Development of transcript-associated microsatellite markers for diversity and linkage mapping studie
Development of transcript-associated microsatellite markers for diversity and linkage mapping studies in hop (Humulus lupulus L.)
Jernej Jakse ?Natasa Stajner ?Zlata Luthar ?
Jean-Marc Jeltsch ?Branka Javornik
Received:13February 2010/Accepted:12June 2010/Published online:2July 2010óSpringer Science+Business Media B.V.2010
Abstract Data mining of gene sequences available from various projects dealing with the development of expressed sequence tags (ESTs)can contribute to the discovery of new microsatellite markers.Our aim was to develop new microsatellite markers in hop isolated from an enriched cDNA library and from coding GenBank sequences and to test their suitability in hop diversity studies and for construction of a linkage map.In a set of 614coding GenBank sequences,72containing microsatellites were found (11.7%);the most frequent were trinucleotide repeats (54.0%)followed by dinucleotide repeats (34.5%).Addition-ally,11sequences containing microsatellites were isolated from an enriched cDNA library.A total of 34primer pairs were designed,29based on GenBank sequences and ?ve on sequences from the cDNA enriched library.Twenty-seven (79.4%)coding microsatellites were successfully ampli?ed and used in diversity and linkage mapping studies.Eleven
primer pairs ampli?ed 12coding microsatellite loci suitable for mapping and were placed on female and male linkage maps.We were able to extend previous simple sequence repeat (SSR)female,male and integral maps by 38.8,25.8and 40.0cM,respectively.In the diversity study,36diverse hop genotypes were analyzed.Twenty-four coding microsatellites were polymorphic,17showing co-dominant behavior and 7primer pairs amplifying three or more bands in some hop genotypes.Altogether,143microsatellite DNA fragments were ampli?ed and they revealed a clear separation of hop genotypes according to geograph-ical region,use or breeding history.In addition,a discussion and comparison of results with other plant coding/EST SSR studies is presented.Our results showed that these microsatellite markers can enhance hop diversity and linkage mapping studies and are a comparable marker system to non-coding SSRs.Keywords Hops áHumulus lupulus L.áMicrosatellites áCoding/EST SSRs áSegregation analysis áMapping
Introduction
The hop plant Humulus lupulus L.is a dioecious member of the Cannabaceae family and is thought to originate from Asia (Murakami et al.2006).The species is subdivided into ?ve taxonomic units:the common wild European hop,which has been
Electronic supplementary material The online version of this article (doi:10.1007/s11032-010-9476-3)contains
supplementary material,which is available to authorized users.J.Jakse áN.Stajner áZ.Luthar áB.Javornik (&)
Agronomy Department,Biotechnical Faculty,University of Ljubljana,Jamnikarjeva 101,1000Ljubljana,Slovenia e-mail:branka.javornik@bf.uni-lj.si
J.-M.Jeltsch
Universite
′de Strasbourg,4rue Blaise Pascal,CS 90032,67081Strasbourg Cedex,France
Mol Breeding (2011)28:227–239DOI 10.1007/s11032-010-9476-3
introduced elsewhere(var.lupulus),wild Japanese hop(var.cordifolius)and North American hops—var. lupuloides,var.neomexicanus and var.pubescens, being native to the eastern,western and midwestern parts of North America,respectively(Small1978). Only the female plants are economically important. They are grown for unfertilized ripe female in?ores-cences—hop cones,which are used mainly for beer brewing and are also interesting as a rich source of pharmaceuticals(Chadwick et al.2006).Commercial hop production is based on the use of hop cultivars derived by clonal selection or by control crosses.
Progress has been made in recent years in the development of microsatellite markers for hop,either for genotyping/diversity studies(Brady et al.1996; Jakse et al.2002;Hadonou et al.2004;Stajner et al. 2005)or for mapping purposes(Jakse et al.2008). They have mainly been developed from anonymous genomic regions using enriched isolation techniques. It was long believed that microsatellite sequences are associated only with the non-coding part of the genome but numerous studies have now shown that microsatellites are also associated with transcribed or coding DNA and regulatory elements(reviewed in Varshney et al.2005).Simple sequence repeat(SSR) markers derived from expressed sequence tag(EST) sequences might be more conserved and thus have a higher rate of transferability than non-coding micro-satellite markers(Scott et al.2000).For example, more than70%of EST microsatellites detected polymorphisms in several Medicago species(Eujayl et al.2004).In contrast,the conserved nature of EST microsatellites may limit their polymorphism. Although EST markers exhibit a lower level of polymorphism compared to non-coding microsatel-lites,their simplicity of development and ease of technical optimization makes them a favoured type of marker over non-coding microsatellites.
Mapping of microsatellites from ESTs can provide a map location for genes of known function or indicating gene-rich regions.The attraction of targeting ESTs for the detection of sequence polymorphism and mapping is that the identi?ed polymorphic markers may corre-spond directly to genes controlling agronomic traits of interest(Varshney et al.2005).For example,the waxy gene in rice has been found to contain a(CT)n microsatellite,whose length polymorphism is associ-ated with amylose content(Ayers et al.1997;Jayamani et al.2007).The discovery and use of EST-or cDNA-based microsatellites has been reported for several plant species,including grape(Scott et al.2000), apricot(Decroocq et al.2003),clover(Barrett et al. 2004),sugarcane(Cordeiro et al.2001),wheat(Eujayl et al.2002)and rye(Khlestkina et al.2004).In hop, seven EST microsatellites have been applied in a diversity assessment of hop germplasm and genotype identi?cation study(Bassil et al.2008).
In this paper,we report on(1)searching for the presence of microsatellite repeats in hop EST data and in a developed SSR-enriched cDNA library,and (2)the identi?cation and characterization of a set of EST microsatellite markers and evaluation of their informativeness for a variability study on a set of H.lupulus genotypes and their utilization for linkage mapping.
Materials and methods
Hop DNA samples
A total of36hop samples were included in the analysis,comprising26hop cultivars representing a broad range of available cultivated germplasm,and 10wild hops from Europe,Japan and North America (Table1).
An F1mapping family of a cross between the cultivar Wye Target and breeding line2/1comprising 144plants was used to test for segregation of the developed markers.The mapping family,segregating for many morphological and agronomic traits,is maintained at the Slovenian Institute of Hop Research and Brewing,Zˇalec,Slovenia.
DNA was isolated from young leaf tissue during the vegetation period or from dormant buds from rhizomes taken during the winter/early spring season using a common CTAB procedure(Kump and Javornik1996).DNA was quanti?ed by means of ?uorimetry(DynaQuant200,GE Healthcare)and dilutions in sterile water at8ng/l l were made. Coding microsatellite sequences
Two sources of hop coding sequences were used, publicly available EST/cDNA sequences deposited in GenBank,and a constructed cDNA library,enriched for microsatellite repeats.
GenBank sequences extraction
A set of614hop ESTs,cDNA or gene sequences were extracted from GenBank(accession numbers CO65 3411–CO654000,CD527119–CD527124,AB015430, AB053487,AB061020,AB061021,AB061022AB061022,AF147497,AF268889,AJ304877,AJ430353, AJ876882,AY263153,AY644677,AY687338,AY68 7339,AY745883,AY772257,AY795910,AY849555) in a total length of193,511bp.A FASTA format?le of sequences was searched for the presence of SSR motifs using PERL script MISA(Thiel et al.2004),in which
Table1Thirty-six hop genotypes used in the study,comprising26diverse hop cultivars and10wild hops from Europe,Japan and North America
Name/genotype Description(pedigree/site of collection,use,country of origin)
Aurora Hybrid of Northern Brewer,higher alpha and European aroma(dual purpose),Slovenia Bor Selection of hybrid progenies of Northern Brewer cross,Czech Republic
Brewer’s Gold Wild Manitoba BB19open pollinated,higher alpha,England
Chinook Cross between Petham Golding and USDA63012,high alpha,USA
Cluster Selection,higher alpha,USA
Ellupulo No information available,Spain
Fuggle Old English cultivar from19th century,pleasant European aroma,England
Galena Brewer’s Gold seedling,high alpha,USA
Ging Dao Do Hua Selection from Late Cluster,higher alpha,China
Glacier Cross between Elsasser F and8685-014M,excellent aroma,USA
Hallertauer Mittelfruher German landrace,very?ne aroma
Keyworths Midseason Selected in19th century,pleasant aroma,England
Kirin2Clonal selection of Shinshuwase(hybrid of Saazer),Japan
Kitamidori Seedling selection from Kirin Brewery,higher alpha,Japan
Magnum Hybrid Galena9German male hop,high alpha acid content,Germany
Merkur A cross between Hallertauer Magnum and81/8/13,high alpha variety,Germany Northern Brewer Canterbury Golding crossed with a male seedling from Brewer’s Gold,higher alpha,UK Paci?c Gem Cross between Late Cluster and male of Fuggle,high alpha,New Zealand
Perle Seedling selection of Northern Brewer,moderately high alpha acids,Germany
Saazer Selection of Czech landrace,European pleasant aroma
Savinjski Golding Slovenian clonal selection of Fuggle,which was introduced in the Savinja valley
at the end of the19th century,European aroma,Slovenia
Serebrianka Considered landrace,aroma,Russia
Southern Brewer Seedling selection of Fuggle,European aroma,South Africa
Topaz Cross between J78and29/70/54,high alpha,Australia
Wye Challenger German Zattler-OP9No.Brewer-downy mildew res.male(17/54/2)9(1/61/57)
Wye Target Hybrid of Northern Brewer,high alpha acids content,England
AH7Wild hop from Serbia
2/1Slovenian wild male,European germplasm
9/2Slovenian wild male,European germplasm
No3-38Japanese wild male hop,carries resistance to aphids
K11Wild hop from Caucasus
R15Wild hop from Russia
752.001Humulus lupulus var.neomexicanus,selected from the wild in Utah,USA
1,008.010Humulus lupulus var.lupuloides,collected from the wild in Saskatchewan,Canada
1,020.001Humulus lupulus var.pubescens,collected from the wild in Missouri,USA
1,355.001Humulus lupulus var.neomexicanus,male,collected from the wild in Colorado,USA
SSRs were de?ned as a minimum of?ve repeats long for dinucleotide motifs(10bp)and four repeats long for higher classes of microsatellites(3–8bp unit length).
cDNA enriched library construction
Total RNA was isolated from cultivar Savinjski Golding tissue cultured in vitro using RNAgents denaturating solution(Promega,USA)according to the manufacturer’s instructions.cDNA was obtained using a SuperScript II Reverse Transcriptase kit (Invitrogen)in20l l reaction volume comprising19 First-Strand buffer,10mM DTT,0.5mM of each dNTP,5l g of total RNA,500ng of oligo(dT)12–18-A primer and200U of SuperScriptII reverse transcrip-tase.The reaction was incubated at42°C for50min and inactivated at70°C for15min.One microlitre of cDNA reaction was used in the20-l l cDNA-PCR step,which included19PCR buffer,2.5mM MgCl2, 0.2mM of each dNTP,0.8l M dT18?A primer, 0.8l M reverse primer mix consisting of a mixture of random decamer primers,and0.8U of Taq polymer-ase(Promega).DNA was ampli?ed with35cycles using the following cycling conditions:94°C for30s, 38°C for1min and72°C for2min.The PCR reactions were then pooled,DNA precipitated,quan-ti?ed by spectrophotometer and further used in the microsatellite enrichment and analysis step,as described previously(Jakse and Javornik2001). Enrichment was performed for di-(AC,AG)and trinucleotide repeats(ACA,AGA)and DNA frag-ments were TA cloned in pGEM-T Easy Vector (Promega,USA).Colonies were screened according to Jakse and Javornik(2001)and55positive clones were selected from the libraries for sequencing. Plasmids were isolated from overnight cultures using GenCatch Plasmid DNA Mini-Prep Kit(Epoch Biolabs,USA)and sequenced by either T7or SP6 universal primers using Big Dye chemistry on an ABI3730XL analyzer by the Macrogen sequencing service(Seoul,Korea).Editing and assembly of the sequences was performed using Codon Code Aligner 1.6.1software(CodonCode Corp.,USA). Sequence comparison
The chosen sequences containing coding microsatel-lites were compared with BLASTX,BLASTN or TBLASTX algorithms at a5910-5cutoff e value using netblast executables(Altschul et al.1990).The highest hit to any known protein(BLASTX algorithm, nr database)or any known EST(BLASTN or TBLASTX algorithm,EST_others database)revealed the highest hits to already known sequences(Electronic Supplementary Material Table1).Searches were done only with known coding sequences(proteins or ESTs) to con?rm that sequences were truly expressed.A putative function was already designated for six hop cDNAs:chs_h1,chs2,chs3and chs4are members of the chalcone synthase genes(Matousˇek et al.2002; Okada et al.2004),hch1is a coding sequence of the endochitinase precursor gene and MYB-CTT5is a hop transcription factor(Matousˇek et al.2005).
Marker development and genotyping
Primer pairs were developed for29sequences from GenBank searches and?ve from cDNA sequences obtained from the enriched library using PRIMER3 software(Rozen and Skaletsky2000)(Supplementary Table1).Special care was taken to search all sequences for homologies with known annotated genes using the BLASTX algorithm to reveal any possible intron–exon boundaries.Putative intron–exon splice sites were eliminated from primer devel-opment(data not shown).The shorter primer of the pair was elongated for the M13(-21)18bp sequence (50-TGTAAAACGACGGCCAGT-30)for economic ?uorescent labeling(Schuelke2000),except that forward primers of pairs chs_H1,chs2,chs3,chs4 and hch1were CY5-labeled at their50ends.Developed primer pairs were?rst tested for PCR ampli?cation according to the described methodology(Jakse et al. 2008)and then ampli?ed on the whole sample of36 hop genotypes or eight plants from the mapping family after optimization.PCR conditions were as follows: total volume10l l with20ng of hop DNA,19PCR buffer(10mM Tris–HCl,50mM KCl, 1.5mM MgCl2pH8.3,Fermentas,Lithuania),0.2mM of each dNTP(Fermentas,Lithuania),0.2l M of each locus speci?c primer and,in the case of‘‘tailed primer pairs’’also0.075l M of M13primer50-labeled with CY5dye and0.25U of Taq polymerase(Fermentas, Lithuania).Ampli?cation was performed using a touchdown cycle protocol:initial denaturation at 94°C for5min,followed by5cycles of45s at 94°C,30s at the initial annealing temperature(65,62, 60,57or55°C),which was lowered by1°C in each
subsequent cycle,followed by25cycles with the reported annealing temperature(Supplementary Table1)and ending with an8-min extension step at 72°C.Samples were denaturated using formamide-bluedextran loading dye(10mg/ml blue dextrane in formamide)and separated on denaturating7% polyacrylamide gels(19:1,7M urea)using an ALFexpressII automated sequencer with a limiting power of electrophoresis of15W and constant temperature of55°C.Fragment lengths were deter-mined with the aid of an external standard(50–500bp, GE Healthcare,USA)and with in-house ampli?ed internal standards using Allele Locator1.03software.
Microsatellite polymorphisms were scored as binary data(presence/absence),due to the multi-allelic pattern of seven loci.The genetic similarity among hop samples was calculated based on a binary matrix using Dice’s similarity index(Nei and Li1979).Cluster analysis was generated from the similarity matrix by the unweighted pair group method using the arithmetic averages(UPGMA)algorithm.The goodness of?t of the clustering results to the data matrix was also estimated.All calculations were performed by the computer program NTSYSpc2.02(Rohlf1998).For further statistical data analysis of coding microsatellite loci showing co-dominant properties,Cervus3.0.3 software(Kalinowski et al.2007)was used to calculate the observed number of alleles(n),observed and expected heterozygosities(H o and H e)and the poly-morphism information content value(PIC). Mapping analysis of markers
Loci showing polymorphism in the parents of the mapping family were ampli?ed on F1progenies.The EST microsatellite markers revealed different pat-terns of segregation:1:1for markers segregating only in the male or in the female parent(types ab9aa and aa9ab),1:2:1for markers segregating in both parents(type ab9ab),and more informative 1:1:1:1segregation in markers segregating in both parents with three alleles(type ab9ac).
Chi-squared values were calculated for each marker to detect any deviation(P B0.05)of segregation from the expected Mendelian ratio.Finally,both distorted and undistorted markers were included in map construction.Map construction was carried out using the JoinMap3.0program(Van Ooijen and Voorrips 2001)implementing the pseudotestcross strategy (Grattapaglia and Sederoff1994).Segregating EST microsatellite markers were added to a previously constructed microsatellite map of hop loci(unpub-lished),which was based on104microsatellite and2 sequence tagged site segregating polymorphisms.The female map was based on91polymorphisms and spanned161.6cM,the male map was based on51 markers and spanned156.8cM,while the integral map covered183.8cM.The mapping criteria were6.0for the minimum LOD score and0.35for the maximum recombination fraction.Within each linkage group (LG),the optimal order of markers was determined using a ripple value of1.0and a jump threshold of5.0. The Kosambi mapping function was applied to convert recombination data to map distances.LGs were graphically presented with MapChart 2.1software (Voorrips2002).
Results
EST SSRs,enriched cDNA library construction and sequence comparison
According to the search parameters(minimum?ve repeats for dinucleotide and four repeats for other higher motifs),the analysis of614hop EST/cDNA sequences revealed72microsatellite-containing sequences(11.7%).Altogether,87microsatellite markers were found in72sequences.Eight sequences had more than one repeat present,separated by more than30bp of nucleotides.In terms of the class of repeats,trinucleotide repeats were the most common (47repeats),followed by dinucleotide repeats(30 repeats).Ten microsatellite sites were found for higher classes of repeats:one for4n,three for5n and six for6n.Three out of four possible dinucleotide repeats were present in the sequences(without the GC/CG repeat)and seven out of ten possible trinucleotide repeats(without ACG/CGT and AGC/ CGT and CCG/CGG repeats).The longest dinucle-otide repeat AG/CT had11units and the longest triucleotide was AAG/CTT with eight repeats (Table2).The majority of the repeats were perfect ones,with only one repeat type.
The usefulness of enrichment isolation techniques for the isolation of genomic microsatellites in hop has been shown in numerous studies to date(Hadonou et al.2004;Stajner et al.2005;Jakse et al.2008).In
this work,we also used double-stranded cDNA for the isolation of microsatellites.Fifty-?ve plasmids were chosen for sequencing,25from GT,14from GA and16from trinucleotide repeat libraries.After an editing and assembly step,11unique sequences remained(20%of the starting sequences,average length260bp,min/max length115/602bp),all of them harboring microsatellite repeats.The longest repeat was dinucleotide microsatellite TC,with32 units,and trinucleotide AAC,with15and8repeats in compound assembly.The shortest repeat present was (AAC)8.All11sequences were submitted to Gen-Bank and accession numbers GU726884–GU726894 were assigned to them.
BLAST searches were used as a tool for compar-ison of the sequences used for marker development with known coding(protein or EST)sequences (Supplementary Table1).Seventeen sequences showed signi?cant hits to known proteins,four to the EST database using the BLASTN algorithm and an additional four to the EST database using the TBLASTX algorithm.Nine out of34sequences showed no signi?cant hit with the employed searches. Only one sequence out of11isolated using the enrichment procedure showed a signi?cant hit to the EST database using the TBLASTX algorithm.
EST microsatellite marker development
Primers were developed for29EST/GenBank sequences containing microsatellite repeats and for?ve cDNA sequences isolated with the enrichment procedure(Sup-plementary Table1).The coding SSR markers were designated according to the GenBank accession number and the microsatellite repeats according to the name of the gene(e.g.,CHS_H1,chs2,chs3,chs4,hch1)and according to the sequence clone from the cDNA enriched library(e.g.,GA4-cDNA-P18).Twenty-seven developed primer sets(79%success rate)revealed an ampli?cation ability and seven primer pairs(CO653685-ATC5, CO653701-TCT7,CO653801-TGA4CAC6,CO65380 3-CT8,GA5-cDNA-B24,GA5-cDNA-L12and3n4-cDNA-D21)failed to amplify any amplicon,even at low stringency PCR conditions.
Mapping
In the mapping study,27primer pairs were further tested for segregation in an F1full sib family.Of27 primers,11primer pairs showed segregation of12loci suitable for mapping,15were monomorphic and one was aa9bb type and not informative in the F1cross (Supplementary Table1).For one primer pair(GA5-cDNA-L5),the ampli?cation of two loci was con-?rmed and both of them were segregating,so they were labeled GA5-cDNA-L5-1and GA5-cDNA-L5-2, respectively.Twelve coding microsatellite loci revealed different patterns of segregation.Seven loci showed the1:1type for markers segregating in the female or male parent(ab9aa and aa9ab),three of them segregating in the male and four in the female parent.The same two alleles present in both parents (ab9ab)revealed the1:2:1type of segregation, which was the case for three loci.The remaining two loci showed the most informative type of segregation, 1:1:1:1,with markers segregating in both parents with three alleles(ab9ac)(Table3).At the5%level,?ve loci were distorted:both loci with1:1:1:1segregation, two loci with1:2:1segregation and one with1:1 segregation(Table3).
For linkage analysis,12coding microsatellite markers were added to a previously developed microsatellite linkage map for hop(unpublished).
Table2Frequency distribution and number of discovered microsatellites in hop EST sequences for di-and trinucleotide types of repeats
Repeat Length Sum 4567891011
AC/GT–123 AG/CT–42111110 AT/AT–8611117
Total dinucleotides30 AAC/
GTT
314
AAG/
CTT
8221114
AAT/
ATT
1212
ACC/
GGT
213
ACT/
ATG
314
AGG/
CCT
112
AGT/
ATC
448
Total trinucleotides47
The female map,with8linkage groups(LGs),was based on91non-coding microsatellite loci,the male map,with7LGs,was based on51,while the integral map had9LGs,with103markers included.The maps spanned161.6,156.8cM and183.8cM, respectively.Of the twelve EST microsatellite mark-ers,nine were suitable for linkage analysis in the female and eight in the male parent.All markers were successfully added to the existing SSR framework maps;the female map was extended by38.8cM,the male map by25.8cM,and the integral map by 40.0cM.In the female map,nine markers were placed in?ve different LGs,four of them in LG1and two in LG5.Another three markers were placed on LGs2,3and7.In the male linkage map,?ve EST markers were placed in LG1and three in LG7.LG1 was also extended by two additional SSR markers not previously included in the map.Two female LGs (LG1and LG5)and one male LG(LG1)were signi?cantly extended,while in male LG7,two markers saturated a region not previously covered with non-coding SSRs.In the integral map,new markers were included in?ve LGs.The whole integral linkage map is shown in Fig.1. Genotyping and variability analysis
Twenty-seven working primer pairs were further used for a variability study in a diverse group of36hop genotypes.Three loci were monomorphic(CO653 790-AC6,CO653955-AAC5and CO653610-ATC4) and17of them revealed the expected co-dominant pattern(one allele for a homozygote or two for a heterozygote hop plant).The number of alleles per locus in the17with a co-dominant pattern ranged from two(seven loci)to seven alleles(four loci),with an average of4.2alleles per locus,which yielded70 different co-dominant alleles.For the17co-dominant loci,three different values for evaluation of marker polymorphism and informativeness were calculated (observed and expected heterozygosities and PIC) (Table4).Seven primer pairs exhibiting multi-allelic pro?les,where more than two DNA fragments were ampli?ed per genotype,showed a higher number of ampli?ed bands,ranging from four up to20,totally amplifying73different bands with an average of10.4 fragments per primer pair.The highest number of DNA fragments—?ve in one hop plant—was ampli-?ed with primer pairs GA5-cDNA-L5and CHS_H1; linkage analysis for the?rst one revealed ampli?cation of two loci with this primer pair.With the other?ve primer pairs,three(two primer pairs)or four DNA fragments(three primer pairs)were ampli?ed at most in one plant.Although the co-dominant properties of such loci are hindered,their application for?nger-printing or use in variability studies is still possible. Moreover,these loci with several different fragments ampli?ed showed high variability(Table4and Sup-plementary Table1).
The average value of Dice’s similarity coef?cient calculated on143polymorphisms among all cultivars was0.504±0.133.The highest similarity coef?cient, 1.0,was between cultivars Fuggle and Savinski Golding,known ecotypes,and the lowest,0.222,
Table3Segregation of coding microsatellite markers in hop F1family Wye Target9breeding line2/1
*Signi?cant at P\0.05Locus Parental
genotypes
Predicted
segregation
Observed
segregation
v2value
CO653584-TTGTT4aa9ab1:182:61 3.084
C0653713-ATC5ab9ab1:2:151:73:1716.574* CO653778-GA9ab9aa1:174:670.348 CO653885-TA6ab9aa1:175:690.250 CO653923-ATA4aa9ab1:119:12578.028* CO653941-GA7ab9aa1:177:660.846 CO653957-TCCCA4ab9ac1:1:1:117:28:40:5119.118* CO653996-TGAG4ab9ab1:2:131:69:40 1.186 chs4ab9aa1:173:670.257 GA4-cDNA-P18aa9ab1:170:730.063 GA5-cDNA-L5-1ab9ac1:1:1:10:14:51:6788.788* GA5-cDNA-L5-2ab9ab1:2:114:54:6442.242*
between German landrace cultivar Hallertauer Mittel-fruher and wild American hop1,008.01.Cluster-ing analysis based on Dice’s similarity coef?cient revealed three major groups(Fig.2).Samples clus-tered in accordance with their geographical origin (American or European hops),use of the cultivars (aroma or bitter hops)and breeding history.The?rst group consisted of16hop samples,12cultivars and four wild ones,all of them belonging to aroma hops or European germplasm.The second group contained14 cultivars with high alpha acids,containing proportions of North American germplasm.Two wild accessions are adjacent to both groups:wild male hop No.3–38 from Japan and wild female plant K11from Georgia. In the third group,four wild North American hop accessions clustered,representing all three North
American varieties:var.neomexicanus(1,355.001and 752.001),var.lupuloides(1,008.010)and var.pubes-cens(1,020.001).The clustering results were con-?rmed by a high cophenetic correlation(r=0.86). Discussion
This study reports the development of EST/coding microsatellite markers in hop and assessment of their suitability for mapping purposes and genetic vari-ability studies.The development of such markers is much simpler when public EST resources are avail-able for the desired species,allowing omission of the tedious step of microsatellite library preparations and further sequencing.
Of the614hop EST/cDNA sequences extracted from GenBank,72sequences containing microsatel-lites were identi?ed,which represented11.7%of all screened ones.The revealed microsatellite frequency is in agreement with data obtained in pepper(Yi et al. 2006),chickpea(Choudhary et al.2009)and eucalyp-tus(Yasodha et al.2008),in which10.2,11.5and 12.9%,respectively,of ESTs harbored an SSR repeat. However,there are also data on lower and higher frequencies of obtained EST SSRs in other plant species,ranging from as low as1.4%in asparagus (Caruso et al.2008),4%in oil palm(Singh et al.2008), 6%in melon(Gonzalez-Ibeas et al.2007)and Lolium (Asp et al.2007),up to14%in strawberry(Lewers et al.2005),17%in apple(Newcomb et al.2006)and 22%in blackberry(Lewers et al.2008).The discovery and frequencies of microsatellites containing ESTs is affected,as well as by the sequence itself,by the software used for searching and by the de?nition of the minimum microsatellite repeat length(Varshney et al. 2005).When our basic search parameters were changed by an additional repeat unit for de?nition of di-and trinucleotide repeats,the frequency of discov-ered hop ESTs containing SSRs decreased from11.7 to5.9%(data not shown).
The frequency distribution of different types of microsatellite motifs con?rmed earlier observations that trinucleotide motifs are the most abundant,which was the case in our set of hop coding sequences (54.0%).Similar frequencies have been reported in other plant species(Morgante et al.2002;Yi et al. 2006).Trinucleotide repeats are necessary for the maintenance of an open reading frame(ORF)of the coding regions,while other types of repeat can disrupt/distort the ORF(Li et al.2004).The higher frequency of hexanucleotide repeats in our dataset (6.9%)can also be explained by the correct ORF having been maintained.Among the dinucleotide repeats,the most abundant were AT repeats,account-ing for56.7%of all dinucleotides,followed by AG repeats(33.3%)(Table2).This is in contrast with other published data,in which AG repeats were the most abundant in a large data set of dicot ESTs (Kumpatla and Mukhopadhyay2005).The most frequent microsatellites among trinucleotides were
Table4Number of scored alleles/DNA fragments(n),size range,observed and expected heterozygosities(H o and H e)and PIC values over24polymorphic hop coding microsatellite loci used in the variability study
Marker n Size range H o H e PIC
CO653502-CTT87186–2490.0560.2100.204 CO653584-TTGTT44a165–215/
CO653624-TCT611a190–230/
CO653631-AG62161–1630.0000.1550.141 CO653699-ATC52221–2240.1940.2220.195 CO653711-AAG58237–2570.5280.7850.740 CO653713-ATC54186–2070.6670.6750.595 CO653778-GA97136–1580.6940.7690.731 CO653875-AT65a230–247/
CO653878-TC62237–2390.1110.1060.099 CO653885-TA69a155–181/
CO653923-ATA42238–2440.1110.2000.178 CO653941-GA73233–2370.1940.2860.249 CO653957-TCCCA47218–2360.7500.7410.696 CO653984-CTT42172–1870.9440.5060.374 CO653996-TGAG42190–1940.3330.2820.239 chs_H112a219–258/
chs25205–2250.3060.3990.367 chs35215–2360.3530.6220.563 chs42384–3900.1000.0970.091 hch17141–2080.2590.7530.702 MYB-CTT53225–2300.4170.4460.365 GA4-cDNA-P1812a181–222/
GA5-cDNA-L520a213–284/
Min20.0000.0970.091 Max70.9440.7850.740 Average 4.20.3590.4300.383 a Loci with multi-allelic pro?les were excluded from calculations of average,min and max number of alleles and H o,H e and PIC values
AAG and AAT repeats,accounting for29.8and 25.5%of all trinucleotide repeats,respectively.This is in agreement with other published data,in which AAG repeats were the most frequent,followed in some species by AAT repeats(Kumpatla and Mukhopadhyay2005;Yi et al.2006).
Microsatellite primer pairs were developed for36 of the72sequences containing SSRs.Of them,27 successfully ampli?ed PCR fragments from the hop genomic DNA(Supplementary Table1),which is a 75%success rate for the developed primer pairs.The obtained rate is in agreement with other plant EST-microsatellite studies,in which rates between60and 90%have been reported(Varshney et al.2005).We observed a higher success rate compared to published results for hop genomic SSRs,in which53%(Stajner et al.2005),57.8%(Jakse et al.2008)and38.5% (Hadonou et al.2004)of the developed primer sets gave a successful ampli?cation pattern.All primer sets produced PCR products in the expected size range.These27primer sets were further used in a mapping and genetic diversity study.
Our diversity analysis results revealed that70% (17)of the working primer pairs showed classical co-dominant inheritance with one or two alleles present,while26%of the primer pairs(seven) ampli?ed3–5DNA bands in some hop genotypes. Although diversity indices cannot be calculated for
such loci,the genotyping data can still be used as binary data in genetic relationship analyses(Fig.2). The average number of alleles for17co-dominant loci was4.2,which is two times lower than that reported for genomic hop microsatellites,in which an average10.6(Stajner et al.2005)and9.7(Jakse et al. 2008)number of alleles were reported,but similar to four alleles per locus in the study by Hadonou et al. (2004).However,Bassil et al.(2008)reported a higher average number of alleles detected in seven hop genic SSR loci,which can be explained by the diverse North American hop germplasm analyzed in their study.In our study,only four native North American hops were analyzed,although they con-tributed to allelic richness since,of143DNA fragments ampli?ed with24primer pairs(Table4), 22of them(15.4%)were unique to these four hops.In contrast,American wild hops were also monomor-phic at four loci.The geographical distribution of microsatellite alleles in hops is very well known and documented(Jakse et al.2004;Stajner et al.2008; Bassil et al.2008).The average observed(0.388)and expected(0.434)heterozygosities and PIC value (0.383)were lower than reported for non-coding hop microsatellites(Jakse et al.2004,2008).The lower level of polymorphism for coding SSR markers is explained by the higher DNA sequence conserva-tion in the transcribed regions(Varshney et al.2005). Despite the lower polymorphism of coding microsat-ellites,it was ef?cient in distinguishing32analyzed hop genotypes,showing the usefulness of hop genic SSRs for successful discrimination of hop genotypes. Moreover,the strong bands,distinct allelic peaks and fewer stutter bands(Varshney et al.2005)make EST/ coding microsatellite markers a suitable choice for hop discrimination analyses.
Analysis of relatedness of36genotypes based on Dice’s coef?cient showed three main groups of genotypes(Fig.2).In general,the geographical origin (European or North American germplasm)and use of the cultivars(aroma or bitter hop)or the breeding history characteristic of breeding centres is repre-sented.Similar observations of clustering in hop diversity analyses have also been obtained with other marker systems,such as RAPD markers(Sustar-Vozlic and Javornik1999),microsatellites(Stajner et al.2008)or AFLPs(Seefelder et al.2000a), showing the suitability of the developed coding microsatellites for diversity and pedigree analysis.
The suitability of the developed coding SSR markers was also tested for linkage analysis in segregating a hop family.Eleven primer pairs (30.6%of developed primer pairs)showed polymor-phism in the mapping family.This?gure is slightly lower than data for non-coding hop microsatellites,in which43.7%of developed markers segregated in the same cross(Jakse et al.2008).In other plant species, various reports on the number of polymorphic EST markers in segregating families can be found,from as high as93.5%in Actinidia(Fraser et al.2004),to a similar number of polymorphic markers as we found in hop in a segregating pepper family(29.2%)(Yi et al.2006)and a lower number of mapped markers for an extensive soybean map(9.7%)(Hisano et al. 2007).Nevertheless,the ease of developing coding microsatellites and their association with the tran-scribed part of the hop genome makes them desirable for mapping.An extension of map length(female 38.8cM,male25.8cM and integral40.0cM)was observed when EST microsatellite markers were added to the existing SSR map,which can be explained by the fact that recombination may be more frequent in gene-rich regions than in non-coding regions and that non-coding microsatellites may be less frequent in gene-rich or euchromatic regions(Yi et al.2006).Distortion of segregation for coding microsatellite markers was observed for?ve loci(45.5%of all markers).Segregation distortion of markers is well documented in already developed hop maps(Seefelder et al.2000b;Cerenak et al.2006) and very variable rates have been reported in numerous plant linkage studies,usually described as gametic,zygotic and post-zygotic selections(Ky et al.2000).
To sum up,we developed EST/coding microsat-ellite markers for hop that add new markers for diversity studies and mapping.Our results showed that hop sequences in public databases are a useful source of microsatellite markers.Recent transcrip-tome sequencing projects in hop(Nagel et al.2008; Wang et al.2008)have yielded more EST sequences, which can be used for further development of gene-based markers.
Acknowledgments This study was funded by the Slovenian Research Agency,contract no.P4-0077,and by the Proteus bilateral France–Slovenia programme,contract no.BI-FR/08-09-PROTEUS-006.
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