Gilliland, Trevor J.

Provan, J.

Wallace, Maragret

English

University of Kentucky

University of Kentucky UKnowledge International Grassland Congress Proceedings XXI International Grassland Congress / VIII International Rangeland Congress Molecular Markers for Classification of Seed Collections of Lolium Cultivars Maragret Wallace Queens University Belfast, UK J. Provan Queens University Belfast, UK Trevor J. Gilliland Queens University Belfast, UK Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Plant Sciences Commons, and the Soil Science Commons This document is available at https://uknowledge.uky.edu/igc/21/14-1/8 The XXI International Grassland Congress / VIII International Rangeland Congress took place in Hohhot, China from June 29 through July 5, 2008. Proceedings edited by Organizing Committee of 2008 IGC/IRC Conference Published by Guangdong People's Publishing House This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in International Grassland Congress Proceedings by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. 瞯 ]556　 瞯 　 Multifunctional Grasslands in a Changing World 　 Volume Ⅱ 　Grasslands/Rangelands Production Systems——— Seed Science and TechnologyMolecular markers for classification of seed collections of Lolium cultivarsM . W allace1 ,2 , J . Provan1 and T . J . Gilliland , 1 ,21 School o f Biological Sciences , Queens University Bel f ast , Northern I reland , 2 A gri‐Food & Biosciences Institute ,Crossnacreev y , Northern I reland , maragret .w allace＠ af bini .gov .ukKey words : Lolium ,taxonomy ,microsatellites ,SNPsIntroduction Molecular based methodologies have been successfully used by several workers to expose discriminating levels of diversitybetween ryegrass seed accessions but cannot be used for registering new varieties as they do not protect against plagiarism . They can ,however , assist in assessing relatedness for Essential Derivation relationships ( Roldan‐Ruiz et al ２０００ ) , and so identify varieties ashighly similar pairs . If more widely related groupings could be identified , in which all varieties still conform to a shared seed sampleprofile , then discrimination of new varieties from all members of a group may only require one group representative to be sown intraditional field based identification trials .Materials and methods Seed of twenty four ryegrass ( Lolium spp) varieties representative of the diversity in commerce , were geneticallyscreened by sequencing three chloroplast genome regions ( trnT‐trnL intergenic spacer , trnL intron and trnL‐trnF intergenic spacer ;Taberlet et al . １９９１) , to identify microsatellite polymorphisms ( method : Provan et al . ２００１ ) and single nucleotide polymorphisms( SNPs) , using the allele‐specific PCR (AS‐PCR) approach ( Provan et al . ２００８) .　 　 Table 1 A lignment o f three regions o f the chlorop lastgenome sequenced in ２４ cultiv ars . １ BX４２１ ,２ Tewera , ３Barsilo , ４ Brutus , ５ Glds Manawa , ６ Foy le , ７Abercomo , ８ Lemtal , ９ Danergo , １０ Aberel f , １１Loretta , １２ Numan , １３ Bealy , １４ Bronsyn , １５ Moy , １６Aberdart , １７ Aberelan , １８ Aberstar , １９ Fennema , ２０Kent IndigN , ２１ Melle , ２２ Ty rone , ２３ Veritas , ２４Herbal . Sites in bold show SN Ps .trnT‐trnL trnL intron trnL‐trnF２ .２ 厖５ 葺６ 5３ 寣３ 滗３ <３ 摀３ 腚３ C３ 殮１ １ W１ �１  １ ^１ 抖３ 种３ .６ 厖２ 葺５ 5５ 寣５ 滗５ <６ 摀６ 腚６ C７ 殮６ 蝌７ I８ 　８ �８ P８ è９ ９ W９ ９  ９ ^９ 抖６ 种１ .１ 厖２ 葺１ 5７ 寣８ 滗９ <０ 摀１ 腚２ C７ 殮２ 蝌９ I０ 　１ �２ P３ è０ １ W２ ３  ４ ^５ 抖１ bC G C G T T T T T ‐ ‐ G A A A T A T C ‐ ‐ ‐ ‐ ‐２ bC G C G T T T T T ‐ ‐ G A A A T A T C ‐ ‐ ‐ ‐ ‐３ bC G C G T T T T T ‐ ‐ G A A A T A T C ‐ ‐ ‐ ‐ ‐４ bC G C G T T T T T ‐ ‐ G A A A T A T C ‐ ‐ ‐ ‐ ‐５ bA A C ‐ T T T T T T ‐ G G ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐６a C G C G T T T T ‐ ‐ ‐ G A A A T A T C ‐ ‐ ‐ ‐ ‐６b C G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C ‐ ‐７ bC G C G T T T T T ‐ ‐ G A ‐ ‐ ‐ ‐ ‐ C ‐ ‐ ‐ ‐ ‐８ bC G C G T T T T T ‐ ‐ G A ‐ ‐ ‐ ‐ ‐ C ‐ ‐ ‐ ‐ ‐９ bC G C G T T T T T ‐ ‐ G A ‐ ‐ ‐ ‐ ‐ C ‐ ‐ ‐ ‐ ‐１０ tC G C G T ‐ ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C C１１ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C ‐ ‐１２ tC G A G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐１３ tC G C ‐ A T T T T ‐ ‐ G A ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐１４ tC G A G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C ‐ ‐１５ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐１６ tC G C G T ‐ ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C C１７ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐１８ tC G C G T ‐ ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C C１９ tC G C G T ‐ ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C C２０ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐２１ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐２２ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐２３ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐２４ tC G C G T T ‐ ‐ ‐ ‐ ‐ T A ‐ ‐ ‐ ‐ ‐ C C C C C ‐Results and discussion Sequencing of the trnT‐L‐F regionidentified both single nucleotide polymorphisms ( SNPs ) andmicrosatellite length polymorphism . Both of these clearlydelineated L . perenne ( １０‐２４ ) , which have the shortermicrosatellite motif lengths in the trnL intron and the longermicrosatellite motif lengths in the trnL‐trnF intergenic region ,as well as a diagnostic SNP at position ３７７ in the trnL intron ,from the L multi f lorum complex (１‐９) . This complex includedone Festulolium (１) , one Westerwold ( ２ ) , four L . perenne xL . multi f lorum hybrids ( ３‐６ ) and three L . multi f lorum ( ７‐９ ) . A notable exception was Bealey ( １３) , which displayed a L .multi f lorum type cytoplasm , suggesting that in the breeding ofthis L . perenne , the final pollycross was constructed uponItalian or hybrid mothers . The hybrid Foyle (６a , ６b) containedboth perennial and Italian cytoplasms , indicating that maternalplants from each species existed in the final multicross , whereasall the other hybrids were created only from Italian mothers .Interestingly , Foyle expresses a very strong perennial‐likephenotype . The three New Zealand varieties ( ５ , １１ , １４ ) allexpressed unique SNPs in contrast to the European or Americanmaterial , possibly indicating a genepool provenance .Conclusions While diagnostic species patterns were identified ,markers for unique variety types were also observed , indicating thepotential of the combined SNP and chloroplast microsatelliteapproach to reveal detail on breeding lineages from seed samples .Examination of more similarly diverse regions should divide varietiesinto similar genotypes within species .ReferencesProvan , J . , Powell , W . and Hollingsworth , P . M . ２００１Chloroplast microsatellites : new tools for studies in plantecology and systematics . T rends in Ecology and Evolution１６ , １４２‐１４７ .Provan , J . , Beatty , G .E . , Hunter , A .M . , McDonald , R .A . , McLaughlin , E . , Preston , S .J . and Wilson , S . ２００８ .Restricted gene flow in fragmented populations of a wind‐pollinated tree" Conserv ation Genetics DOI : １０ .１００７ / s１０５９２‐００７‐９４８４‐yRoldan‐Ruiz , I . , Calsyn , E . , Gilliland , T .J . , Coll , R . , Van Eijk , M .J .T . and De Loose , M . ２０００ Estimating genetic conformitybetween related ryegrass ( Lolium) varieties , II . AFLP‐based markers . Molecular Breeding , ６ , ５９３‐６０２ .

Molecular Markers for Classification of Seed Collections of \u3ci\u3eLolium\u3c/i\u3e Cultivars

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Molecular Markers for Classification of Seed Collections of \u3ci\u3eLolium\u3c/i\u3e Cultivars

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