因，一是EST数据库中不同物种间基因的差异导致SSR分布的差异；二是SSR检索的软件和标准不同导致最终筛选结果的差异。本研究所得菠萝的EST中，二、三、六核苷酸是主导类型，分别占SSR总数的42.61%，29.25%，20.13%。菠萝二核苷酸重复中AG/TC是比较常见的优势基元类型，在亚麻^[19]、甜瓜^[22]、苹果^[3]、花生^[20]中也都存在这一规律；而三核苷酸的重复基元比较分散，优势基元AGC/TCG仅占三核苷酸重复类型的13.98%，六核苷酸SSR在总的EST中有128条，而六核苷酸的重复基元有80种，种类虽丰富，数量却不多。

本研究共设计了636对菠萝EST-SSR引物，合成的30对引物中有27对引物有良好的扩增结果，可以预见对剩余引物的筛选有较高成功率地保证。菠萝EST-SSR出现频率高，而且类型丰富，可为今后凤梨科Bromeliaceae植物分类、遗传作图、遗传多样性分析、功能基因定位等方面研究奠定良好基础。

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重复类型	SSR数量	所占总SSR比例/%	出现频率/%	分布密度/kb
二核苷酸	271	42.61	4.79	17.34
三核苷酸	186	29.2'	3.29	2'.27
四核苷酸	22	3.46	0.39	213.64
五核苷酸	29	4.56	0.51	162.07
六核苷酸	128	20.13	2.26	36.72
总计	636	100	11.24	7.39

重复类型	重复频率/%
AAC/TTG	3.06
AAG/TTC	4.90
AAT/TTA	2.76
ACA/TGT	2.76
ACG/TGC	3.37
AGA/TCT	7.96
AGC/TCG	13.78
AGG/TCC	3.37
ATC	0.61
ATG/TAC	1.07
ATT/TAA	2.76
CAA	0.61
CAC	1.07
CAG/GTC	7.04
CCA	0.61
CCG/GGC	1.53
CCT/GGA	4.90
CGA/GCT	3.37
CGC/GCG	1.53
CGT/GCA	4.90
CTA/GAT	3.37
CTC/GAG	5.51
CTG/GAC	6.43
CTT/GAA	7.96
GTA	0.61
TCA	2.76
TGG	0.61

SSR位置	登录号	产物长度/bp	重复基元	T/℃	上游引物 (5'→3')	下游引物 (5'→3')	基因功能	扩增带数
ACSSR1	DT338344.1	390	(GCT)7/(TGG)7	59.8	CGTTCAAGTTAGCA-GAAGCTGA	CAGAAACACTTCCA-CAACCAAA	水分胁迫诱导蛋白	7
ACSSR2	DT339079.1	290	(CCTCTT)3/(CTTCCG)3	60.2	A-GATCGGGTTCTTCACTCT TCA	AGACAAG-GTTCTTCTTTCGCAC	甲酰转移酶	4
ACSSR4	DT339555.1	399	(GA)16	60.1	AAGCCAAAGCAGA-CAAAAGAAG	TAGAACACCCAACTG-GTTCAAA	未知蛋白	7
ACSSR6	DT337477.1	388	(CT)17	60.0	CAGCAGATTTCGCAGGT-GT	CTTGGCGCT-GTTGATAATGTAG	未知蛋白	2
ACSSR7	C0732232.1	393	(AG)16	60.4	AAGAGCAAGAGAGGAG-TAGGGG	GAGAAAATTACAGGT-GTTGGCG	未知蛋白	4
ACSSR8	DT335862.1	394	(TA) 9	60.0	TCAAGTTTGGTGGCTTC-CTAAT	TTGTGTTGGCTTAT-GTCGTTTC	未发现的序列	5
ACSSR9	DT335902.1	397	(CT) 21	59.1	GGGATGCTCCAGTTTCT-GAT	ATTTATGATGGCGAC-CTTCTTC	衰老相关蛋白	6
ACSSR10	C0731041.1	396	(AT) 12	59.9	GTACGCGGTCTTCTACT-GCC	GACTCCATGTTCACAC-GACAAT	假定的脱水反应蛋白质RD22前体	6
ACSSR11	DT337447.1	398	(GA)11	60.3	TTAACTCC-CATCTTCGTTTTGG	CTAGAGAGACCATA-CATCCCGC	未发现的序列	1
ACSSR12	DT338461.1	395	(TC) 10	60.0	TTTCAGAGCTTTAGAG-GATGGC	GGGTATCTTTGGCTA-CATCGAC	未知蛋白	3
ACSSR13	DT338327.1	379	(AG)15	60.7	GGGGAAGAAGACGAA-GAAGAAG	AAGCTATGCGAAGTA-GACAGGC	未知蛋白	3
ACSSR14	DT338672.1	394	(CT)15	60.2	GGT-GTCTCAGGTCAGGTAG-GAG	CGAAGTTTGC-CTTTTGTTTGAC	拟30s核糖体蛋白S9	5
ACSSR15	DT338506.1	398	(CTA)12	60.7	GAATCC-CCAGCATAGTTTCCTT	ATGGCCTTTTC-CTCTCTCTCTC	未发现的序列	3
ACSSR16	DT337566.1	396	(TCT)8	59.8	CTCTTC-CTCTTCTTCGTCTTCG	CACTGAAGTTTCTC-CTCAGCAA	核糖体L11相似蛋白	6
ACSSR17	DT336908.1	393	(CAG)9	60.0	CTTGTCCATTAGCACCA-CAAAA	CCTCCTCCTCCTCCTCT-GTTAT	未知蛋白	5
ACSSR18	DT338459.1	387	(GAA)6	60.5	CAGATCGAGGACCA-GATGAAAT	GCACC-CAAATTCTTTTGTAAGC	未知蛋白	3
ACSSR19	DT336929.1	383	(CAG)8	60.0	AGGTTATCCACCG-CAAACTCTA	GCTGTTCTTGGTCCT-GCTTATT	未发现的序列	3
ACSSR20	C0731978.1	378	(GAC)6	60.1	CGGTAACTGTAATTGT-GCAGGA	AGTTTGGGCTCTTGAAA ACATC	拟新生多肽α链	3
ACSSR21	DT337637.1	383	(TCT)11	59.9	AAGGCAACGTAGAGAG-GTGAAG	AAACCACACAAGGGA-GAAAAGA	细胞质膜质子泵	6
ACSSR22	DT337211.1	351	(AAAC)6	60.3	ATGAACCCTTCC-CTTCTCCTTA	CTCCTTCCCACAAAAT-CAAAAC	未发现的序列	4
ACSSR23	C0731699.1	341	(GCGA)5	61.1	GGAGGGTCTCAT-CAAGGGATAC	GACAAATCTCGATCTC-CACCTC	PoP3肽	4
ACSSR24	DT336569.1	392	(AAAGA)5	59.8	GATCTCTTGCTC-CAACTGGTTT	ATCTTCGATTGT-GAAAACTGGC	抑制蛋白[菠萝]	3
ACSSR25	C0730830.1	391	(GATTA)4	59.9	GTTCTCCATTGCTTCTC-GATCT	AGGGCATACTACCAT-CAACAGG	菠萝绿色成熟果实cDNA库的菠萝cD-NA克隆	4
ACSSR26	DT336115.1	397	(CCTCTT)3	59.6	AAGATCGGGTTCAT-CACTCTTC	ACCAAAACAGCATC-TATCTGCC	未知蛋白	4
ACSSR27	DV190745.1	395	(AGAAGC)3	59.9	TGCAATTCTGT-GAGTTCTTCGT	GAAACACTGCTCC-CTTGTCTCT	未发现的序列	4
ACSSR29	DT337199.1	391	(CTTCCG)4	60.1	GATCGGGTTCTTCACTTT TCAG	CCTG-CACTTTCTTTTGCTTCTT	未知蛋白	3
ACSSR30	DT338688.1	387	(AGCTCT)3	59.8	TACACCACCACCTTTTC-CTTTT	CTTGTTCTCTCCCTCTC-CTCCT	未发现的序列	4

Development of novel EST-SSR markers in pineapple (Ananas comosus) by data mining from public EST sequences

doi: 10.11833/j.issn.2095-0756.2017.02.018