Article(id=1302212379450827519, tenantId=1146029695717560320, journalId=1301849931339890755, issueId=1302212221539472091, articleNumber=null, orderNo=null, doi=10.3969/j.issn.2095-1191.2026.06.014, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1771516800000, receivedDateStr=2026-02-20, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1788401225197, onlineDateStr=2026-09-03, pubDate=1782316800000, pubDateStr=2026-06-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1788401225197, onlineIssueDateStr=2026-09-03, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1788401225197, creator=13701087609, updateTime=1788401225197, updator=13701087609, issue=Issue{id=1302212221539472091, tenantId=1146029695717560320, journalId=1301849931339890755, year='2026', volume='57', issue='6', pageStart='1625', pageEnd='1956', issueExtLink='null', onlineDate='null', pubDate='1782316800000', pubDateStr='2026-06-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1788401187547, creator='13701087609', updateTime=1788405081323, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1302228553291034731, tenantId=1146029695717560320, journalId=1301849931339890755, issueId=1302212221539472091, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1302228553291034732, tenantId=1146029695717560320, journalId=1301849931339890755, issueId=1302212221539472091, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1774, endPage=1784, ext={EN=ArticleExt(id=1302212379782177536, articleId=1302212379450827519, tenantId=1146029695717560320, journalId=1301849931339890755, language=EN, title=SSR loci characterization and molecular marker development based on Anthurium andraeanum L. transcriptomes, columnId=1302212259774747620, journalTitle=Journal of Southern Agriculture, columnName=Crop Genetics & Breeding·Germplasm Resources·Biotechnology, runingTitle=null, highlight=null, articleAbstract=
Objective

This study aimed to discover SSR loci of Anthurium andraeanum L. through transcriptome sequencing and develop polymorphic EST-SSR molecular markers,providing reliable tools for evaluating germplasm resources,constructing genetic maps,and molecular breeding in Anthurium andraeanum L.

Method

Transcriptome sequencing of spathes of Anthurium andraeanum L. cultivars of five colors using Illumina,and MISA was employed to screen SSR loci,analyze their types,frequency,and distribution characteristics. EST-SSR primers were designed using Primer 3.0. A total of 120 pairs of primers were randomly selected. After preliminary screening of three representative cultivars,PCR amplification was conducted for 22 Anthurium andraeanum L. cultivars,with calculation of polymorphism information content (PIC). Finally,the effectiveness of EST-SSR markers was validated by unweighted pair group method with arithmetic mean (UPGMA) using NTSYSpc 2.10e.

Result

Transcriptome sequencing of Anthurium andraeanum L. generated 22381 unigenes,with the total length of 34872210 bp,from which 11324 SSR loci were detected,distributed across 7705 unigenes,with an occurrence frequency of 50.60% and an average distribution distance of 3.08 kb. The predominant repeat motifs were mononucleotide (24.55%),dinucleotide (46.41%),and trinucleotide (27.69%),with the predominant nucleotide repeat type of A/T (21.59%),AG/CT (37.73%),and AC/GT (6.30%). SSR locus lengths were primarily between 12-20 bp,with repeat numbers predominantly ranging from 5 to 12. A total of 7705 primers containing SSR loci were designed for unigenes,7456 pairs of EST-SSR primers were obtained. Polymorphic detection was performed for the 120 pairs of EST-SSR primers,in which 107 pairs successfully amplified (with the success rate of 89.17%),with 75 pairs of primers showing polymorphism. A total of 20 pairs of primers were further selected,and the DNA of 22 Anthurium andraeanum L. cultivars was taken as templates for validation,yielding 16 pairs of highly polymorphic primers (PIC>0.5000). These primers demonstrated excellent discriminatory capability for the 22 Anthurium andraeanum L. cultivars that were classified into four groups,consistent with traditional morphological classification.

Conclusion

SSR loci of Anthurium andraeanum L. transcriptomes exhibit high abundance and polymorphism,which can be used for efficient molecular marker development. The developed EST-SSR markers show characteristics such as high polymorphism and stability,providing effective tools for germplasm evaluation,genetic map construction,and breeding assisted by molecular markers,which can facilitate genetic elucidation and cultivar improvement of major ornamental traits of Anthurium andraeanum L.

, authors=Lin-shan ZHONG, Feng-qin YAO, Bo ZHANG, Wei-ting CHEN, Hui-feng LIN, Fa-zhuang LIN, authorsList=Lin-shan ZHONG, Feng-qin YAO, Bo ZHANG, Wei-ting CHEN, Hui-feng LIN, Fa-zhuang LIN, authorCompany=null, correspAuthors=Fa-zhuang LIN, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1302212380855919367, articleId=1302212379450827519, tenantId=1146029695717560320, journalId=1301849931339890755, language=CN, title=红掌转录组SSR位点表征及分子标记开发, columnId=1302212260148040678, journalTitle=南方农业学报, columnName=作物遗传育种·种质资源·分子生物学, runingTitle=null, highlight=null, articleAbstract=
目的

通过转录组测序挖掘红掌SSR位点,开发多态性EST-SSR分子标记,为红掌种质资源评价、遗传图谱构建及分子育种提供可靠的工具支撑。

方法

利用Illumina对5个不同花色的红掌品种佛焰苞进行转录组测序,再采用MISA对SSR位点进行筛选,并统计其类型、频率及分布特征。利用Primer 3.0设计EST-SSR引物,随机选取120对引物,经3个代表性品种初筛后,对22个红掌品种进行PCR扩增,并计算多态性信息含量(PIC)。最后,基于NTSYSpc 2.10e的非加权组平均法(UPGMA)进行聚类分析,验证EST-SSR标记的有效性。

结果

红掌转录组测序获得22381条Unigenes(总长34872210 bp),从中检测出11324个SSR位点,分布于7705条Unigenes中,出现频率为50.60%,平均分布距离为3.08 kb。SSR位点中优势重复基序为单核苷酸(24.55%)、二核苷酸(46.41%)和三核苷酸(27.69%),核苷酸优势重复类型为A/T(21.59%)、AG/CT(37.73%)、AC/GT(6.30%)。SSR位点长度主要集中在12~20 bp,重复次数以5~12次为主。对含有SSR位点的7705个Unigenes设计引物,共获得7456对EST-SSR引物。随机筛选120对EST-SSR引物进行多态性检测,其中107对成功扩增(成功率89.17%),其中75对引物具有多态性,进一步筛选条带清晰的20对引物,并以22个红掌品种DNA为模板对其进行有效验证,获得16对高多态性引物(PIC>0.5000),这些引物对22个红掌品种表现出良好的区分能力,可将红掌品种分为四大类群,与传统形态学分类结果一致。

结论

红掌转录组SSR位点丰度和多态性均较高,可作为高效分子标记开发来源。开发的EST-SSR分子标记具有多态性高、稳定性好等特点,为红掌种质资源评价、遗传图谱构建和分子标记辅助育种提供了有效工具,有助于后续红掌重要观赏性状的遗传解析和品种改良。

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钟琳珊(1990-),https://orcid.org/0009-0005-6920-1999,主要从事花卉遗传育种研究工作,E-mail:

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林发壮(1985-),https://orcid.org/0009-0000-6088-1813,副研究员,主要从事花卉遗传育种研究工作,E-mail:
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Plant Science176(3):397-405., articleTitle=EST-SSR sequences revealed the relationship of D-genomein diploid and tetraploid species in Gossypium, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1302212381090800392, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, xref=1, ext=[AuthorCompanyExt(id=1302212381099189001, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, companyId=1302212381090800392, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1Flower Research Institute,Sanming Academy of Agricultural Sciences/Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area,Sanming,Fujian 365051,China), AuthorCompanyExt(id=1302212381107577610, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, companyId=1302212381090800392, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1三明市农业科学研究院花卉研究所/福建省(山区)作物遗传改良与创新利用重点实验室,福建 三明 365051)])], figs=[ArticleFig(id=1302212388275643188, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Fig. 1, caption=Distribution of SSR length of Anthurium andraeanum L.transcriptomes, figureFileSmall=S+gW4COzUj2xQ4rP+BZKIQ==, figureFileBig=xK8RSAE02JIsutNsJhXVrQ==, tableContent=null), ArticleFig(id=1302212388451803957, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=图1, caption=红掌转录组中SSR长度分布, figureFileSmall=S+gW4COzUj2xQ4rP+BZKIQ==, figureFileBig=xK8RSAE02JIsutNsJhXVrQ==, tableContent=null), ArticleFig(id=1302212388648936246, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Fig. 2, caption=Preliminary screening of amplification of partial EST-SSR primers, figureFileSmall=MNZrDYM/CrzIEy+dzlTK4A==, figureFileBig=mpuqOceorsYXp48Ze48qvg==, tableContent=null), ArticleFig(id=1302212388858651447, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=图2, caption=部分EST-SSR引物扩增产物电泳初筛

M:DL500 DNA Marker;1~32:EST-SSR引物扩增产物

, figureFileSmall=MNZrDYM/CrzIEy+dzlTK4A==, figureFileBig=mpuqOceorsYXp48Ze48qvg==, tableContent=null), ArticleFig(id=1302212388942537528, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Fig. 3, caption=Cluster analysis of 22 Anthurium andraeanum L. cultivars, figureFileSmall=KAyfsv2nDpY1ggWBbeyIGQ==, figureFileBig=Pc4fU4OsvZ+OVasXDItotA==, tableContent=null), ArticleFig(id=1302212389026423609, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=图3, caption=22个红掌品种的聚类分析

图中材料编号与表1中的品种名对应

, figureFileSmall=KAyfsv2nDpY1ggWBbeyIGQ==, figureFileBig=Pc4fU4OsvZ+OVasXDItotA==, tableContent=null), ArticleFig(id=1302212389085143866, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 1, caption=

Materials of Anthurium andraeanum L. cultivars for polymorphism analysis

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 Code名称 Name来源 Source佛焰苞花色 Spathe color
1绿如意 Green Jade三明市农业科学研究院红色(47A)
2红火鹤 Scherzeranum昆明安祖花园艺有限公司红色(45A)
3原种花烛 Wild Anthurium市场收集绿色(146B)
4红布加迪 Bugatti Red广东瑞恩科技有限公司红色(46B)
5梦幻 Princess Amalia Brilliant昆明安祖花园艺有限公司复色(红—白—绿)
6皇冠 Royal Champion昆明安祖花园艺有限公司红色(N45C)
7阿里克斯 Princess Alexia Red广东瑞恩科技有限公司红色(45B)
8白王子 White King昆明安祖花园艺有限公司白色(NN155B)
9明农玄红 Mingnong Xuanhong三明市农业科学研究院红色(46A)
10祥梦 Xiangmeng广州花卉研究中心红色(45B)
11利维姆 Livium昆明安祖花园艺有限公司复色(紫红—白)
12奥运粉俏 Red Miracle昆明安祖花园艺有限公司红色(53C)
13宝贝橙Baby Orange昆明安祖花园艺有限公司橙色(44B)
14明农秋月 Mingnong Qiuyue三明市农业科学研究院复色(红—绿)
15红唇 Princess Alexia Elegance昆明安祖花园艺有限公司复色(白—红)
16樱桃红 Cherry Red广州花卉研究中心粉色(54B)
17彩虹冠军 Rainbow Champion广东瑞恩科技有限公司复色(红—橙)
18恋歌 Liange三明市农业科学研究院红色(N45C)
19卡瓦丽 Cavalli昆明安祖花园艺有限公司紫色(64B)
20埃索多 Esudo昆明安祖花园艺有限公司红色(52A)
21明尼苏达 Minnesota昆明安祖花园艺有限公司红色(44A)
22福运 Fuyun广州花卉研究中心红色(45B)
), ArticleFig(id=1302212389164835643, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表1, caption=

红掌EST-SSR多态性分析品种材料

, figureFileSmall=null, figureFileBig=null, tableContent=
编号 Code名称 Name来源 Source佛焰苞花色 Spathe color
1绿如意 Green Jade三明市农业科学研究院红色(47A)
2红火鹤 Scherzeranum昆明安祖花园艺有限公司红色(45A)
3原种花烛 Wild Anthurium市场收集绿色(146B)
4红布加迪 Bugatti Red广东瑞恩科技有限公司红色(46B)
5梦幻 Princess Amalia Brilliant昆明安祖花园艺有限公司复色(红—白—绿)
6皇冠 Royal Champion昆明安祖花园艺有限公司红色(N45C)
7阿里克斯 Princess Alexia Red广东瑞恩科技有限公司红色(45B)
8白王子 White King昆明安祖花园艺有限公司白色(NN155B)
9明农玄红 Mingnong Xuanhong三明市农业科学研究院红色(46A)
10祥梦 Xiangmeng广州花卉研究中心红色(45B)
11利维姆 Livium昆明安祖花园艺有限公司复色(紫红—白)
12奥运粉俏 Red Miracle昆明安祖花园艺有限公司红色(53C)
13宝贝橙Baby Orange昆明安祖花园艺有限公司橙色(44B)
14明农秋月 Mingnong Qiuyue三明市农业科学研究院复色(红—绿)
15红唇 Princess Alexia Elegance昆明安祖花园艺有限公司复色(白—红)
16樱桃红 Cherry Red广州花卉研究中心粉色(54B)
17彩虹冠军 Rainbow Champion广东瑞恩科技有限公司复色(红—橙)
18恋歌 Liange三明市农业科学研究院红色(N45C)
19卡瓦丽 Cavalli昆明安祖花园艺有限公司紫色(64B)
20埃索多 Esudo昆明安祖花园艺有限公司红色(52A)
21明尼苏达 Minnesota昆明安祖花园艺有限公司红色(44A)
22福运 Fuyun广州花卉研究中心红色(45B)
), ArticleFig(id=1302212389257110332, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 2, caption=

Information of SSR loci of Anthurium andraeanum L.transcriptomes

, figureFileSmall=null, figureFileBig=null, tableContent=
类型 Type数值 Value
Unigenes总数
Total number of unigenes
22381
Unigenes总长度(bp)
Total length of unigenes
34872210
SSR位点数
Number of SSR locus
11324
包含SSR位点的Unigenes数
Number of unigene containing SSR locus
7705
包含2个以上SSR位点的Unigenes数
Number of unigene containing more than two SSR loci
2499
含有复合型SSR位点的Unigenes数
Number of unigene containing compound SSR locus
1434
), ArticleFig(id=1302212389324219197, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表2, caption=

红掌转录组SSR位点信息

, figureFileSmall=null, figureFileBig=null, tableContent=
类型 Type数值 Value
Unigenes总数
Total number of unigenes
22381
Unigenes总长度(bp)
Total length of unigenes
34872210
SSR位点数
Number of SSR locus
11324
包含SSR位点的Unigenes数
Number of unigene containing SSR locus
7705
包含2个以上SSR位点的Unigenes数
Number of unigene containing more than two SSR loci
2499
含有复合型SSR位点的Unigenes数
Number of unigene containing compound SSR locus
1434
), ArticleFig(id=1302212389416493886, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 3, caption=

Type,number,and distribution frequency of SSR of Anthurium andraeanum L. transcriptomes

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
数目
Number
占比(%)
Proportion
分布频率(%)
Distribution frequency
分布距离(kb)
Distribution distance
分布长度(bp)
Distribution length
单核苷酸 Mononucleotide278024.5512.4212.5411.77
二核苷酸 Dinucleotide525546.4123.486.6422.30
三核苷酸 Trinucleotide313627.6914.0111.1218.59
四核苷酸 Tetranucleotide900.790.40387.4722.53
五核苷酸 Pentanucleotide190.170.081835.3831.54
六核苷酸 Hexanucleotide440.390.20792.5572.69
总计 Total11324100.0050.60--
均值 Average---3.0818.90
), ArticleFig(id=1302212389491991359, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表3, caption=

红掌转录组中SSR类型、数量及分布频率

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
数目
Number
占比(%)
Proportion
分布频率(%)
Distribution frequency
分布距离(kb)
Distribution distance
分布长度(bp)
Distribution length
单核苷酸 Mononucleotide278024.5512.4212.5411.77
二核苷酸 Dinucleotide525546.4123.486.6422.30
三核苷酸 Trinucleotide313627.6914.0111.1218.59
四核苷酸 Tetranucleotide900.790.40387.4722.53
五核苷酸 Pentanucleotide190.170.081835.3831.54
六核苷酸 Hexanucleotide440.390.20792.5572.69
总计 Total11324100.0050.60--
均值 Average---3.0818.90
), ArticleFig(id=1302212389580071744, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 4, caption=

Distribution of SSR motifs of Anthurium andraeanum L. transcriptomes

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
基序
Motif
重复次数
Repeat number
出现频率(%)
Occurrence frequency
重复类型
Repeat type
基序
Motif
重复次数
Repeat number
出现频率(%)
Occurrence frequency
单核苷酸 MononucleotideA/T244521.59AATCC/ATTGG10.01
C/G3352.96ACGAG/CGTCT10.01
二核苷酸 DinucleotideAC/GT7136.30AGAGC/CTCTG10.01
AG/CT427337.73AGAGG/CCTCT20.02
AT/AT2442.15AGATC/ATCTG10.01
CG/CG250.22AGCTC/AGCTG10.01
三核苷酸 TrinucleotideAAC/GTT740.65AGGCG/CCTCG10.01
AAG/CTT5524.87AGGGG/CCCCT10.01
AAT/ATT600.53六核苷酸 HexamerAAAAAC/GTTTTT10.01
ACC/GGT2832.50AAAGGC/CCTTTG10.01
ACG/CGT1641.45AACAGC/CTGTTG10.01
ACT/AGT270.24AACCAG/CTGGTT10.01
AGC/CTG5484.84AACGAG/CGTTCT10.01
AGG/CCT7826.91AAGAGG/CCTCTT10.01
ATC/ATG1711.51AAGCCG/CGGCTT10.01
CCG/CGG4754.19AAGCGG/CCGCTT20.02
四核苷酸 TetranucleotideAAAC/GTTT20.02AAGCTG/AGCTTC10.01
AAAG/CTTT140.12AAGGAG/CCTTCT20.02
AAAT/ATTT20.02AAGGGC/CCCTTG10.01
AACC/GGTT10.01ACACCC/GGGTGT10.01
AAGC/CTTG20.02ACCAGT/ACTGGT10.01
AAGG/CCTT50.04ACCATC/ATGGTG10.01
AATC/ATTG20.02ACCCCC/GGGGGT10.01
ACAG/CTGT50.04ACCGCC/CGGTGG10.01
ACAT/ATGT60.05ACCTGG/AGGTCC10.01
ACCC/GGGT20.02ACGAGG/CCTCGT30.03
ACGC/CGTG50.04ACGCAG/CGTCTG10.01
ACGG/CCGT20.02ACGGCG/CCGTCG10.01
ACGT/ACGT10.01ACGGGG/CCCCGT10.01
ACTC/AGTG50.04AGAGCG/CGCTCT10.01
AGAT/ATCT90.08AGAGGC/CCTCTG10.01
AGCG/CGCT100.09AGCCCC/CTGGGG20.02
AGGC/CCTG20.02AGCGGC/CCGCTG10.01
AGGG/CCCT70.06AGCGGG/CCCGCT10.01
ATCC/ATGG60.05AGGCGG/CCGCCT30.03
ATCG/ATCG10.01AGGGCG/CCCTCG10.01
ATGC/ATGC10.01AGGGGC/CCCCTG10.01
五核苷酸 PentanucleotideAAAAC/GTTTT30.03ATCCCG/ATCGGG10.01
AAAAG/CTTTT20.02ATCGCC/ATGGCG30.03
AAAGG/CCTTT10.01ATCGGC/ATGCCG10.01
AAGAG/CTCTT30.03CCCCCG/CGGGGG20.02
AAGGG/CCCTT10.01CCCGGG/CCCGGG10.01
), ArticleFig(id=1302212389655569217, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表4, caption=

红掌转录组中SSR基序的分布

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
基序
Motif
重复次数
Repeat number
出现频率(%)
Occurrence frequency
重复类型
Repeat type
基序
Motif
重复次数
Repeat number
出现频率(%)
Occurrence frequency
单核苷酸 MononucleotideA/T244521.59AATCC/ATTGG10.01
C/G3352.96ACGAG/CGTCT10.01
二核苷酸 DinucleotideAC/GT7136.30AGAGC/CTCTG10.01
AG/CT427337.73AGAGG/CCTCT20.02
AT/AT2442.15AGATC/ATCTG10.01
CG/CG250.22AGCTC/AGCTG10.01
三核苷酸 TrinucleotideAAC/GTT740.65AGGCG/CCTCG10.01
AAG/CTT5524.87AGGGG/CCCCT10.01
AAT/ATT600.53六核苷酸 HexamerAAAAAC/GTTTTT10.01
ACC/GGT2832.50AAAGGC/CCTTTG10.01
ACG/CGT1641.45AACAGC/CTGTTG10.01
ACT/AGT270.24AACCAG/CTGGTT10.01
AGC/CTG5484.84AACGAG/CGTTCT10.01
AGG/CCT7826.91AAGAGG/CCTCTT10.01
ATC/ATG1711.51AAGCCG/CGGCTT10.01
CCG/CGG4754.19AAGCGG/CCGCTT20.02
四核苷酸 TetranucleotideAAAC/GTTT20.02AAGCTG/AGCTTC10.01
AAAG/CTTT140.12AAGGAG/CCTTCT20.02
AAAT/ATTT20.02AAGGGC/CCCTTG10.01
AACC/GGTT10.01ACACCC/GGGTGT10.01
AAGC/CTTG20.02ACCAGT/ACTGGT10.01
AAGG/CCTT50.04ACCATC/ATGGTG10.01
AATC/ATTG20.02ACCCCC/GGGGGT10.01
ACAG/CTGT50.04ACCGCC/CGGTGG10.01
ACAT/ATGT60.05ACCTGG/AGGTCC10.01
ACCC/GGGT20.02ACGAGG/CCTCGT30.03
ACGC/CGTG50.04ACGCAG/CGTCTG10.01
ACGG/CCGT20.02ACGGCG/CCGTCG10.01
ACGT/ACGT10.01ACGGGG/CCCCGT10.01
ACTC/AGTG50.04AGAGCG/CGCTCT10.01
AGAT/ATCT90.08AGAGGC/CCTCTG10.01
AGCG/CGCT100.09AGCCCC/CTGGGG20.02
AGGC/CCTG20.02AGCGGC/CCGCTG10.01
AGGG/CCCT70.06AGCGGG/CCCGCT10.01
ATCC/ATGG60.05AGGCGG/CCGCCT30.03
ATCG/ATCG10.01AGGGCG/CCCTCG10.01
ATGC/ATGC10.01AGGGGC/CCCCTG10.01
五核苷酸 PentanucleotideAAAAC/GTTTT30.03ATCCCG/ATCGGG10.01
AAAAG/CTTTT20.02ATCGCC/ATGGCG30.03
AAAGG/CCTTT10.01ATCGGC/ATGCCG10.01
AAGAG/CTCTT30.03CCCCCG/CGGGGG20.02
AAGGG/CCCTT10.01CCCGGG/CCCGGG10.01
), ArticleFig(id=1302212389726872386, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 5, caption=

Distribution of repeat number of SSR motifs of Anthurium andraeanum L. transcriptomes

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
重复次数 Repeat number总计
Total
56789101112131415161718192021222324>25
单核苷酸
Mononucleotide
1287551286170135104734331242191091262780
二核苷酸
Dinucleotide
947730675550368393382115126114132124978971674944311515255
三核苷酸
Trinucleotide
148372442727867634481275342301233136
四核苷酸
Tetranucleotide
571872131190
五核苷酸
Pentanucleotide
122211119
六核苷酸 Hexanucleotide2510421244
总计Total1577170111709586191722990676297270223208171131116927761563217711324
占比(%) Proportion13.9315.0210.338.465.4715.218.745.972.622.381.971.841.511.161.020.810.680.540.490.281.56
), ArticleFig(id=1302212389806564163, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表5, caption=

红掌转录组中SSR基序的重复次数分布

, figureFileSmall=null, figureFileBig=null, tableContent=
重复类型
Repeat type
重复次数 Repeat number总计
Total
56789101112131415161718192021222324>25
单核苷酸
Mononucleotide
1287551286170135104734331242191091262780
二核苷酸
Dinucleotide
947730675550368393382115126114132124978971674944311515255
三核苷酸
Trinucleotide
148372442727867634481275342301233136
四核苷酸
Tetranucleotide
571872131190
五核苷酸
Pentanucleotide
122211119
六核苷酸 Hexanucleotide2510421244
总计Total1577170111709586191722990676297270223208171131116927761563217711324
占比(%) Proportion13.9315.0210.338.465.4715.218.745.972.622.381.971.841.511.161.020.810.680.540.490.281.56
), ArticleFig(id=1302212389882061636, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=EN, label=Table 6, caption=

Sequence information of 20 pairs of polymorphic primers

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
Number
引物
Primer
上游引物
Forward primer
下游引物
Reverse primer
重复单元
Repeat unit
序列长度(bp)
Sequence length
PIC
1DN422435'-TTTCCCCTCCCCACTAGACT-3'5'-TAGGTGAGGCGCTTCAGAGT-3'(GA)92670.7769
2DN282985'-GTGGAACCTTCTGTCTCCCA-3'5'-TACAGATGATGGGGCTCTCC-3'(GA)102760.8347
3DN292315'-AATCAAGTGAATTGGGGCTG-3'5'-TAGCAACAGCAACAGCAACC-3'(GCT)82400.7066
4DN277455'-TGCAGCCTCCTAGTTCCATT-3'5'-CGGCGTAGGAAGAAACAGAG-3'(TGC)81550.7562
5DN328245'-ACTCTCCTCATCCACCCAGA-3'5'-TGTTCTTGTTCTTCCTCGGG-3'(GCA)72100.7479
6DN321845'-TAAACGTCTCCGAACCCAAC-3'5'-GTGGGAACAGGAGAGACCAG-3'(CTG)71400.6736
7DN303115'-AGAAGAGGAAGCTTGGCCTC-3'5'-GGTCCTTGGAGTAGGGTTCC-3'(AGC)72370.7107
8DN273435'-CCTGATCCTGCACAGTGAGA-3'5'-GACGTGGAGGACGAGCAG-3'(GCA)71260.4901
9DN464495'-AGGGGTGGAATCGGTACTTC-3'5'-AGAACAGTGGTAAGAGCGGC-3'(CTTG)52230.8058
10DN374545'-CGGACAGGAAGGAATTCAAA-3'5'-CCTCAATGCTCCCTCCTCTT-3'(AGAC)51420.7479
11DN317295'-CAACTCTCTCAACACCTGCG-3'5'-AGGTGCAGAGACATCTTGGG-3'(CTCG)51280.7521
12DN336475'-CTTCAGCAGTACGGCTCCTC-3'5'-CAAAAGAAGAACCAAACGCC-3'(CAG)62710.2500
13DN307965'-CGCATCCAAATGGTATCCTC-3'5'-TGGAAGTGATCCTGTTGACG-3'(TCC)62160.7975
14DN302505'-CGCCAATTCCAGATCTCATT-3'5'-GTACCTCAGGCTCATGCCTC-3'(TCT)61530.7397
15DN300745'-GGAGAGGTGGTAGGATGGGT-3'5'-CACAGTTGTAACACGTCCCG-3'(GGC)62280.8264
16DN166895'-TGGAACCGGAGGAAGATGTA-3'5'-CAACAACATTGAGACGGTCG-3'(TGC)61950.7355
17DN306545'-AGCTGGGTTTTGTTCCCTTT-3'5'-TAGTAATCGACAGGACGGGG-3'(GAG)81810.4298
18DN307975'-TCATCCTCCAGATCTCCCAC-3'5'-TCGAGGATGGAGATGACCTT-3'(CAG)61690.7107
19DN310755'-TTCACCATTCTACGCATCCA-3'5'-GATCTCGGAGGAAGGAGAGG-3'(CCA)3CT(ACC)42590.3182
20DN366935'-ACGACAAGTGTTCCTACCCG-3'5'-CCGTCCTCGTGTTGGTTTAT-3'(GGAT)51300.6570
), ArticleFig(id=1302212389957559109, tenantId=1146029695717560320, journalId=1301849931339890755, articleId=1302212379450827519, language=CN, label=表6, caption=

20对多态性引物序列信息

, figureFileSmall=null, figureFileBig=null, tableContent=
序号
Number
引物
Primer
上游引物
Forward primer
下游引物
Reverse primer
重复单元
Repeat unit
序列长度(bp)
Sequence length
PIC
1DN422435'-TTTCCCCTCCCCACTAGACT-3'5'-TAGGTGAGGCGCTTCAGAGT-3'(GA)92670.7769
2DN282985'-GTGGAACCTTCTGTCTCCCA-3'5'-TACAGATGATGGGGCTCTCC-3'(GA)102760.8347
3DN292315'-AATCAAGTGAATTGGGGCTG-3'5'-TAGCAACAGCAACAGCAACC-3'(GCT)82400.7066
4DN277455'-TGCAGCCTCCTAGTTCCATT-3'5'-CGGCGTAGGAAGAAACAGAG-3'(TGC)81550.7562
5DN328245'-ACTCTCCTCATCCACCCAGA-3'5'-TGTTCTTGTTCTTCCTCGGG-3'(GCA)72100.7479
6DN321845'-TAAACGTCTCCGAACCCAAC-3'5'-GTGGGAACAGGAGAGACCAG-3'(CTG)71400.6736
7DN303115'-AGAAGAGGAAGCTTGGCCTC-3'5'-GGTCCTTGGAGTAGGGTTCC-3'(AGC)72370.7107
8DN273435'-CCTGATCCTGCACAGTGAGA-3'5'-GACGTGGAGGACGAGCAG-3'(GCA)71260.4901
9DN464495'-AGGGGTGGAATCGGTACTTC-3'5'-AGAACAGTGGTAAGAGCGGC-3'(CTTG)52230.8058
10DN374545'-CGGACAGGAAGGAATTCAAA-3'5'-CCTCAATGCTCCCTCCTCTT-3'(AGAC)51420.7479
11DN317295'-CAACTCTCTCAACACCTGCG-3'5'-AGGTGCAGAGACATCTTGGG-3'(CTCG)51280.7521
12DN336475'-CTTCAGCAGTACGGCTCCTC-3'5'-CAAAAGAAGAACCAAACGCC-3'(CAG)62710.2500
13DN307965'-CGCATCCAAATGGTATCCTC-3'5'-TGGAAGTGATCCTGTTGACG-3'(TCC)62160.7975
14DN302505'-CGCCAATTCCAGATCTCATT-3'5'-GTACCTCAGGCTCATGCCTC-3'(TCT)61530.7397
15DN300745'-GGAGAGGTGGTAGGATGGGT-3'5'-CACAGTTGTAACACGTCCCG-3'(GGC)62280.8264
16DN166895'-TGGAACCGGAGGAAGATGTA-3'5'-CAACAACATTGAGACGGTCG-3'(TGC)61950.7355
17DN306545'-AGCTGGGTTTTGTTCCCTTT-3'5'-TAGTAATCGACAGGACGGGG-3'(GAG)81810.4298
18DN307975'-TCATCCTCCAGATCTCCCAC-3'5'-TCGAGGATGGAGATGACCTT-3'(CAG)61690.7107
19DN310755'-TTCACCATTCTACGCATCCA-3'5'-GATCTCGGAGGAAGGAGAGG-3'(CCA)3CT(ACC)42590.3182
20DN366935'-ACGACAAGTGTTCCTACCCG-3'5'-CCGTCCTCGTGTTGGTTTAT-3'(GGAT)51300.6570
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红掌转录组SSR位点表征及分子标记开发
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钟琳珊 , 姚凤琴 , 张博 , 陈玮婷 , 林辉锋 , 林发壮 *, *
南方农业学报 | 作物遗传育种·种质资源·分子生物学 2026,57(6): 1774-1784
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南方农业学报 |作物遗传育种·种质资源·分子生物学 2026 , 57 (6) : 1774 -1784
红掌转录组SSR位点表征及分子标记开发
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钟琳珊 , 姚凤琴, 张博, 陈玮婷, 林辉锋, 林发壮*, *
作者信息
  • 1三明市农业科学研究院花卉研究所/福建省(山区)作物遗传改良与创新利用重点实验室,福建 三明 365051
通讯作者:
林发壮(1985-),https://orcid.org/0009-0000-6088-1813,副研究员,主要从事花卉遗传育种研究工作,E-mail:
作者简介:

钟琳珊(1990-),https://orcid.org/0009-0005-6920-1999,主要从事花卉遗传育种研究工作,E-mail:

SSR loci characterization and molecular marker development based on Anthurium andraeanum L. transcriptomes
Lin-shan ZHONG , Feng-qin YAO, Bo ZHANG, Wei-ting CHEN, Hui-feng LIN, Fa-zhuang LIN
Affiliations
  • 1Flower Research Institute,Sanming Academy of Agricultural Sciences/Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area,Sanming,Fujian 365051,China
出版时间: 2026-06-25 doi: 10.3969/j.issn.2095-1191.2026.06.014
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目的

通过转录组测序挖掘红掌SSR位点,开发多态性EST-SSR分子标记,为红掌种质资源评价、遗传图谱构建及分子育种提供可靠的工具支撑。

方法

利用Illumina对5个不同花色的红掌品种佛焰苞进行转录组测序,再采用MISA对SSR位点进行筛选,并统计其类型、频率及分布特征。利用Primer 3.0设计EST-SSR引物,随机选取120对引物,经3个代表性品种初筛后,对22个红掌品种进行PCR扩增,并计算多态性信息含量(PIC)。最后,基于NTSYSpc 2.10e的非加权组平均法(UPGMA)进行聚类分析,验证EST-SSR标记的有效性。

结果

红掌转录组测序获得22381条Unigenes(总长34872210 bp),从中检测出11324个SSR位点,分布于7705条Unigenes中,出现频率为50.60%,平均分布距离为3.08 kb。SSR位点中优势重复基序为单核苷酸(24.55%)、二核苷酸(46.41%)和三核苷酸(27.69%),核苷酸优势重复类型为A/T(21.59%)、AG/CT(37.73%)、AC/GT(6.30%)。SSR位点长度主要集中在12~20 bp,重复次数以5~12次为主。对含有SSR位点的7705个Unigenes设计引物,共获得7456对EST-SSR引物。随机筛选120对EST-SSR引物进行多态性检测,其中107对成功扩增(成功率89.17%),其中75对引物具有多态性,进一步筛选条带清晰的20对引物,并以22个红掌品种DNA为模板对其进行有效验证,获得16对高多态性引物(PIC>0.5000),这些引物对22个红掌品种表现出良好的区分能力,可将红掌品种分为四大类群,与传统形态学分类结果一致。

结论

红掌转录组SSR位点丰度和多态性均较高,可作为高效分子标记开发来源。开发的EST-SSR分子标记具有多态性高、稳定性好等特点,为红掌种质资源评价、遗传图谱构建和分子标记辅助育种提供了有效工具,有助于后续红掌重要观赏性状的遗传解析和品种改良。

红掌  /  转录组  /  EST-SSR分子标记  /  开发
Objective

This study aimed to discover SSR loci of Anthurium andraeanum L. through transcriptome sequencing and develop polymorphic EST-SSR molecular markers,providing reliable tools for evaluating germplasm resources,constructing genetic maps,and molecular breeding in Anthurium andraeanum L.

Method

Transcriptome sequencing of spathes of Anthurium andraeanum L. cultivars of five colors using Illumina,and MISA was employed to screen SSR loci,analyze their types,frequency,and distribution characteristics. EST-SSR primers were designed using Primer 3.0. A total of 120 pairs of primers were randomly selected. After preliminary screening of three representative cultivars,PCR amplification was conducted for 22 Anthurium andraeanum L. cultivars,with calculation of polymorphism information content (PIC). Finally,the effectiveness of EST-SSR markers was validated by unweighted pair group method with arithmetic mean (UPGMA) using NTSYSpc 2.10e.

Result

Transcriptome sequencing of Anthurium andraeanum L. generated 22381 unigenes,with the total length of 34872210 bp,from which 11324 SSR loci were detected,distributed across 7705 unigenes,with an occurrence frequency of 50.60% and an average distribution distance of 3.08 kb. The predominant repeat motifs were mononucleotide (24.55%),dinucleotide (46.41%),and trinucleotide (27.69%),with the predominant nucleotide repeat type of A/T (21.59%),AG/CT (37.73%),and AC/GT (6.30%). SSR locus lengths were primarily between 12-20 bp,with repeat numbers predominantly ranging from 5 to 12. A total of 7705 primers containing SSR loci were designed for unigenes,7456 pairs of EST-SSR primers were obtained. Polymorphic detection was performed for the 120 pairs of EST-SSR primers,in which 107 pairs successfully amplified (with the success rate of 89.17%),with 75 pairs of primers showing polymorphism. A total of 20 pairs of primers were further selected,and the DNA of 22 Anthurium andraeanum L. cultivars was taken as templates for validation,yielding 16 pairs of highly polymorphic primers (PIC>0.5000). These primers demonstrated excellent discriminatory capability for the 22 Anthurium andraeanum L. cultivars that were classified into four groups,consistent with traditional morphological classification.

Conclusion

SSR loci of Anthurium andraeanum L. transcriptomes exhibit high abundance and polymorphism,which can be used for efficient molecular marker development. The developed EST-SSR markers show characteristics such as high polymorphism and stability,providing effective tools for germplasm evaluation,genetic map construction,and breeding assisted by molecular markers,which can facilitate genetic elucidation and cultivar improvement of major ornamental traits of Anthurium andraeanum L.

Anthurium andraeanum L.  /  transcriptome  /  EST-SSR molecular marker  /  development
钟琳珊, 姚凤琴, 张博, 陈玮婷, 林辉锋, 林发壮. 红掌转录组SSR位点表征及分子标记开发. 南方农业学报, 2026 , 57 (6) : 1774 -1784 . DOI: 10.3969/j.issn.2095-1191.2026.06.014
Lin-shan ZHONG, Feng-qin YAO, Bo ZHANG, Wei-ting CHEN, Hui-feng LIN, Fa-zhuang LIN. SSR loci characterization and molecular marker development based on Anthurium andraeanum L. transcriptomes[J]. Journal of Southern Agriculture, 2026 , 57 (6) : 1774 -1784 . DOI: 10.3969/j.issn.2095-1191.2026.06.014
【研究意义】红掌(Anthurium andraeanum L.)为天南星科花烛属多年生常绿草本植物,别名火鹤、安祖花,原产中南美洲等热带地区,其花型独特(佛焰苞、肉穗花序)、花色丰富、叶形美观、花期长,是全球重要的高档热带盆花和切花,经济价值极高。目前,我国已成为全球红掌种植量和消费规模最大的市场(林发壮等,2020b)。然而,作为舶来品,红掌的优质种源长期被国外企业把控。近年来,国内陆续选育出一些新品种(姚凤琴等,2022周辉明等,2022陆锦萍等,2023霍文雨等,2024),但栽培品种多为二倍体,种间高度杂合,导致杂交育种周期长、难度大、效率低。准确鉴定品种、解析亲缘关系并筛选遗传距离远的亲本材料,是提高杂交育种成功率、加速国产红掌品种选育进程的关键环节(吴立强等,2011)。分子标记技术为此提供了有力工具,其中SSR因其具有共显性遗传、多态性信息含量(PIC)高、重复性好、在基因组中分布广泛且均匀等优点,已成为植物遗传研究中应用最广泛的分子标记之一(Zhu et al.,2009)。然而,传统的SSR分子标记开发存在周期长、成本高、效率低等问题。随着高通量测序技术的发展,转录组数据的获取变得更加容易和高效,根据转录组测序数据,开发出基于表达序列标签(EST)的SSR新型分子标记(EST-SSR),不仅能避免基因组DNA中非编码区的干扰,还直接与功能基因相关联(王娟娟等,2016)。【前人研究进展】目前红掌分子标记的开发及应用研究已取得一定进展,已有较多研究者采用多种分子标记技术对红掌种质进行遗传多样性分析、亲缘关系鉴定及分子育种。早期的研究主要基于传统分子标记技术(RAPD、SRAP等),如朱佳文(2005)采用RAPD分子标记对7个花色相近的红掌品种进行遗传多样性分析,结果表明红掌栽培品种具有丰富的遗传多样性,但品种间遗传关系较近;孟鹤(2011)从已公布引物中筛选出21对多态性高的SRAP引物用于红掌亲缘关系研究;王呈丹等(2013)利用SRAP引物对33个红掌品种进行分析,筛选出26对多态性引物,扩增出314条多态性条带,并发现有2个红掌品种(粉冠军和新粉冠军)的亲缘关系最近。随着分子标记技术的发展,ISSR分子标记因其更高的多态性和稳定性被广泛应用于红掌研究中。户帅雅等(2020)利用已公布的引物筛选出10条多态性ISSR引物,对56个红掌品种进行遗传多样性分析,结果发现红掌品种间遗传多样性较高,多态性比率达95.96%,且聚类结果与佛焰苞颜色存在显著相关性。随后,高通量测序技术的兴起推动了红掌分子标记研究进入新阶段,如Wang和Chuang(2013)基于NCBI数据库中已有的花烛属表达序列设计SSR引物,用于鉴定红掌变种;郁永明等(2015)基于红掌品种Alabama的转录组数据开发SSR分子标记,筛选出22对多态性好的EST-SSR引物;蒋锦雷(2016)基于香型红掌品种(Mystral)和无香型红掌品种(Alabama)转录组数据,开发筛选出14对多态性EST-SSR引物,为红掌花香性状的分子标记辅助选择提供了工具;陈艳梅(2018)利用简化基因组测序(SLAF-seq)技术对红掌F1代群体进行测序,并开发SNP分子标记,筛选出42484个多态性SLAF标签用于遗传图谱构建。与其他分子标记相比,SSR分子标记在红掌研究中表现出优势:(1)SSR分子标记多态性高,能够有效区分亲缘关系较近的品种(Zhang et al.,2014);(2)SSR分子标记稳定性好,试验结果重复性高(Varshney et al.,2005);(3)基于转录组开发的EST-SSR分子标记与功能基因直接相关,有助于重要性状基因的定位和克隆(Gupta et al.,2003)。然而,当前红掌SSR分子标记的开发仍面临一些挑战:现有SSR分子标记数量仍显不足,难以满足高密度遗传图谱构建的需求(于翠等,2012);不同研究者开发的SSR分子标记缺乏系统性和可比性(Wang and Chuang,2013);大多数SSR分子标记未与重要观赏性状建立明确关联(蒋锦雷,2016)。【本研究切入点】近年来,随着测序技术的快速发展和成本降低,基于全基因组和转录组测序开发SSR分子标记已成为新趋势。虽然已开发出一定数量的红掌SSR分子标记,但已报道的SSR分子标记多基于单一品种转录组开发,通用性较差。针对红掌EST-SSR分子标记仍显不足的现状,亟需开发新型、丰富的EST-SSR分子标记。【拟解决的关键问题】通过对5个不同花色红掌品种的佛焰苞组织进行转录组测序,系统挖掘转录组序列中的SSR位点信息,并筛选出多态性EST-SSR引物,旨在开发出丰富、高效的红掌EST-SSR分子标记,为深入开展红掌种质资源遗传多样性分析、高精度遗传图谱构建及分子标记辅助育种工作提供关键的工具支撑。
供试材料为7个花色且包含原生种、观叶品种和观花品种等22份红掌种质(表1),用于EST-SSR引物验证及聚类分析。
三明市农业科学院花卉所红掌课题组对不同佛焰苞颜色红掌品种的苞片进行Illumina高通量深度测序。从转录组测序数据中取5个红掌品种[阿拉巴马(红色)、粉阿拉巴马(粉色)、白马王子(白色)、明农红歌(橙色)、明农倾城(紫色)]盛花期的佛焰苞片。委托杭州联川生物技术股份有限公司进行转录组测序,并通过denovo方法拼装得到Unigenes,用作后续SSR位点筛选及引物设计的基础数据。
采用MISA程序查找SSR位点(https://webblast.ipk-gatersleben.de/misa/),参数设置标准:单核苷酸(Mononucleotide)、二核苷酸(Dinucleotide)、三核苷酸(Trinucleotide)、四核苷酸(Tetranucleotide)、五核苷酸(Pentanucleotide)和六核苷酸(Hexanucleotide)最少重复次数分别为10、6、5、5、5和5。采用Primer 3.0设计SSR引物,序列长度为8~30 bp,GC含量为40%~60%,理论退火温度(Tm)为55.0~65.0 ℃,预期产物长度为100~300 bp。
从引物组中随机选取120对长度为18~24 bp且碱基重复次数2~4次的SSR引物,委托福州尚亚生物技术有限公司合成。以种源类型、株型、叶形和花色均存在明显差异的3个红掌品种原种花烛(原生种,大株型,阔卵圆形叶,绿色)、宝贝橙(商业杂交种,小株型,窄卵圆形叶,橙色)和卡瓦丽(商业杂交种,中等株型,中等卵圆形叶,紫色)为样品,对SSR引物进行筛选。将22个红掌品种(表1)对筛选出的引物进行验证。22个红掌品种佛焰苞苞片颜色采用英国皇家园艺学会标准比色卡(RHS)进行比对。红掌基因组DNA提取采用OMEGA植物基因组提取试剂盒(上海索宝生物科技有限公司,R6733),采用1%琼脂糖电泳检测和BioDrop超微量蛋白核酸分析仪检测DNA质量和浓度,然后用无菌ddH2O将DNA样品浓度稀释至20 ng/μL,于-20 ℃保存备用。PCR反应体系、扩增程序及电泳检测参照周辉明等(2024)的方法。
首先,采用人工读带的方法,将SSR扩增条带进行二元化编码,即有条带记为1,无条带记为0,构建0/1矩阵。随后,根据Anderson等(1993)的方法计算引物的PIC。最后,利用NTSYSpc2.10e进行遗传关系分析,计算出品种间的戴斯相似系数(Dice similarity coefficient,DSC),并通过 SAHN程序的非加权组平均法(UPGMA)进行系统聚类,生成亲缘关系树状图。
经转录组测序获得22381条Unigenes,总长度为34872210 bp。利用MISA软件对所有Unigenes进行SSR位点分析,结果发现有7705条Unigenes中共检测出11324个SSR位点,SSR发生频率为34.43%,SSR出现频率为50.60%;其中有5206条Unigenes仅包含1个SSR位点,2499条Unigenes含有2个及以上SSR位点,另外有1434条Unigenes含有复合型SSR位点(表2)。
单核苷酸、二核苷酸和三核苷酸是红掌转录组中的主要重复基序,占SSR总数的98.65%,其中以二核苷酸重复类型占比最高,为46.41%,三核苷酸和单核苷酸重复类型次之,分别占27.69%和24.55%。四核苷酸、五核苷酸和六核苷酸重复类型SSR数量少,三者总和仅占1.35%。从分布距离来看,不同重复类型核苷酸的分布距离各不相同,SSR位点数目越多,分布距离越小,最大的平均分布距离为1835.38 kb,最小的平均分布距离为6.64 kb,平均分布距离为3.08 kb(表3)。分布距离的均值是用Unigene总长度(34872210 bp)除以SSR位点数(11324个)计算得出。分布长度的均值是用各类型的数量作权重来计算,即各类型总长度之和(214030.66 bp)除以总位点数(11324个)计算得出。
表4可知,从红掌转录组SSR基序类型看,11324个SSR位点共包含84种重复基序类型,单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复各有2、4、10、21、13和34种;从SSR重复频率看,单核苷酸重复单元有2种类型,其中A/T类型的重复基序出现频率最多,有2445个(21.59%),C/G类型仅有335个(2.96%)。二核苷酸重复单元有4种类型,其中AG/CT类型出现频率最高,为37.73%(4273次),其次是AC/GT类型6.30%(713次)和AT/AT类型2.15%(244次),出现频率最低的类型为CG/CG,仅为0.22%(25次)。三核苷酸重复单元有10种类型,其中AGG/CCT类型出现频率最高,为6.91%(782次),其次是AAG/CTT类型4.87%(552次)、AGC/CTG类型4.84%(548次)和CCG/CGG类型4.19%(475次),其他类型较少。四核苷酸重复单元有21种,AAAG/GTTT、AGCG/CGCT和AGAT/ATCT类型占主导地位,出现频率分别为0.12%(14次)、0.09%(10次)和0.08%(9次),其他类型均较少。五核苷酸重复单元有13种类型,其中AAAAC/GTTTT和AAAGAG/CTCTT类型出现频率最多,均为0.03%(各3次),其次是AAAAG/CTTTT和AGAGG/CCTCT类型,均为0.02%(各2次),其余类型均为1次。六核苷酸重复单元最多,有34种类型,其中ACGAGG/CCTCGT、ATCGCC/ATGGCG和AGGCGG/CCGCCT类型的出现频率最高,均为0.03%(各3次),其他类型均不超过2次。
SSR长度的变异源于重复单元次数的差异。本研究发现,红掌转录组SSR的重复类型较为丰富,涵盖了单核苷酸~六核苷酸的所有类型。SSR位点的基序重复次数分布在5~70次,主要集中在5~12次。单核苷酸、二核苷酸和三核苷酸分别重复10~70次、6~42次和10~23次,其余核苷酸重复为5~18次。比较不同重复次数的SSR数量,其中重复10次数量的SSR数量最多,为1722个(15.21%);其次重复6次的SSR数量为1701个(15.02%)、重复5次的SSR数量为1577个(13.93%)、重复7次的SSR数量为1170个(10.33%)、重复11次的SSR数量为990个(8.74%)、重复8次的SSR数量为958个(8.46%)、重复12次的SSR数量为676个(5.97%)、重复9次的SSR数量为619个(5.47%),重复13次以上的SSR数量很少,表现为随着重复次数的增加,SSR数量逐渐减少(表5)。
SSR长度变化是其多态性变化的重要因素,红掌转录组重复序列长度为10~385 bp,平均长度为24.99 bp。长度小于12 bp的SSR占比为16.11%(1593个),长度为12~20 bp 的SSR占比最高,为48.12%(4759个),长度为21~30 bp的SSR占比为16.91%(1672个),长度为31~40 bp的SSR占比为6.71%(664个),长度为40 bp以上的SSR占比较少,为12.10%(1197个)(图1)。
对含有SSR位点的7705个Unigenes进行引物设计,成功设计出7456对EST-SSR引物。为进一步验证红掌EST-SSR引物的可用性和有效性,从中随机选出长度为18~24 bp且碱基重复次数2~4的EST-SSR引物120对,以种源类型、株型、叶形和花色均存在明显差异的3个红掌品种(原种花烛、宝贝橙和卡瓦丽)的DNA为模板进行PCR扩增验证,结果(图2)显示,有107对引物能扩增出条带,扩增成功率为89.17%,其中75对引物能在3个红掌品种中表现出多态性,多态性比率为62.5%,从中筛选出20对条带清晰的EST-SSR引物。
利用22个红掌品种对20对EST-SSR引物进行有效性验证,结果(表6)显示,PIC为0.2500~0.8347,平均为0.6830。其中,16对引物具有高度多态性(PIC>0.5000),4对具有中度多态性(0.2500≤PIC≤0.5000)。利用16对高多态性SSR引物对22个红掌品种进行聚类分析,结果(图3)显示,在DSC为0.38处,供试种质聚类为四大类群,Ⅰ类群为观叶品种绿如意;Ⅱ类群为商品化观花红掌品种;Ⅲ类群为原生花烛品种红火鹤;Ⅳ类群为原生花烛品种原种花烛,其中,Ⅲ、Ⅳ类群原生花烛种质独立成簇,与栽培品种遗传差异较大;观叶品种绿如意(Ⅰ类)与其余观花品种(Ⅱ类)明显分离。在Ⅱ类群内,当DSC为0.69时,复色品种梦幻、明农秋月、红唇、彩虹冠军进一步聚类为同一亚群,表明这4个复色品种间具有较高的遗传相似性。16对EST-SSR引物对红掌种质的聚类分析结果与传统形态学分类一致,证实该引物组合适用于红掌种质的亲缘关系推断。此结果亦支持了基于转录组数据开发SSR分子标记技术路径在红掌种质中的可行性。
由于红掌尚无参考基因组且属非模式物种,利用其转录组数据开发SSR分子标记是实现高效标记开发的首选途径(Unamba et al.,2015)。本研究对5个不同花色红掌品种佛焰苞进行转录组分析,获得22381条Unigenes(总长34872210 bp),SSR位点检测结果显示,34.43%的Unigenes(7705条)含有SSR位点,共发现11324个SSR位点。SSR的出现频率为50.60%(平均每1.98 kb Unigenes序列含1个SSR),整体分布密度约为每3.08 kb含1个SSR,表明转录组序列中存在丰富的SSR多态性位点,其出现频率高于前人基于红掌转录组序列的SSR分析结果:郁永明等(2015)发现品种Alabama的SSR出现频率为12.17%;蒋锦雷(2016)发现品种Mystral和Alabama的SSR出现频率为13.33%;林发壮等(2020b)发现品种Alabama的SSR出现频率为18.13%。不同研究间SSR出现频率的差异可能与测序技术、转录组数据来源品种差异和SSR筛选标准有关(Bliss and Suzuki,2012)。与其他植物相比,本研究中红掌SSR的出现频率(50.60%)高于三角梅(44.91%)(赵彤等,2019)、牡丹(25.52%)(贺丹等,2019)、菊花(45.71%)(丁红旭,2020)、兰属植物(20.84%)(孙叶等,2020)、非洲菊(24.65%)(林发壮等,2020a)、石蒜(14.74%)(李青竹等,2021)、野鸦椿(28.20%)(钟琳珊等,2022)、南酸枣(13.02%)(徐梦阳等,2025),但低于桐花树(68.55%)(赵宇航,2019)、美国红枫(55.75%)(郭聪,2021)等植物。整体来看,红掌转录组SSR出现频率处于较高水平。
红掌转录组中SSR重复基元较为丰富,单核苷酸~六核苷酸重复基元均有出现,优势重复基元为二核苷酸重复类型,占SSR总数的46.41%,其次为三核苷酸重复类型(27.69%)和单核苷酸重复类型(24.55%),三者共占SSR总数的98.65%;四核苷酸、五核苷酸和六核苷酸重复类型数量最少,分别占0.79%、0.17%和0.39%,与锥栗(顾光仕等,2020)、芹菜(陈昌龙等,2020)、枇杷(蒋爽等,2021)、美国红枫(郭聪,2021)、灰毡毛忍冬(刘思思等,2021)等大多植物由二核苷酸重复类型占主导地位的研究结果一致。而已有研究报道表明,也有部分植物SSR的主要类型是单核苷酸重复类型,例如柿(杜改改等,2017)、黄麻(陶爱芬等,2019)、三角梅(赵彤等,2019)、非洲菊(林发壮等,2020a)和黑枸杞(黄兴发等,2021),也有少数植物是六核苷酸重复类型(李炎林等,2014)、三核苷酸重复类型(李青竹等,2021)。另外,作为双子叶植物的红掌SSR中二核苷酸重复单元出现频率最高的类型为AG/CT(37.73%),其次是单核苷酸重复单元类型A/T(21.59%),以这2个优势重复类型为主的双子叶植物还有李府贡枣(周军永,2019)、三角梅(赵彤等,2019)、锥栗(顾光仕等,2020)、非洲菊(林发壮等,2020a)、南方红豆杉(蒋路园等,2024)等。本研究中红掌转录组的核苷酸重复类型及其优势重复基元与其他红掌转录组研究相似,红掌以单核苷酸、二核苷酸和三核苷酸SSR基序为高频重复单元,而四核苷酸、五核苷酸和六核苷酸SSR基序占少数的分布可能是自身物种特异性差异及自然选择的结果。
研究发现,SSR位点的重复次数(≥12次)与序列长度(≥20 bp)共同构成多态性阈值(Thao et al.,2013),而短重复基序(如单核苷酸、双核苷酸)因选择压力较低,变异速率显著高于长基序,进而产生更高多态性(Zhang et al.,2005张婷婷等,2023)。红掌转录组SSR位点长度大于20 bp有3533个(35.72%),重复次数12次以上的SSR位点有2587个(22.85%),其中大多数位点属于二核苷酸重复,表明这些SSR位点理论上具有高多态性。为验证上述SSR位点的实际应用价值,本研究随机选取120对EST-SSR引物进行扩增验证,结果显示扩增成功率为89.17%,多态性比率达62.5%,证实基于红掌转录组开发的SSR引物具有较好的扩增效率和多态性。进一步以条带清晰的20对引物对22个红掌品种进行聚类分析,筛选出16对高度多态性引物(PIC>0.5000),其聚类结果(图3)与供试材料的形态学分类高度吻合:22个品种在DSC为0.38处被划分为四大类群,其中两类原生花烛种质(红火鹤和原种花烛)始终独立于所有栽培品种之外,提示野生种质与商品化品种之间存在较大的遗传分化,可能与长期人工选育对栽培品种遗传背景的定向塑造有关;观叶品种绿如意与观花品种的明显分离,则从分子层面印证了以叶型和花型为目标的选育策略对品种遗传结构的深远影响。值得关注的是,在观花品种中,4个复色品种(梦幻、明农秋月、红唇、彩虹冠军)在DSC为0.69处聚为同一亚群,表明这些品种间存在较近的亲缘关系,推测这些品种共享部分育种亲本或花色调控相关的遗传背景。这一亚群聚类信息可为后续复色花色的遗传解析及定向杂交组合的亲本选配提供分子依据。需要指出的是,本研究样本量较少,后续需扩大种群规模验证标记普适性;SSR标记尚未与具体性状(如花香、抗病性)建立连锁关系。后续将整合更多种质资源完善EST-SSR分子标记体系,构建高密度遗传图谱定位重要农艺性状QTL,再结合功能基因组学挖掘调控花色、花香的关键基因,有效推动红掌分子育种从分子标记开发向功能基因挖掘的跨越,加速红掌新品种选育进程。
红掌转录组SSR位点丰度和多态性均较高,可作为高效分子标记开发来源。开发的EST-SSR标记具有多态性高、稳定性好等特点,为红掌种质资源评价、遗传图谱构建和分子标记辅助育种提供了有效工具,有助于后续红掌重要观赏性状的遗传解析和品种改良。

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2026年第57卷第6期
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doi: 10.3969/j.issn.2095-1191.2026.06.014
  • 接收时间:2026-02-20
  • 首发时间:2026-09-03
  • 出版时间:2026-06-25
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  • 收稿日期:2026-02-20
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    1三明市农业科学研究院花卉研究所/福建省(山区)作物遗传改良与创新利用重点实验室,福建 三明 365051

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林发壮(1985-),https://orcid.org/0009-0000-6088-1813,副研究员,主要从事花卉遗传育种研究工作,E-mail:
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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