Article(id=1276597991574213070, tenantId=1146029695717560320, journalId=1235980609244409860, issueId=1276597973173801322, articleNumber=null, orderNo=null, doi=10.3969/j.issn.1000-2561.2024.07.007, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1690819200000, receivedDateStr=2023-08-01, revisedDate=1694707200000, revisedDateStr=2023-09-15, acceptedDate=null, acceptedDateStr=null, onlineDate=1782294279235, onlineDateStr=2026-06-24, pubDate=1721836800000, pubDateStr=2024-07-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1782294279235, onlineIssueDateStr=2026-06-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1782294279235, creator=13701087609, updateTime=1782294279235, updator=13701087609, issue=Issue{id=1276597973173801322, tenantId=1146029695717560320, journalId=1235980609244409860, year='2024', volume='45', issue='7', pageStart='1303', pageEnd='1520', issueExtLink='null', onlineDate='null', pubDate='1721836800000', pubDateStr='2024-07-25', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1782294274847, creator='13701087609', updateTime=1782294274847, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=1359, endPage=1366, ext={EN=ArticleExt(id=1276597991880397264, articleId=1276597991574213070, tenantId=1146029695717560320, journalId=1235980609244409860, language=EN, title=Identification of Germplasm Resources and Construction of Molecular Identity Card for Rhynchostylis, columnId=1236256430060261740, journalTitle=Chinese Journal of Tropical Crops, columnName=Germplasm Resources, Genetics & Breeding, runingTitle=null, highlight=null, articleAbstract=

Rhynchostylis is an epiphytic orchid belonging to the Orchidaceae family. It possesses high ornamental and economic value. In this study, based on the collection of 10 different germplasm resources from three common species of Rhynchostylis, 21 pairs of SSR polymorphic primers were selected using fluorescent-labeled capillary electrophoresis for allele variation detection. Among them, 13 primer pairs exhibited high polymorphism, resulting in a total of 47 amplified alleles. The average number of observed alleles (Na) was 2.238, and the average number of effective alleles (Ne) was 1.860. The observed heterozygosity (Ho) of the 21 loci ranged from 0 to 0.778, with a mean of 0.421, while the expected heterozygosity (He) ranged from 0.148 to 0.574, with a mean of only 0.340. The Shannon index (I) ranged from 0.096 to 0.987, with a mean of 0.567. The clustering results and PCoA analyses of the 10 germplasm were consistent with the traditional botanical classification, and all the germplasm could be classified into three taxa by species. On this basis, four pairs of core primers were selected to construct a molecular ID for each germplasm. This study lays a foundation for the collection, conservation, identification, development and utilization of the germplasm resources of Rhynchostylis.

, authors=null, authorsList=Wenfang XIAO, Zuo LI, Heming CHEN, Fubing LYU, authorCompany=null, correspAuthors=Fubing LYU, 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=1276597994170487254, articleId=1276597991574213070, tenantId=1146029695717560320, journalId=1235980609244409860, language=CN, title=钻喙兰属种质资源鉴评与分子身份证构建, columnId=1236256430219645304, journalTitle=热带作物学报, columnName=种质资源与遗传育种, runingTitle=null, highlight=null, articleAbstract=

钻喙兰属为兰科的一类附生型兰花,具有较高的观赏价值和经济价值。本研究基于前期收集的钻喙兰属3个常见种的10份不同种质资源,采用荧光标记毛细管电泳进行等位变异检测,筛选出21对SSR多态性引物,其中13对引物位点呈高多态性,共扩增出47个等位基因,平均观测等位基因数(Na)为2.238,平均有效等位基因数(Ne)为1.860。21个位点的观测杂合度(Ho)为0~0.778,均值为0.421,期望杂合度(He)为0.148~0.574,均值仅0.340,Shannon指数(I)为0.096~0.987,均值为0.567。10份种质的聚类结果和PCoA分析与传统植物学分类一致,可将10份种质按种划分成3个类群。在此基础上筛选出4对核心引物构建每份种质的分子身份证。本研究为后续钻喙兰属种质资源的收集保存、鉴评和开发利用奠定基础。

, authors=

肖文芳(1985—),女,硕士,助理研究员,研究方向:花卉遗传育种。

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* 吕复兵(LYU Fubing),E-mail:
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肖文芳(1985—),女,硕士,助理研究员,研究方向:花卉遗传育种。

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肖文芳(1985—),女,硕士,助理研究员,研究方向:花卉遗传育种。

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Effects of three kinds of mycorrhizal fungi on growth of Rhynchostylis gigantea seedling[J]. Guangdong Agricultural Sciences, 2017, 44(4): 46-52. (in Chinese), articleTitle=Effects of three kinds of mycorrhizal fungi on growth of Rhynchostylis gigantea seedling, refAbstract=null), Reference(id=1276598290380619787, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, doi=null, pmid=null, pmcid=null, year=2007, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[28], rfOrder=44, authorNames=International Union for the Protection of New Varieties of Plants, journalName=null, refType=null, unstructuredReference=International Union for the Protection of New Varieties of Plants. Guidelines for DNA-profiling: molecular marker selection and database construction//BMT Guidelines[C]. 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Genetic diversity analysis and core collection of Cymbidium ensifolium germplasm resources[J]. Acta Horticulturae Sinica, 2022, 49(1): 175-186. (in Chinese), articleTitle=Genetic diversity analysis and core collection of Cymbidium ensifolium germplasm resources, refAbstract=null), Reference(id=1276598290573557774, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, doi=null, pmid=null, pmcid=null, year=2022, volume=39, issue=3, pageStart=562, pageEnd=570, url=null, language=null, rfNumber=[30], rfOrder=47, authorNames=王黎, 周琪, 高燕会, journalName=浙江农林大学学报, refType=null, unstructuredReference=王黎, 周琪, 高燕会. 石蒜属种间杂交种的鉴定和分子身份证构建[J]. 浙江农林大学学报, 2022, 39(3): 562-570., articleTitle=石蒜属种间杂交种的鉴定和分子身份证构建, refAbstract=null), Reference(id=1276598290649055247, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, doi=null, pmid=null, pmcid=null, year=2022, volume=39, issue=3, pageStart=562, pageEnd=570, url=null, language=null, rfNumber=[30], rfOrder=48, authorNames=WANG L, ZHOU Q, GAO Y H, journalName=Journal of Zhejiang A & F University, refType=null, unstructuredReference=WANG L, ZHOU Q, GAO Y H. Construction of molecular identification card of Lycoris interspecific hybrids[J]. Journal of Zhejiang A & F University, 2022, 39(3): 562-570. (in Chinese), articleTitle=Construction of molecular identification card of Lycoris interspecific hybrids, refAbstract=null)], funds=[Fund(id=1276598281144763357, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, awardId=2024E04J1267, language=CN, fundingSource=广州市科技计划项目(2024E04J1267), fundOrder=null, country=null), Fund(id=1276598281371255774, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, awardId=202127TD, language=CN, fundingSource=广东省农业科学院学科团队建设项目(202127TD), fundOrder=null, country=null), Fund(id=1276598281446753247, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, awardId=2022-NBN-00-015, language=CN, fundingSource=广东名优花卉种质资源库运行维护项目(2022-NBN-00-015), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1276598267492303799, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, xref=null, ext=[AuthorCompanyExt(id=1276598267504886712, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, companyId=1276598267492303799, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences / Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization, Guangzhou, Guangdong 510640, China), AuthorCompanyExt(id=1276598267517469625, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, companyId=1276598267492303799, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=广东省农业科学院环境园艺研究所/广东省园林花卉种质创新综合利用重点实验室,广东广州 510640)])], figs=[ArticleFig(id=1276598277990646741, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=EN, label=Fig. 1, caption=Dendrogram by UPGMA method and molecular identity card of 10 Rhynchostylis germplasms, figureFileSmall=f/W9UDothrsrsF3+wnajwA==, figureFileBig=xVUZicu4f0B5we9iBztU+g==, tableContent=null), ArticleFig(id=1276598278078727126, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=CN, label=图1, caption=10个钻喙兰属种质的UPGMA遗传聚类图及分子身份证, figureFileSmall=f/W9UDothrsrsF3+wnajwA==, figureFileBig=xVUZicu4f0B5we9iBztU+g==, tableContent=null), ArticleFig(id=1276598278468797399, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=EN, label=Fig. 2, caption=Principal coordinate analysis of 10 Rhynchostylis germplasms based on 21 SSR markers, figureFileSmall=KBXjV7qIZ4TITwEX+Y1Pog==, figureFileBig=TigdLzndKr3/nn2GOLBmVg==, tableContent=null), ArticleFig(id=1276598278804341720, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=CN, label=图2, caption=基于21对SSR引物的10份钻喙兰属种质的主坐标分析, figureFileSmall=KBXjV7qIZ4TITwEX+Y1Pog==, figureFileBig=TigdLzndKr3/nn2GOLBmVg==, tableContent=null), ArticleFig(id=1276598278875644889, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=EN, label=Tab. 1, caption=

Traits of tested germplasm resources

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.学名Scientific name花色Flower color花朵大小Flower size/cm花朵数Flower number花梗姿态Peduncle attitude花香Flower scent广州地区花期Flowering time in Guangzhou
R01海南钻喙兰R. gigantea ‘Shen’s Suns’浅黄绿底紫红斑3.9×3.725半下垂浓香1—2月
R02海南钻喙兰R. gigantea白底紫红大斑3.5×3.025半下垂浓香1—2月
R03海南钻喙兰细斑变型R. gigantean f. maculate白底浅紫红细点3.1×3.228半下垂浓香1—2月
R04海南钻喙兰橘色变种R. gigantea var. vivaphandhuli纯橘色3.0×2.920半下垂浓香1—2月
R05海南钻喙兰纯白变种R. gigantea f. alba纯白3.2×3.220半下垂浓香1—2月
R06海南钻喙兰全红变型R. gigantea f. Chang Daeng纯紫红3.2×2.220半下垂浓香1—2月
R07立梗狐尾兰R. coelestis白底尖端蓝紫色1.9×2.323直立浓香5—6月
R08立梗狐尾兰纯白变型R. coelestis f. alba纯白2.3×2.019直立浓香5—6月
R09立梗狐尾兰粉色变型R. coelestis f. rosea白底尖端淡粉色1.6×1.921直立浓香5—6月
R10钻喙兰R. retusa白底浅紫红点2.2×1.9105半下垂基本无香5—6月
), ArticleFig(id=1276598278938559450, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=CN, label=表1, caption=

供试种质资源性状

, figureFileSmall=null, figureFileBig=null, tableContent=
编号No.学名Scientific name花色Flower color花朵大小Flower size/cm花朵数Flower number花梗姿态Peduncle attitude花香Flower scent广州地区花期Flowering time in Guangzhou
R01海南钻喙兰R. gigantea ‘Shen’s Suns’浅黄绿底紫红斑3.9×3.725半下垂浓香1—2月
R02海南钻喙兰R. gigantea白底紫红大斑3.5×3.025半下垂浓香1—2月
R03海南钻喙兰细斑变型R. gigantean f. maculate白底浅紫红细点3.1×3.228半下垂浓香1—2月
R04海南钻喙兰橘色变种R. gigantea var. vivaphandhuli纯橘色3.0×2.920半下垂浓香1—2月
R05海南钻喙兰纯白变种R. gigantea f. alba纯白3.2×3.220半下垂浓香1—2月
R06海南钻喙兰全红变型R. gigantea f. Chang Daeng纯紫红3.2×2.220半下垂浓香1—2月
R07立梗狐尾兰R. coelestis白底尖端蓝紫色1.9×2.323直立浓香5—6月
R08立梗狐尾兰纯白变型R. coelestis f. alba纯白2.3×2.019直立浓香5—6月
R09立梗狐尾兰粉色变型R. coelestis f. rosea白底尖端淡粉色1.6×1.921直立浓香5—6月
R10钻喙兰R. retusa白底浅紫红点2.2×1.9105半下垂基本无香5—6月
), ArticleFig(id=1276598280914076635, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=EN, label=Tab. 2, caption=

Characteristics of 21 polymorphic microsatellite makers

, figureFileSmall=null, figureFileBig=null, tableContent=
引物Primer重复基元SSR motif引物序列(5'-3')Primer sequence (5'-3')片段大小Fragment size/bp检出率Detection rate/%观测等位基因数Na有效等位基因数Ne观测杂合度Ho期望杂合度He多态性信息含量PICShannon指数I
2-N10002234(AAC)5F:CCTGCAGGTTAAACAACAACAA;
R:ATTTAGACCGTGGGAAGCCT
213~227703.0002.7460.3330.4690.8180.866
5-N1001614(GAA)7F: TTTGGTGGAGGATGGAGAAG;
R: GCTTCCTTGAGGTGAAGAGC
97~1061002.0001.5900.3330.2130.5510.381
7-N1001895(TCT)8F: ATCGCCATTTTTGTGCTCTT;
R: ATTGAAATGATGGCTTTCGC
172~192902.0001.6670.2220.2220.3540.414
14-N1007590(CT)5F: TGACATGGAAGTTATGGCATTC;
R: TGTCGCAATTCTATGTCAGGA
115~1281002.6671.8140.6670.4310.5310.709
22-N10009521(AT)9F: TGAAGAAAAGGTCTAACAAGCAGA;
R: TGAGTTAAAGGAAGAAATGGCCT
126~1361002.3331.6860.5000.3380.5660.559
25-N101022(ATT)5F: TGGACCACCTATCTCAAGGC;
R: ACGACATGCTCATGATTCCA
128~1331002.0001.6000.3330.3150.4360.501
28-N1001301(ATT)19F: TCCTTCCAACATTTATTGCTCC;
R: TCACATCCTTCAATATTGCCC
135~163901.6671.5560.3330.2080.8200.347
28-N1012378(AAG)7F: GGAAGAAGGAGGAGGTGAGG;
R: TCCTAGAGAAAGAGCTTGGAGC
167~1791002.3332.0910.6670.3980.3470.653
37-N3601419(AAG)8F: AATGGAAAATCTGGCAGCAC;
R: TCTCCATCACTTCAATGGCA
181~1901002.0001.8570.6670.3700.6920.568
42-N8802928(TA)8F: CCCAAAACTTTACTTTTCCAGC;
R: CTGTAAAAGCGTACCTGGCA
96~1221002.6672.2270.4440.5320.3750.843
43-N5725511(TA)6F: CTGTAAAAGCGTACCTGGCG;
R: AGGACCCCCAACATACAACA
120~148902.6672.0430.4440.3940.7630.684
45-N1438823(GAA)5F: ATTTGGCTCAAATCCACCAA;
R: ATACCTGAAAGCATGGTGCC
163~1941002.0001.8570.6670.3700.6280.568
68-N2965390(AAT)6F: TCAATGATATCACATTCCTTGTACG;
R: GCTAGGATTTTGTTAATTATTGTGC
98~1121001.3331.0600.0560.0510.4090.096
74-N8009753(TTC)10F: TGTTTGCAGGGAAATTTGTTTCA;
R: TCTTCAACTTATGTGATTGCAGG
105~1321003.3332.7670.6670.5740.4600.987
82-N100023(AG)6F: AGGGTGGGAAGGGAGATAAA;
R:CTCTGCCTTCTCCATCTCTCA
154~1711002.0001.7950.7780.4260.6320.612
88-N1001036(AG)5F: AGGGAGGGAGTGAGAGAGGA;
R: TCCCGCTTTCTATCGCTATC
143~1611002.0001.4710.3890.2640.7190.420
88-CB033847(AT)31F: GCTCCGCTAGCTCTGACAGA;
R: CATCCGAGCTGATGAAAGGT
134~1401002.6672.2600.6110.4580.5310.770
89-N100105(TA)5F: TTTCCTGCTCTCCATTCCTG;
R: TGTCATCTTCTCACACTCCCA
97~1011002.3331.6490.3330.3290.5530.564
90-CB034920(TGA)12F: TGTTGGTCTTCGGAAAGAAGTAT;
R: AGCGCAGATAATAAAATCCTACC
137~1671002.6672.1960.1670.3940.3650.701
91-N1001523(AG)8F: TGATGGAATGAAGTTGAGGGA;
R: GGCACATATGCTTTGTGGTG
116~1361002.0001.8670.2220.2410.5260.443
92-CK858704(GA)6F: GAGGCTGAGCAAAAGATTATGAG;
R: CGAATCATCGGATTCTTACTCTT
160~1631001.3331.2670.0000.1480.4200.212
均值2.2381.8600.4210.3400.5470.567
), ArticleFig(id=1276598280993768412, tenantId=1146029695717560320, journalId=1235980609244409860, articleId=1276597991574213070, language=CN, label=表2, caption=

21对多态性引物相关信息

, figureFileSmall=null, figureFileBig=null, tableContent=
引物Primer重复基元SSR motif引物序列(5'-3')Primer sequence (5'-3')片段大小Fragment size/bp检出率Detection rate/%观测等位基因数Na有效等位基因数Ne观测杂合度Ho期望杂合度He多态性信息含量PICShannon指数I
2-N10002234(AAC)5F:CCTGCAGGTTAAACAACAACAA;
R:ATTTAGACCGTGGGAAGCCT
213~227703.0002.7460.3330.4690.8180.866
5-N1001614(GAA)7F: TTTGGTGGAGGATGGAGAAG;
R: GCTTCCTTGAGGTGAAGAGC
97~1061002.0001.5900.3330.2130.5510.381
7-N1001895(TCT)8F: ATCGCCATTTTTGTGCTCTT;
R: ATTGAAATGATGGCTTTCGC
172~192902.0001.6670.2220.2220.3540.414
14-N1007590(CT)5F: TGACATGGAAGTTATGGCATTC;
R: TGTCGCAATTCTATGTCAGGA
115~1281002.6671.8140.6670.4310.5310.709
22-N10009521(AT)9F: TGAAGAAAAGGTCTAACAAGCAGA;
R: TGAGTTAAAGGAAGAAATGGCCT
126~1361002.3331.6860.5000.3380.5660.559
25-N101022(ATT)5F: TGGACCACCTATCTCAAGGC;
R: ACGACATGCTCATGATTCCA
128~1331002.0001.6000.3330.3150.4360.501
28-N1001301(ATT)19F: TCCTTCCAACATTTATTGCTCC;
R: TCACATCCTTCAATATTGCCC
135~163901.6671.5560.3330.2080.8200.347
28-N1012378(AAG)7F: GGAAGAAGGAGGAGGTGAGG;
R: TCCTAGAGAAAGAGCTTGGAGC
167~1791002.3332.0910.6670.3980.3470.653
37-N3601419(AAG)8F: AATGGAAAATCTGGCAGCAC;
R: TCTCCATCACTTCAATGGCA
181~1901002.0001.8570.6670.3700.6920.568
42-N8802928(TA)8F: CCCAAAACTTTACTTTTCCAGC;
R: CTGTAAAAGCGTACCTGGCA
96~1221002.6672.2270.4440.5320.3750.843
43-N5725511(TA)6F: CTGTAAAAGCGTACCTGGCG;
R: AGGACCCCCAACATACAACA
120~148902.6672.0430.4440.3940.7630.684
45-N1438823(GAA)5F: ATTTGGCTCAAATCCACCAA;
R: ATACCTGAAAGCATGGTGCC
163~1941002.0001.8570.6670.3700.6280.568
68-N2965390(AAT)6F: TCAATGATATCACATTCCTTGTACG;
R: GCTAGGATTTTGTTAATTATTGTGC
98~1121001.3331.0600.0560.0510.4090.096
74-N8009753(TTC)10F: TGTTTGCAGGGAAATTTGTTTCA;
R: TCTTCAACTTATGTGATTGCAGG
105~1321003.3332.7670.6670.5740.4600.987
82-N100023(AG)6F: AGGGTGGGAAGGGAGATAAA;
R:CTCTGCCTTCTCCATCTCTCA
154~1711002.0001.7950.7780.4260.6320.612
88-N1001036(AG)5F: AGGGAGGGAGTGAGAGAGGA;
R: TCCCGCTTTCTATCGCTATC
143~1611002.0001.4710.3890.2640.7190.420
88-CB033847(AT)31F: GCTCCGCTAGCTCTGACAGA;
R: CATCCGAGCTGATGAAAGGT
134~1401002.6672.2600.6110.4580.5310.770
89-N100105(TA)5F: TTTCCTGCTCTCCATTCCTG;
R: TGTCATCTTCTCACACTCCCA
97~1011002.3331.6490.3330.3290.5530.564
90-CB034920(TGA)12F: TGTTGGTCTTCGGAAAGAAGTAT;
R: AGCGCAGATAATAAAATCCTACC
137~1671002.6672.1960.1670.3940.3650.701
91-N1001523(AG)8F: TGATGGAATGAAGTTGAGGGA;
R: GGCACATATGCTTTGTGGTG
116~1361002.0001.8670.2220.2410.5260.443
92-CK858704(GA)6F: GAGGCTGAGCAAAAGATTATGAG;
R: CGAATCATCGGATTCTTACTCTT
160~1631001.3331.2670.0000.1480.4200.212
均值2.2381.8600.4210.3400.5470.567
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钻喙兰属种质资源鉴评与分子身份证构建
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肖文芳 , 李佐 , 陈和明 , 吕复兵 *
热带作物学报 | 种质资源与遗传育种 2024,45(7): 1359-1366
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热带作物学报 |种质资源与遗传育种 2024 , 45 (7) : 1359 -1366
钻喙兰属种质资源鉴评与分子身份证构建
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肖文芳, 李佐, 陈和明, 吕复兵*
作者信息
  • 广东省农业科学院环境园艺研究所/广东省园林花卉种质创新综合利用重点实验室,广东广州 510640
通讯作者:
* 吕复兵(LYU Fubing),E-mail:
Identification of Germplasm Resources and Construction of Molecular Identity Card for Rhynchostylis
Wenfang XIAO, Zuo LI, Heming CHEN, Fubing LYU*
Affiliations
  • Environmental Horticulture Research Institute, Guangdong Academy of Agricultural Sciences / Guangdong Key Lab of Ornamental Plant Germplasm Innovation and Utilization, Guangzhou, Guangdong 510640, China
出版时间: 2024-07-25 doi: 10.3969/j.issn.1000-2561.2024.07.007
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钻喙兰属为兰科的一类附生型兰花,具有较高的观赏价值和经济价值。本研究基于前期收集的钻喙兰属3个常见种的10份不同种质资源,采用荧光标记毛细管电泳进行等位变异检测,筛选出21对SSR多态性引物,其中13对引物位点呈高多态性,共扩增出47个等位基因,平均观测等位基因数(Na)为2.238,平均有效等位基因数(Ne)为1.860。21个位点的观测杂合度(Ho)为0~0.778,均值为0.421,期望杂合度(He)为0.148~0.574,均值仅0.340,Shannon指数(I)为0.096~0.987,均值为0.567。10份种质的聚类结果和PCoA分析与传统植物学分类一致,可将10份种质按种划分成3个类群。在此基础上筛选出4对核心引物构建每份种质的分子身份证。本研究为后续钻喙兰属种质资源的收集保存、鉴评和开发利用奠定基础。

钻喙兰属  /  SSR  /  分子身份证

Rhynchostylis is an epiphytic orchid belonging to the Orchidaceae family. It possesses high ornamental and economic value. In this study, based on the collection of 10 different germplasm resources from three common species of Rhynchostylis, 21 pairs of SSR polymorphic primers were selected using fluorescent-labeled capillary electrophoresis for allele variation detection. Among them, 13 primer pairs exhibited high polymorphism, resulting in a total of 47 amplified alleles. The average number of observed alleles (Na) was 2.238, and the average number of effective alleles (Ne) was 1.860. The observed heterozygosity (Ho) of the 21 loci ranged from 0 to 0.778, with a mean of 0.421, while the expected heterozygosity (He) ranged from 0.148 to 0.574, with a mean of only 0.340. The Shannon index (I) ranged from 0.096 to 0.987, with a mean of 0.567. The clustering results and PCoA analyses of the 10 germplasm were consistent with the traditional botanical classification, and all the germplasm could be classified into three taxa by species. On this basis, four pairs of core primers were selected to construct a molecular ID for each germplasm. This study lays a foundation for the collection, conservation, identification, development and utilization of the germplasm resources of Rhynchostylis.

Rhynchostylis  /  SSR  /  molecular ID
肖文芳, 李佐, 陈和明, 吕复兵. 钻喙兰属种质资源鉴评与分子身份证构建. 热带作物学报, 2024 , 45 (7) : 1359 -1366 . DOI: 10.3969/j.issn.1000-2561.2024.07.007
Wenfang XIAO, Zuo LI, Heming CHEN, Fubing LYU. Identification of Germplasm Resources and Construction of Molecular Identity Card for Rhynchostylis[J]. Chinese Journal of Tropical Crops, 2024 , 45 (7) : 1359 -1366 . DOI: 10.3969/j.issn.1000-2561.2024.07.007
钻喙兰属(Rhynchostylis Blume)为兰科、树兰亚科、万代兰族、指甲兰亚族的一类单轴生长型附生兰花,具花多且密的总状花序,因此又得名狐狸尾兰。该属目前仅发现和鉴定5个原生种,包括钻喙兰(R. retusa)、海南钻喙兰(R. gigantea)、立梗狐尾兰(R. coelestis)、R. rieferiR. cymifera,主要分布于我国的云南省、贵州省、海南岛,以及印度、斯里兰卡和东南亚各国[1]。该属植物株型紧凑、花繁色艳且绝大多数具浓烈芳香,栽培管理简单,耐热性强,易杂交,具有较高的观赏价值和开发应用前景。从19世纪中叶开始,陆续有钻喙兰属的种间杂交种、与近缘兰科植物之间的属间杂交种在英国皇家园艺学会(Royal Horticultural Society)登录,截至2023年6月,登录的钻喙兰属原生种F1代杂交组合已达402个。泰国作为野生钻喙兰属植物分布的中心地区之一,是最早开展钻喙兰属植物育种、繁殖及栽培研究的国家之一,培育的品种在世界各地十分畅销。我国最早于19世纪末20世纪初由台湾企业从泰国引种栽培钻喙兰属植物,并进行育种和栽培的相关研究,目前已培育出四倍体海南钻喙兰等极具特色的新品种,成为国内外各大兰展的获奖热门品种[2]。但我国大陆地区的市场尚未打开,钻喙兰属植物鲜为消费者所知。
除传统杂交育种工作外,钻喙兰属植物的相关研究主要集中在组培快繁、栽培管理和野生资源分布及鉴定等方面[3-7],分子生物学方面仅有极少量利用不同分子标记或简化基因组测序针对单独的种进行遗传多样性等方面的分析。针对海南钻喙兰,目前仅利用SRAP标记技术分析来自海南岛22个居群地的144份野生资源的遗传多样性[8],发现海南钻喙兰的核型公式为2n=2x=36m+ 2sm[9],并利用RT-PCR结合RACE技术从海南钻喙兰中分离得到2个仅在生殖器官中表达的B类MADS-box基因RgAP3RgPI[10];针对钻喙兰,目前仅利用2个RAPD引物对包括钻喙兰在内的9个不同的兰科原生种进行了遗传相似性分析[11],利用13条RAPD引物和7条ISSR引物对采自印度7个不同地区的钻喙兰进行遗传多样性分析[12],并利用ddRAD技术对包括钻喙兰在内的4个兰花原生种进行简化基因组测序和遗传多样性分析[13];针对立梗狐尾兰构建了包含10 600个BAC克隆的人工细菌染色体[14]以供后续进行基因组结构分析。
基于钻喙兰属植物本身的优良特性,及其与蝴蝶兰(Phalaenopsis Blume)、万代兰(Vanda)、指甲兰(Aerides falcata Lindl.)等能够进行远缘杂交创制跨属杂交新种质的优势,近年来受到越来越多育种工作者的关注,急需系统性开展钻喙兰属种质资源的开发、保护、保存与鉴评工作。TP-M13-SSR(simple sequence repeat with tailed primer M13)技术结合了SSR分子标记技术和荧光测序技术,具有重复性高、结果准确等优点,在较大程度上解决了传统凝胶电泳分析通量较低、扩增产物检测流程繁琐、数据记录工作量大等一系列问题[15]。目前TP-M13-SSR已在茶花[16]、百合[17]、桂花[18]、荷花[19]等各类观赏花卉的种质资源鉴定和指纹图谱构建方面得到应用,在兰科植物中已成功应用于蝴蝶兰的突变体鉴定[20]、建兰(Cymbidium ensifolium)的核心种质构建[21]、石斛兰(Dendrobium Sw.)原生种的鉴评[22]等方面。
为充分保护和利用钻喙兰属资源的多样性,本研究利用自主开发的钻喙兰属基因组SSR引物和兰科通用SSR引物,对钻喙兰属常见3个种的10份种质资源进行分析鉴评,从DNA水平揭示钻喙兰属种质资源的亲缘关系,分析其遗传多样性并建立种质资源的分子身份证,为钻喙兰属种质资源的保护及开发利用提供理论依据。
供试品种为广东省农业科学院环境园艺研究所收集保存的10份钻喙兰属原生种资源,具体性状信息见表1,均种植于广东省农业现代化科技示范区广东省农业科学院环境园艺研究所温室大棚内,于2020年5月采集根尖,于液氮中速冻15 min后放入超低温冰箱,备用。
根据简化基因组信息开发筛选出12对钻喙兰属内和12对钻喙兰属与蝴蝶兰属通用引物(待发表),在不同样品中进行单重PCR扩增,反应体系总体积为10 µL,其中含50 ng/µL DNA模板1.2 µL,10×Buffer Ⅰ缓冲液1.0 µL,5 U/µL的TaKaRa HS Taq酶0.1 µL,5 µmol/L的上下游引物各0.3 µL,2.5 mmol/L的dNTP 0.8 µL,5 µmol/L的TP-M13 0.5 µL,去离子水5.8 µL。反应程序为:95 ℃预变性5 min;95 ℃变性30 s,60 ℃退火30 s,72 ℃延伸30 s,30个循环;95 ℃变性30 s,53 ℃退火30 s,72 ℃延伸30 s,10个循环;60 ℃,30 min。
在96孔板中,每孔加入筛选引物的扩增产物1.0 μL,ROX-500分子量内标和甲酰胺混合液(0.5∶8.5)9 μL,95 ℃变性3 min后,利用ABI 3730XL检测仪进行检测,1 kV电压进样10 s,电泳15 kV,30 min。将Data Collection软件收集的原始数据文件导入到GeneMapper 3.2软件中进行分析,将各峰值的位置与其泳道中的分子量内标予以比较,计算目标DNA片段的准确大小。各荧光标记座位上独立进行3次重复的毛细管电泳检测,取3次重复的平均值并四舍五入取整,作为试验材料在该座位上的数据。
根据扩增数据对10份钻喙兰属种质进行遗传多样性分析。运用GenAlEx 6.503软件计算引物的等位基因数(Na)、有效等位基因数(Ne)、Shannon指数(I)、观测杂合度(Ho)和期望杂合度(He),并绘制PCoA图,利用PowerMarker V3.25软件计算引物的多态性信息含量(PIC),利用NTSYS 2.10软件进行聚类作图。并根据引物的PIC及其在钻喙兰属内不同种间的通用性筛选核心引物,构建每份种质的分子身份证。
钻喙兰属目前已发现和鉴定的原生种共5个,其中3个发现较早、分布广泛、杂交利用率较高,3个种均已有杂交后代登录,另2个原生种分布范围狭窄、发现较晚,尚无育种利用和针对性研究。
广泛分布于中国海南岛和中国黄海附近岛屿,缅甸仰光和东高附近以及北部的其他地方,泰国东部地区、西南地区的北碧府,新加坡及附近岛屿,老挝的万象平原和波罗芬高地,柬埔寨的Camchay山,越南胡志明市和广治省附近,婆罗洲,印度尼西亚的阿南巴斯岛。该种的主要特点:花序下垂;花较大,花朵平均直径为3.4 cm;唇瓣片前部明显3裂;距狭圆锥形,底端锐尖;蕊柱足不明显;芳香;花期冬季到春季[23]。山采株的花色主要为白底红点、白底红粗斑及纯白色变种。目前不同品系间已杂交选育出纯红色变形,且台湾地区利用粗斑带橙色的突变个体已选育出橙色海南钻喙兰,并利用橙色个体与白色变种进行杂交选育偏绿色的海南钻喙兰。截至2023年6月,该种在英国皇家园艺学会登录的杂交F1代有186个,其中海南钻喙兰作为父本的有156个,作为母本的有30个,可见其作为父本的杂交育性显著高于作为母本的杂交育性。
广泛分布于泰国北部、东北部、西部、西南部的山区和巴蜀省的低海拔地区,柬埔寨南部,越南大叻附近的山区,老挝。该种的主要特点:花序挺立斜生;花较小,花朵平均直径为2.3 cm;唇瓣片前部明显3裂;距狭圆锥形,底端锐尖下弯;蕊柱足明显;芳香;花期春季到夏季[23]。山采株的花色为白底带蓝紫色晕、蓝紫色唇瓣,也有白底带紫红色晕的变形和纯白色的变形。目前中国台湾地区利用多倍体育种技术已选育出四倍体的立梗蓝狐尾。截至2023年4月,该种已在英国皇家园艺学会登录的杂交F1代有174个,其中立梗狐尾兰作为母本的有137个,作为父本的有37个,其作为父本的杂交育性显著高于作为母本的杂交育性。
广泛分布于中国云南省西南部和贵州省,斯里兰卡,印度北部和南部,泰国,马来西亚,爪哇岛,菲律宾吕宋岛的巴丹省、布拉干省、甲米地和黎刹省。该种的主要特点:花序下垂;花较小,花朵平均直径为2.2 cm;唇瓣片不明显3裂;距囊状,两侧压扁,底端钝圆;蕊柱足明显;无香或淡香;花期冬季到春季[23]。山采株的花色为白底紫红点,已选育出纯白色变形。截至2023年4月,该种已在英国皇家园艺学会登录的杂交F1代有46个,其中钻喙兰作为母本的仅有2个,作为父本的有44个,其作为父本的杂交育性同样显著高于作为母本的杂交育性。
仅分布于菲律宾。2009年发现并鉴定为钻喙兰属新种,尚无育种利用等针对性的相关研究。该种的主要特点:花序下垂;花较小;唇瓣狭窄且小,急剧上翘;距较短;芳香[24]
仅分布于印度西南部Nilambur的常绿森林中。2018年发现并鉴定为钻喙兰属新种,尚无育种利用等针对性的相关研究。该种的主要特点:花序下垂;花较小;斜菱形花瓣;方形渐变成倒卵形的唇瓣,先端微凹;长且尖的距;球形花粉块;无香;花期春季到夏季[25]
本研究共采用6个不同的海南钻喙兰原生种、3个不同的立梗狐尾兰原生种和1个山采株色钻喙兰原生种,涵盖钻喙兰属常见3个种中目前发现和鉴定的所有变种和变型,具体性状见表1。除花色外,海南钻喙兰与钻喙兰、立梗狐尾兰最主要的性状区别在于花梗的下垂与直立,而钻喙兰区别于海南钻喙兰和立梗狐尾兰的性状主要在于花香及花朵数,钻喙兰基本无香但花朵数可多达百朵,海南钻喙兰和立梗狐尾兰均具浓香但花朵数较少。
采用自主开发的12对钻喙兰属特异引物和12对钻喙兰属与蝴蝶兰属通用引物,对来自3个种的10份钻喙兰属种质进行检测,其中3对引物扩增后不具多态性,其余21对引物共扩增出47个等位基因(表2),每对引物扩增出1~3个等位基因,Na均值为2.238,Ne均值为1.860。24个位点的Ho为0~0.778,均值为0.421,He为0.148~ 0.574,均值仅0.340,I介于0.096~0.987之间,均值为0.567,表明供试种质的杂合程度极低,遗传多样性不高,这与供试种质均为原生种或其变型和变种有关。21对引物中有13对引物位点呈高多态性(PIC>0.5)[26],证明本研究所筛选出的多数引物适用于钻喙兰属种质鉴评。
根据Nei’s遗传距离,对10份钻喙兰属种质资源构建UPGMA遗传关系聚类图(图1),如图1所示,10份钻喙兰属种质被分为三大类,同一个种的种质聚为一类,10份钻喙兰属种质的PCoA分析结果显示,PCoA1和PCoA2分别占所有钻喙兰属种质总遗传变异的32.63%和15.30%;PCoA分析同样将10份种质分为三大类,同一个种的种质聚为一类(图2)。聚类结果和PCoA分析结果与钻喙兰属种质植物学分类结果一致。
根据引物多态性信息含量(PIC)及其在钻喙兰属内不同种间的通用性,从1对引物开始逐步增加引物数量筛选出可将供试种质全部区分的引物组合,最终确定引物组合28-N1001301+2-N10002234+43-N5725511能够将所有样品区分开,但这3个引物的通用性均未达到100%,考虑到后续样品的适用性,增加通用性100%的88-N1001036引物(PIC仅次于前3个引物)作为核心引物,最终确定28-N1001301+2-N10002234+ 43-N5725511+8-N1001036作为今后钻喙兰属种质资源鉴定的核心引物组合,其他引物作为备选。
通过数据化编码构建钻喙兰属种质的分子身份证,其构成包括2个部分。第1部分为前2位字母,表示物种信息,钻喙兰属目前共5个种,包括海南钻喙兰(RG)、立梗狐尾兰(RC)、钻喙兰(RR)、R. rieferi(RI)、R. cymifera(RC)。第2部分为后续的数字,代表4对核心引物扩增结果,2-N10002234、28-N1001301、43-N5725511、88-N10010364的扩增片段按分子量大小排列,从小到大以阿拉伯数字1~9标注,若该位点在某个种质中无扩增则记为0,每个引物占2位。如R01海南钻喙兰(R. gigantea ‘Shen’s Suns’)的分子身份证为RG00334411,RG代表其为海南钻喙兰这个种的种质资源,00334411为其通过核心引物扩增转换而来的专属编码。最终构建的10份钻喙兰属种质分子身份证代码均不相同,能够将所有种质区分开,并利用每个种质资源的分子身份证编号,分别制作种质特异的条形码(图1)。
钻喙兰属种质资源虽不丰富,但作为蝴蝶兰属近缘物种,具有易杂交的特性,可与蝴蝶兰进行跨属远缘杂交创制新种质。因钻喙兰属大多数种质具浓香,可作为蝴蝶兰新品种的香味性状亲本来源,目前钻喙兰属常见3个种与蝴蝶兰属不同种质均已有杂交新品种育成。且钻喙兰属种质具有蝴蝶兰不具备的许多优点,如栽培管理简单粗放、抗性强,海南钻喙兰在广东地区不用低温催花可应春节开花,因此钻喙兰属种质极具开发应用潜力。虽然钻喙兰属的育种工作起步较早,1825年已在野外发现钻喙兰这个原生种并命名,1935年在英国皇家园艺学会上登录了第1个钻喙兰与凤蝶兰的跨属杂交种Rhynchanthe Bernardii,1970年在英国皇家园艺学会上登录了第1个海南钻喙兰与钻喙兰间的种间杂交种R. Chorchalood,但与蝴蝶兰属、石斛属、万代兰属等其他大属相比,其进展缓慢,目前国内外仅个别兰场栽培了少量单株作为稀有资源进行销售,资源鉴评等相关研究相对十分匮乏,严重制约了钻喙兰属种质的保护和开发利用。
我国作为钻喙兰属野生资源分布区,海南岛分布大量海南钻喙兰,而云南和贵州则分布有钻喙兰野生资源,但目前针对相关资源仅进行了少量的分布调查[2,7]、组培快繁和栽培管理[5-6,27]等方面的简单研究,资源鉴评方面仅利用SRAP标记技术分析了来自海南岛22个居群地144份野生资源的遗传多样性[7]。SSR分子标记是国际植物新品种保护联盟指定的构建植物DNA指纹图谱的首选方法[28],可检测DNA序列的差异,稳定性强、可重复性好,已广泛应用于兰科植物的种质鉴评[20-22,29]。分子身份证是将目标种质的分子指纹图谱进行编码,形成种质对应的特异编号储存其身份信息,对于数字化管理种质和鉴别具有重要作用,已在石斛兰[22]、石蒜属[30]等花卉中成功应用。本研究基于自有资源圃收集保存的钻喙兰属3个常见种的10份不同种质资源,利用自主开发的SSR引物,对其进行遗传多样性分析、亲缘关系分析和分子身份证构建。根据SSR分子标记分析的遗传距离系数,利用UPGMA方法和PCoA分析可以将供试的10份钻喙兰属种质按照种划分为3个类群,与传统植物分类结果一致。根据引物多态性信息含量及其在钻喙兰属内不同种间的通用性筛选出的4对核心引物能够将10份钻喙兰属种质完全区分开,为每份种质建立了独有的分子身份证。这充分说明本研究开发筛选的引物适用于钻喙兰属种质的鉴评,可用于野生资源的遗传多样性分析、品种资源的分类鉴定、资源圃的分类管理等,为今后钻喙兰属种质的收集保存和鉴定提供理论基础。
  • 广州市科技计划项目(2024E04J1267)
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  • 广东名优花卉种质资源库运行维护项目(2022-NBN-00-015)
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2024年第45卷第7期
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doi: 10.3969/j.issn.1000-2561.2024.07.007
  • 接收时间:2023-08-01
  • 首发时间:2026-06-24
  • 出版时间:2024-07-25
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  • 收稿日期:2023-08-01
  • 修回日期:2023-09-15
基金
广州市科技计划项目(2024E04J1267)
广东省农业科学院学科团队建设项目(202127TD)
广东名优花卉种质资源库运行维护项目(2022-NBN-00-015)
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    广东省农业科学院环境园艺研究所/广东省园林花卉种质创新综合利用重点实验室,广东广州 510640

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* 吕复兵(LYU Fubing),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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