Transfer of simple sequence repeat (SSR) markers from major ... - abcic

q USDA 2005 ISSN 1479-2621

Plant Genetic Resources 3(1); 45– 57 DOI: 10.1079/PGR200461

Transfer of simple sequence repeat (SSR) markers from major cereal crops to minor grass species for germplasm characterization and evaluation M. L. Wang1*, N. A. Barkley1, J.-K. Yu2, R. E. Dean3, M. L. Newman1, M. E. Sorrells2 and G. A. Pederson1 1

USDA-ARS, Plant Genetic Resources Conservation Unit, 1109 Experiment Street, Griffin, GA 30 223, USA, 2Department of Plant Breeding and Genetics, Cornell University, 240 Emerson Hall, Ithaca, NY 14850, USA and 3Plant Genetic Resources Conservation Unit, University of Georgia, 1109 Experiment Street, Griffin, GA 30223, USA

Received 5 October 2004; Accepted 30 November 2004

Abstract A major challenge for the molecular characterization and evaluation of minor grass species germplasm is the lack of sufficient DNA markers. A set of 210 simple sequence repeat (SSR) markers developed from major cereal crops (self-pollinated wheat and rice, mainly self-pollinated sorghum and out-crossing maize) were evaluated for their transferability to minor grass species (finger millet, Eleusine coracana; seashore paspalum, Paspalum vaginatum; and bermudagrass, Cynodon dactylon). In total, 412 cross-species polymorphic amplicons were identified. Over half of the primers generated reproducible cross-species or cross-genus amplicons. The transfer rate of SSR markers was correlated with the phylogenetic relationship (or genetic relatedness) of these species. The average transfer rate of genomic SSR markers was different from the average transfer rate of expressed sequence tag (EST)-SSR markers. The level of polymorphism was significantly higher among species (67%) than within species (34%), and was related to the degree of out-crossing for each species. The level of polymorphism detected within species was 57% from self-incompatible species, 39% from out-crossing species and 20% from self-pollinated species. Genomic SSRs detected a higher level of polymorphism than EST-SSRs. The use of transferred polymorphic SSR markers for the characterization and evaluation of germplasm is discussed.

Keywords: germplasm; Poaceae; polymorphism level; SSR marker; transfer rate

Introduction The grass family (Poaceae) contains over 700 genera and 10,000 species, ranging from major cereal crops (including wheat, rice, maize and sorghum) to natural turfgrasses (e.g. bermudagrass and seashore paspalum). Most humans are directly dependent on cereal crops for their diet. Approximately 20% of the Earth’s land surface

* Corresponding author. E-mail: [email protected]

is covered with grasses, and domestic animals are raised on diets consisting partly or wholly of pasture grasses (Shantz, 1954; Kellogg, 2001). The grass family therefore plays a very important role in both agricultural production and environmental sustenance. Major cereal crops have been the focus of grass genome research. Thus, vast amounts of genomic information have been generated for the major cereal crops. In contrast, very limited genomic information for minor grass species is available. Genomic information (e.g. DNA markers) is useful for molecular characterization and evaluation

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of plant genetic resources. Lack of sufficient DNA markers in minor grass species therefore hinders current genetic resources research. Microsatellites (simple sequence repeats, SSRs) are among the most variable types of DNA sequence in plant and animal genomes. Since SSR markers are highly polymorphic, abundant and easy to use, they have become the marker of choice for genetic mapping, paternity testing and population studies (Goldstein and Schlo¨terer, 1999). As DNA markers, SSRs are also highly useful for characterization of plant genetic resources. Comparative genetic mapping of cereal crops has shown that both gene contents and/or gene orders are largely conserved over the evolutionary history of the grasses (Moore et al., 1995; Gale and Devos, 1998) to the extent that Bennetzen and Freeling (1993) suggest the grass genomes represent a ‘single genetic system’. Thus, for conserved genomic regions, map location of a sequence in one species can be used to predict the location of the sequence across much of the grass family. Transfer of SSR markers across species or genera has been reported in several cereal crops such as rice (Zhao and Kochert, 1993), wheat (Ro¨der et al., 1995), sorghum and maize (Brown et al., 1996; Cordeiro et al., 2001) and barley (Thiel et al., 2003). SSR sequences have also been identified within expressed sequence tags (ESTs), and many of these have been exploited to generate ‘EST-SSR’ markers (Kantety et al., 2002) which

have been used for comparative framework mapping in wheat and rice (Yu et al., 2004). However, little research has been conducted on the transferability of SSR markers from major cereal crops to minor grass species. The objectives of this study were to (i) exploit the transferability of SSR markers developed in major cereal crops to minor grass species; (ii) compare the transfer rate of genomic SSR markers and EST-SSR markers; (iii) estimate the polymorphism level of cross-species or cross-genus amplicons; and (iv) evaluate the transferred polymorphic SSR markers on accessions of turfgrass germplasm.

Materials and methods Grass species selection and DNA extraction Twenty-four accessions were used for this study (Table 1). Eight accessions were chosen from four major cereal crop species representing common wheat (Triticum aestivum L.), maize (Zea mays L.), rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.). Sixteen accessions were chosen from nine minor grass species representing bermudagrass (Cynodon dactylon L. Rich, Cynodon transvaalensis Burtt-Davy and Cynodon £ magennisii Hurcombe), seashore paspalum (Paspalum vaginatum O. Swartz, Paspalum lividum Trin. ex Schltdl. and Paspalum remotum J. Remy), durum wheat (Triticum

Table 1. Selected grass species and cultivars Accession no. MP W1 MP W2 MP D1 MP D2 MP D3 MP D4 PI 606331 PI 614900 PI 558532 PI 550473 PI 564163 PI 533839 PI 462383 PI 427234 PI 482594 PI 442480 PI 564236 PI 288216 PI 615161 PI 184339 PI 404874 Grif 15 167 Grif 15 194 PI 508952 a

Species Triticum aestivum Triticum aestivum Triticum durum Triticum durum Triticum durum Triticum durum Oryza sativa Oryza sativa Zea mays Zea mays Sorghum bicolor Sorghum bicolor Eleusine coracana Eleusine coracana Eleusine coracana Eleusine indica Cynodon dactylon Cynodon dactylon Cynodon transvaalensis Cynodon £ magennisii Paspalum lividum Paspalum vaginatum Paspalum vaginatum Paspalum remotum

Cultivar was used in a mapping population.

Cultivar/collector a

ABS 2000 USG 3592a Chahbo 88a Vica IACT12a Bena Cocodrie Earl Mo17a B73a BTX623a IS3620Ca Unnamed Unnamed Mukototsi 102 Unnamed Unnamed Daniela 157 430 HI 10 Sanibel 628-79

Abbreviation

Source

Origin

W1 W2 D1 D2 D3 D4 R1 R2 M1 M2 S1 S2 F1 F2 F3 F4 C1 C2 C3 C4 P1 P2 P3 P4

Jerry Johnson Jerry Johnson Shiaoman Chao Shiaoman Chao Shiaoman Chao Shiaoman Chao Harold Bockelman Harold Bockelman Robert Stebbins Robert Stebbins PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin PGRCU, Griffin

USA USA USA USA USA USA USA USA USA USA USA Nigeria India Nepal Zimbabwe Belgium Australia Madagascar Israel South Africa Uruguay USA USA Bolivia

Transfer of simple sequence repeat markers

turgidum L. Thell. Convar. durum (Desf.) MK) and finger millet (Eleusine coracana L. and Eleusine indica L.). In order to develop DNA markers for mapping genes for pre-harvesting sprouts, four accessions of durum wheat mapping parents from two crosses (D1 £ D2, D3 £ D4) were used: D1 and D3 (resistance to pre-harvesting sprouts), D2 and D4 (susceptible to pre-harvesting sprouts). The selected grass species were from four subfamilies: Ehrhartoideae (rice), Pooideaea (common wheat and durum wheat), Chloridoideae (finger millet and bermudagrass) and Panicoideae (maize, sorghum and seashore paspalum), and their phylogenetic relationship is shown in Fig. 1 (Gaut, 2002). Fresh young leaves from plants were collected into plastic bags and quickly frozen in liquid nitrogen. The frozen leaves were transferred to and stored in a 2 808C freezer for later DNA extraction. DNA was extracted either using the CTAB method (Reichardt and Rogers, 1997) or using E.Z.N.A.w Plant DNA Mini Kits from Omega Bio-Tek (product no. D3486-02). DNA was dissolved in 0.1 £ TE (1 mM Tris, 0.1 mM EDTA, pH 8.0) and then diluted to 10 ng/ml to use as the PCR template.

47

Texas A & M University, personal communication), 30 pairs of sorghum EST-SSR primers were selected by Dr J. K. Yu (Department of Plant Breeding and Genetics, Cornell University) and 30 pairs of sorghum genomic SSR primers were selected from http://sorghumgenome.tamu. edu. To avoid redundancy, all the EST-SSR sequences and genomic SSR sequences were subjected to a BLAST search. All the primers were obtained from Operon (Qiagen, MD, USA, http://www.operon.com). The GeneAmp PCR System 9700 (Applied Biosystems) was employed for PCR. Ten ng of genomic DNA template were used for each reaction in a total volume of 12.5 ml with a final concentration of 1 £ PCR buffer, 0.8 pmol/ ml each primer, 0.2 mM dNTPs, 1.5 mM MgCl2 and 0.05 U/ml Taq DNA polymerase (Promega). All the PCRs were performed using a programme for denaturing at 948C for 4 min; 10 cycles at 948C for 1 min, 508C for 30 s, 728C for 40 s decreasing by increments of 0.58C for annealing with each cycle; 35 cycles at 948C for 1 min, 458C for 30 s, 728C for 40 s; extending at 728C for 10 min and then storing at 48C.

PCR fragment separation and data analysis PCR primer selection and cross-species or cross-genus amplification A set of 210 SSR primers were selected and used in this experiment (Table 2). Fifty pairs of EST-SSR primers each from wheat and rice were selected from http:// wheat.pw.usda.gov/ITMI/EST-SSR and 50 pairs of maize EST-SSR primers were selected from http://www. maizegdb.org. Because fewer sorghum EST-SSR markers were available in the public database (Dr P. E. Klein,

Products from the PCRs were separated on a 3% agarose gel (a 7:6 ratio of MetaPhor agarose from FMC BioProducts and DNA grade agarose from FisherBiotech) in 1 £ TBE buffer by electrophoresis at 150 V for 2 h. The agarose gels were stained with ethidium bromide and visualized with UV light. Separated fragments on the agarose gels were sized by referencing with a 50-bp DNA ladder (Invitrogen). For calculation of the percentage of cross-species amplification, only clear

Fig. 1. A phylogeny of the grasses, featuring five major subfamilies and the earliest branching lineage. Each subfamily is represented by a triangle with height proportional to the number of species in the family. The number of species is taken from Kellogg (2000). The position to each subfamily for finger millet (Eleusine coracana), bermudagrass (Cynodon dactylon) and seashore paspalum (Paspalum vaginatum) was based on references of Hilu and Alice (2001) and Giussani et al. (2001), respectively. The species in bold were the focus of this study.

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Table 2. Primer information and original amplicon sizes Primer name

EST/marker no.

W1* W2 W3 W4* W5* W6* W7 W8 W9 W10 W11 W12* W13 W14* W15 W16 W17* W18 W19 W20* W21* W22 W23 W24 W25 W26* W27 W28* W29 W30 W31* W32 W33 W34* W35 W36 W37 W38 W39* W40* W41 W42 W43* W44 W45* W46 W47 W48* W49* W50* R1 R2 R3 R4 R5 R6 R7 R8 R9 R10

TaLr1130B07A TaLr1130B02A BI480021 BG909793 BG909764 BG909482 BG909325 BG909152 BG908960 BG908922 BG908890 BG908909 BG908875 BG908818 BG908811 BG908791 BG908790 BG908612 BG908603 BG908569 BG909413 BG909459 BI479123 BI479875 BI479902 BI480011 BI480021 BI479580 BI479767 BI479809 BI480549 BG604521 BG605281 BG605310 BG606122 BG604565 BG605476 BG604694 BG604862 BG604939 BG605043 BG605005 BG313284 BG313511 BG262529 BG262603 BG262485 BG274944 BI479561 BG263001 AA751878 AA753906 AA754479 AU029524 AU030789 AU055902 AU062506 AU064296 AU067946 AU075565

F-primer GCCTTCGCCACCAACTTC GGTATTGTGGCTTTCGCATT GAGGCGGCCTACAAGAAGAT GCTAGGGTTTCAGGCTCCTC GCTAGCAACCAGAGACTCAC GTCCAGCTCTCGGTACTTGG CAAGCCCATCCCCATCGCCG GCCTACGCAGACCGACTTCC AGCTTGGCCCTCTTCTCGCC GACGACGACGAGGACGAG CAGCGTAGGGATCTGAATGG GTGGAGAAGCCGAAGATGAC AGACGGCGCGCATGGCGA CGACGATTCCAATCACGAG CACCACGTTCCACGCTACTA GCATGAATTGTGCATATCGAG GTGGCAGGCAGGCAAGCAAG AAGAATAGGAGTTTAGGACC GCAACCGTGCGCGCGACTCC TGTACTACGCCTGCATGCTC CGATCCATGCCATATCACAG TACTAGAGGAGATGCAGACG GCGAGGCTCAACCAAGAG TCCCCAAAGATCCAGATCAC TCCGACGACCACCACCGAC AGGATGACGACCAGGACAAC GAGGCGGCCTACAAGAAGAT CGAGCACGAGAAGCTAAACC GCTCAAGCACAACGAAATCA TGCTGTTCGACATCATCCTC GACCAGATGAAACCGCTGAT CGACGCCATTCCTGAGAATA TACCCGAACCTTCTCGACTC TTCTTCCCTCCGAATTTCCT GATTTGGGGCCTAGGGTTAG GTGTCATCCCTCTGGTCGAT TCTCCCTCCTCAGCTACGAC GTCCTCGTGCTAGGGACCTT CTTGCCCCCTCTCGCCCC GCCGCCCACTCCGACCTCGC CGATCCCTCTGCTGCATT CTCTTCCTCGCAGCAGCA CTACAAGATCGGGGAGGACA TCCTTGGTCACTCTCCAAACTT GAGCTTCAGGGTCAAATGGA ATCCGTGCCATCGTCTACTC GTTCACGCAGCAGCCCACCAG GCGCATACGATACGAGCGAG GCAGTCGAACCTGAGACCAT ACGCCAAGATCATCAACGAC AGGCTGAGAGCCACAACAAT AAGTCCGTCGACAGGATGAG CGCTACCGTTATTCAACCCT AGCAGCACGCGAAACCCT AGCAATGAGGCAGTTCGTCT TGACCAGGGAGAGAGAGGAA CTGGAGCATCCTGATTCCAT CGACGGCACGGTAAAATAAA CGACTTCTCCTGTCCTGACC CATGTTCATCCTCCTCCTCC

R-primer CACGACGTACAAGTACACATGC TACCATTACCGGTTGCGACT GAGGCGGCCTACAAGAAGAT GCTGGCATCTAGGTGCGCCC GTCGTCCTCGTCGTTGTTG TGCATCCAAACAAGCCATGC AAAATTCCACCTGCTTGACG TGAGGATGTACGGGGAGAAG CAGAGGCCAGTGCATTTCTC GACGTCGGTGGTGTAGGTCT CTGCCAGATACAACGGCGTC GTAAGCAAGCGCTCCAGAAT AAGTCTACGCGGGTGAGGT CCCATGGGTGTTGACTCTG GCTGCTGCTCTTCGTGGT TCCAACCATTCCAATCAACA TGACGAGCTCATCGTCGTAG CCTGAGGCGATACAGGATGT CGGCACCTCGTAGTACTGG GTGCTCAAGGACATCGGGTA CCGCTTGTAACTTGGACACA TGGCAATAGCCTGAACCACT GCAAGGCTGGCTGAATCTAC CGTCACCACGATCTTGTTCC ACATGTACCTCGCGCCATC GCTCCTACAAGCCCACACAT ATGCTTATTCTCGCAACACA GGTGTCACCCGCACTAGG CCTTCCCAACTGAAACGAAT CCAGGCATTTTCCTTTCTGA AACCCCTTGTTGCACACCT CTTCCCTTGCTTGCTGACAT CCGAAGTGGAAGAGGTCGTA CAGCATTGGCGACAGACC CATGTCATCCCTCTCCTCGT ATATGCATGCCTTGTTGTTG AGCACGGGCGTCGACATGGC GTGAGCACACAGCAGCAGTT TCCGGACGACATTCTCTCTC TGGTTAGGATGTTGCTGCAC AGAGCTCTCGCTTCCTCGTA CGGCGTGCTTGTAGTGGTAG CTAGTACCTCCCGCACATGG GAACGTGAGAAGATATAT TCCTCGTCCTCATCATCCTC GTTGTTGTTGAGGCTGCTGA CCTCTCCTCCTCCTCGTTCT GCCTCCATTTCTGAGGATGA ATCGTAGAGCTGGAGGACGA GCCGCGATGGCCGCAGTA CTGGTGCCTTCCTCTCAAAG GCTGCTCTTCCTTGTGGCTA CCTAGCTTGTTTGAGACGCC GTGGTTGGGATCGGAATTG AGCATGAAGTGCATGACCC ATTTGATCGCTCGCTCCTTA GGATCCGTCTTCTTCATCCA ATCCCGTCACAGCAAAGAAC ACCTTTGCACCATGCTCTCT GTGTTCAAGAAGCAGCCTCC

Size (bp) 101 98 203 196 246 201 199 209 193 134 177 200 217 195 202 198 177 166 216 206 181 210 187 201 187 208 203 197 204 182 242 196 226 201 197 191 133 197 169 213 196 220 203 209 197 199 211 209 177 203 210 159 192 153 230 260 180 229 200 238

Transfer of simple sequence repeat markers

49

Table 2. Continued Primer name

EST/marker no.

R11 R12 R13 R14 R15 R16 R17 R18 R19 R20 R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 R33 R34 R35 R36 R37 R38 R39 R40 R41 R42 R43 R44 R45 R46 R47 R48 R49 R50 M1 M2 M3 M4* M5* M6 M7 M8* M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19* M20

AU077604 AU085858 AU093677 AU094854 AU101104 AU101425 AU161212 AU163694 AU164433 AU172629 AU172716 AU173294 AU173943 AU174090 AU174143 AW155019 AW155251 AW155354 BE040340 BI305848 BI794914 BI799065 BI801121 BI802494 BI809802 BI811586 BI813627 BM420928 BQ164728 BU673531 C19405 C25113 C25171 C27539 C72465 C73165 CA753426 D15529 D21962 D22307 AW171791 AW061734 D84409 AI691437 AW244179 AI881368 AW067031 AW091461 AW461040 AW067275 AW330738 AW499191 AW231652 AW519917 AW438120 AJ011615 AI948008 AI941906 AW225123 AW062092

F-primer

R-primer

Size (bp)

TGACTGCAATAGTGGATGGC GAGCTCGAATCCACCAAGAA AGCAGCTGGAGGAGGAGAAG TTCCTTTGGTTTCCATCTCG TCGTCGGTTTTGATAGGAAT CTCAGCCGTCCCCTTCTC GCTCGATTTCTCCTCCTCCT GAGTTCTTGCGGAGGATTCA CTGTTCGAGGACGGGAGG GTACACGATCGAGCACCAGA CGCTCCTTGTCTTCCTCATC CCCTTCCCTGTCAAAACAAA ACCGAGGCCTGAACATTTTT GTTCCGCTCCAAAGTCAAAG ACTCTCACTGGCTCGCAGTC ATAATCGGGAGGAGAAACCC TGCTCTCGCTTCTTCTCTCC GCTTCTTCTTCCTCGTCCCT GTGGTTCTTCACCAAGCTCC GCGACAGCACCGGATTATAG AGATCATGTCCCGTACCCAC GCCCAAGGAGGAGGATCTAC TACTACGGCGTCGAGCACAT GGCTGTGCCCAAATACAAGT CCATGCTCGAACGAGTGATA GATCTTGATCTCGAGCCGTC CAGCACACAGCAGCTACAGG GTCATCTACCACACCCAGCC CACCGACGACGACGCTAC GCTTGCACGTGTTTGTGACT CTAAAACCCTAACCCCCGAA CCATGAGCAACATGGTGAAG CTGATCAATCCTTCTGCGTG AGACGACCTCGACAGGAAGA ATCGTTGCCTCGTAACAACC GCCTCCAGCGAAAGTAATCA CCGCCTAGATAGACTTCCCC CTCTCGACCTCGGTGTTGC GAAGCAGCCAACCCAATG TCAACTCAACCGCATATCCC GACCTCTTCCTCGTCGTCTGAGT ATCTCGTCTACCTAACCCACCCTC AGCCTCCTGAGACCTCTCGATT CACAACTCCATCAGAGGACAGAGA CTGCTCCTCCTCACCCCACT TTATGAACGTGGTCGTGACTATGG CTCGATAGCTCTGCTGCTTCCTC TAAAGCTATGATGGCACTTGCAGA CCATCCACCACTAGAAAGAGAGGA TAACTACTACACCACTCGCGCAAA GTGGCATTTTTATCTGCAACACAG CCACAACTCGCTGCTGTCAATA TTAATAGCTACCCGCAACCAAGAA CTGTACATGGATATGGCATTGGTG TTTTCTGCAGGGATAACATTTGTG TTTCATGTGCTTGCAGAGTTTGAC AAGAAAACAGGTAACGGGCATGTA TTCCTCTTTTTAGTAGGGGGTTGG GATTACAACCCACCGGAGTTACAG AAGAAGAAAGAGAAGAAGCACGGG

GTTGAACTCGAACGTCCCTC GTCAGATACAGCGACGACGA GCTCTTCATCAGCGGGTC GCATTGGTACAGGGAGAAGC TGGTGGGATTTTATTGAGGG GGTCTCCTCCTCCAACACC CGTCCTTAACGCAGAGGAAC GTCCTCGTAGAAGAGCGCC CGCTTGAAGATTCCGGTTAG GGAGGAAGACCCAGAAGGAC GAGCTGAGACTTGAGCCAGG TTTGTTTACATTCCCCTGAGC ATGATCCGAATCCAATCCAA TACATGTCCATCCACCCAAA AGAGCGTGTAGGAGAGCAGC GTAGGCCTGCGTGACGAT CGGTCAGGAACTGTCCTCAT AGCTTGCTGCTGATCTTCCT AAACATCCACACCCACACAA GATCTTGGCGGTGTCCTTC ATTTAGCACGTTGTCAGCCC GAGCATGGCCTTGTTGTTTT TCTCCTTGCTGACACTGACG AGCAAGCTTCTGTGGGTGTT GTCGTCTCGGTTGGGGTT GTGTGGGAGCTCGTGGAC CCCAGTGTCCTACCAACCAC CTTGGTCCAACCCGAACTTA GGACCGACCAACAAAGTACC AATCGATCTTCCGTCCTCCT CTAGGCACTTGAGGCTCTCG TACCGGAGGAAGAGGACCTT CCTCGTATCGTGGATGTGAC GCTGTCCACTGCACAAGAAA ACCTGCAACTGCCAGAAGAG TTGATCTCCTCCTCCGACAC CCCCGTAGATCTGCTTGAAC GGTATCTCCTCCCTCTTCGG GAACATGACGGCGAACAAC TATTTGCCTGAAGAATGCCC CACGAGAGGGGTTGTGGAGAT CAGGTGAAGAATCTGGTGAGGTC ACTTCGCCACCTTACATTCTTGA CTGCTACGACATACGCAAGGC CCCGAGGATCTCGTAGTACTTGGT ATATCTGTCCCTCTCCCACCATC CAACACCAGCCCTACCCAGA CATATTTGCCTTTGCCCTTTTGTA TTAATCGATCGAGAGGTGCTTTTC GTGTCGTGTTGGGAGAACATGAG TGTAACTTAATCGTGGGCAGAACC AGGAGGCTGCTGACCCTTCTACT CTGAGCCACAGTACCTTGCTGTT GCATATACACCACCTTGGACAACA ATAGGAGGTGAGGTGAGGAGGAAG GTCATGCAAGTATCCGCTGTCTT TGGAATTCTTCTGACTACCCCAAG AAGATGATGGTGGAGAGGATGAAG GCTCTTCCTAGGTGCAGACAAAGA GGACAGCTCGTATTATAACCTGCG

189 205 237 290 203 266 279 300 179 291 179 196 224 295 200 283 235 227 218 164 276 276 205 254 225 233 269 186 232 241 259 195 247 241 218 278 224 300 298 270 130 148 ? 156 160 149 157 159 140 143 139 153 127 121 157 158 150 142 146 145

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Table 2. Continued Primer name

EST/marker no.

M21 M22* M23 M24 M25 M26* M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41 M42 M43 M44 M45 M46 M47 M48 M49 M50 S1 S2* S3 S4 S5 S6 S7 S8 S9* S10* S11* S12 S13* S14 S15 S16* S17* S18 S19 S20 S21* S22 S23 S24* S25* S26* S27 S28* S29* S30*

AI714614 AJ011614 AW461037 AW498199 AF148498 AF019146 AW163864 AI692068 AI857227 AF073330 AI855352 U81960 AW054269 AI770592 AW017605 AW400071 AW424565 AI833843 AI947716 AW573317 AW057036 AI649893 AI668245 AW257910 AW562406 AI939884 AF043346 AI854917 AI734806 AF057183 CNL160 CNL161 CNL162 CNL163 CNL164 CNL165 CNL166 CNL167 CNL168 CNL169 CNL170 CNL171 CNL172 CNL173 CNL174 CNL175 CNL176 CNL177 CNL178 CNL179 CNL180 CNL181 CNL182 CNL183 CNL184 CNL185 CNL186 CNL187 CNL188 CNL189

F-primer

R-primer

Size (bp)

TTACACCAAGGTCCGAAACAAGAT GAGAGGTCGTCGTCGCTACTG ATCCTCCTCCATATTCTATCGCGT CATGGGACAGCAAGAGACACAG TCGGTCGAGGCATAACTAGCTC CTGTCGTAAGAGCGCCAACAG GAGCTACTCAGCCAAGACGAAAAG CAATGTGTTATTGATTGTACACCGC GTGACGAGATGGTGCAGAAAGAT GTCTACCAGGACGTTTACCTGTGG GAAGTCGCTGATGAGAACGTAACC GTGATACCGGGTAATCTGGTGC CGGCCTATGTAACAATCCCTAGC GCATCTGTAGCCTTTTTGTGTGTG TGATCCAAGAAAAGCAAAGCAAAC CAGAACAACAACCAGAACCAGAAG AGACGAACCCACCATCATCTTTC GAAGGGAAGAAAGGATAGGAACCC GATGTCTCTATGGAACCCAGCAAC AGTACTTTCAGGCAGGGACCTTCT CGGCGAGGATAACATGCAGTA TGGGTGCTAAAACGTAACAACAAA CCTACAAATCAACCATCGATTTCC TGGACTTCGAAAATTCTCTTCAGC GAATTACCTGTTTGGAGCGGAAG AACTCCGAGATCTACGACAACAGC CCGTCTTCTTCAGGGTGTTCC TGGACGATCTGCTTCTTCAGG AATACCAAGCTGCACTCAGAAACC AGCAGGAGTACCCATGAAAGTCC TCACCAGACCACCAGCTTC GTGGGTCGTCATCCCGTA TTGGAGGGAAGAAGACAAGC GCTTCTCAGCTCAAGCATCTG GTGGACGATGGATGGATCA CCGCATTCCTATCCTCTCCT CAGGTGCATAGCAAGTGTGG ATATCCTCTCTCCGGCCACT GGAGCTGAGGATCGAGAGTG GACTCGGACATCGACAACG GAGGGCGTACAGGAAGAACA TCGAGTGCCTCAAGGAGTG CGCAAGGCAACATCAGTATC GGGTCAGTGCTGGACAAGAT GGGTCGATTCTGTTCCTCAA TGCGGATGTAAAGGAGGGTA GCCATGAGCTACCAAGCAG CATCAGCGTGACCGACTACT GTGTCTACGGTGGTGGAGGT ACCATTCCGAAGCTTCCAC CACAATAATGGATCCCACCA CACGGATTGGGATCTCTTTG CTCCACAAACCACCACCAC AGGGAAGGAACCAAGACCAT GGTAGCAGAGAGCGGAGAGG TGGATGAGGGTTTCTTTGGA GGGATTTGGGATTTGGATTT CCAGGAGGCACTCTCTTCAG TAGCAAGCAGAAATCGACCA CACCTCCGTCCTCTTCGAT

TCTTGGAAGGCAAGACTCTACCTG GAGACCAGATTCTTGGAACGGTAA GAAACAGAGCAGGAACCGGAG ACCTTCATCACCTGCAACTACGAC CAGCTTCTTCCACTTCTTCAGCA GTCTGAACGATGAACAGTACACGC TCACTTGCATGAGCAACTTCAGTA ACAGCAGGAGGCAGAGACTGAC CCTGGAGGTGGAAGGAGAGG CCTCAATCCTTTGTGGACAAACAC GCTAGCTAGTGTGAGTTCTTCCGC GATGATGGGTGATCATCGGTTC AAGGGAAAGAATAATCCAACCGTC CTCAGCTTGCAGGTTATCGCTT TGGCAGTCTGTATAGTTGTCCGAA CCATTAGCCATCTTGGCGTT CGCTTGGCATCTCCATGTATATCT AGGCAAGGCTCAGCAGTCAG GAGACGCCTACGAGTACCACAACT AACGCACTTCTTGTAGCTGTAGGG TCTTGAGCTGAACACTGATCTTGG GAGGACGAAGCAGAAATCCTACC GTGCCATCAAAGAGGAATTGGTAG GAGAGGAGGAGCTTCACGAGC GATTTTCTCTGTAGTTCGAGGCTGG GAGGAAGAGTTGGCCAGGATG GTGGAGTTAGTAGGGTCGTTGCAC GAAGGCTTCTTCCTCGAGTAGGTC CGTCAAATCCAGCCTAAGCATC TATCACAGCACGAAGCGATAGATG GAGAACGGGCCAAGGTACT TTTGGCATCACATGACCAGT AGCATGAGACCTGGAAGCAG AAGCAGTCGTGGAAGTGGAG ATCACCACTGCCTCTCACAA TTCCACTGACCACCCATGTA CTGCATGTTTCGTTTGTGCT TCGTTCATCAGCACTTCATCA AGTCGGAACTTTGAGGACGA TCTGCCACATCTTCATCGAC CCGAGAAGGACTTGGTGAAG CAACAACGAAGAAACGACGA TCAGGAGGAGTCGAGCATCT CATAGCAGGGTGAGGAGGTG CAGTCGTCCTCGATCATGTG CAAACCAGCTAATGAGACGATG GTCGTCGTCCTCAGAAGCA GGCTCTTGAACCTCTTGTCG TGGTGGCAGTTGTAGCACTC GCGAGCTCAGAGGAGTTGTT GTAGTCCTCCAGCAGGATCG TTCTCACTAAATTGCGCTACCA CCGGCCTCGTTGTAGTAGAC CTCAGTCACCTCCACCGAAG GATGTCGTTGCGGTTGTAGA GGCTGGACGGACATACAAGT ATCGACTGGGAGAATCAACG GCGACAACAGAACAACGGTA ACCATTGTCCCTCACTCCTG CCTCAAACCTCCCGTTCC

126 123 156 124 ? 160 113 130 148 ? 157 ? 137 140 125 158 157 143 125 125 157 151 142 126 160 123 ? 160 150 ? 159 384 257 266 200 352 371 361 388 298 206 187 155 290 211 170 250 284 219 333 355 347 260 372 305 381 159 266 156 169

Transfer of simple sequence repeat markers

51

Table 2. Continued Primer name

EST/marker no.

SG1 SG2 SG3 SG4 SG5 SG6 SG7 SG8 SG9 SG10 SG11 SG12

Xtxp 279 Xtxp 46 Xtxp 63 Xtxp 201 Xtxp 266 Xtxp 33 Xtxp 114 Xtxp 69 Xtxp 41 Xtxp 21 Xtxp 40 Xtxp 312

ATTCTGACTTAACCCACCCCTAAA GGGCAATCTTGATGGCGACAT CCAACCGCGTCGCTGATG GCGTTTATGGAAGCAAAAT GTTGTCTAGTATAGCAAGGTGGG GAGCTACACAGGGTTCAAC CGTCTTCTACCGCGTCCT ACACGCATGGTTTGACTG TCTGGCCATGACTTATCAC GAGCTGCCATAGATTTGGTCG CAGCAACTTGCACTTGTC CAGGAAAATACGATCCGTGCCAAGT

SG13 SG14 SG15 SG16

Xtxp 168 Xtxp 339 Xtxp 10 Xtxp 287

SG17 SG18 SG19 SG20 SG21 SG22 SG23 SG24 SG25 SG26 SG27 SG28 SG29

Xtxp 289 Xtxp 20 Xtxp 331 Xtxp 141 Xtxp 210 Xtxp 354 Xtxp 105 Xtxp 6 Xtxp 317 Xtxp 176 Xtxp 17 Xtxp 65 Xtxp 303

SG30

Xtxp 225

AGTCAAAACCGCCACAT CCGCACTCTCCACTCT ATACTATCAAGAGGGGAGC GCAAGCGAGCTGACTTAT GTAACGAGA AAGTGGGGTGAAGAGATA TCTCAAGGTTTGATGGTTGG AACGGTTATTAGAGAGGGAGA TGTATGGCCTAGCTTATCT CGCTTTTCTGAAAATATTAAGGAC TGGGCAGGGTATCTAACTGA TGGTATGGGACTGGACGG ATCGGATCCGTCAGATC CCTCCTTTTCCTCCTCCTCCC TGGCGGACATCCTATT CGGACCAACGACGATTATC CACGTCGTCACCAACCAA AATGAGGAAAATATGAAACA AGTACCAA TTGTTGCATGTTGGTTATAG

F-primer

R-primer

Size (bp)

AGCTCATCAATGTCCCAAACC 276 AGGTGTGGCTCGGGGAGAAC 253 GTGGACTCTGTCGGGGCACTG 204 CTCATAAGGCAGGACCAAC 222 ATAATAGTAGATGCGTGTCAAAAGAA 197 CCTAGCTATTCCTTGGTTG 221 CATAATCCCACTCAACAATCC 234 TTGATAATCTGACGCAACTG 188 AAATGGCGTAGACTCCCTTG 278 ACCTCGTCCCACCTTTGTTG 179 GGGAGCAATTTGGCACTAG 138 GTGAACTATTCGGAAGAA 192 GTTTGGAGGAAA GAGAAGGGGAGAGGAGAA 178/182? CGGAACACAGGGAAGG 202 AGTACTAGCCACACGTCAC 145 CAAAGTGCTACTAAACCTAT 367 GCAGGGTGAA CTGCCTTTCCGACTC 290 ACCCATTATTGACCGTTGAG 217 AGTATAATAACATTTTGACACCCA 226 CAACAAGCCAACCTAAA 163 GATGAGCGATGGAGGAGAG 188 GCCTTTTTCTGAGCCTTGA 157 TGTTGACGAAGCAACTCCAAT 291 TCTAGGGAGGTTGCCAT 120 TCAGAATCCTAGCCACCGTTG 162 GGAGAGCCCGTCACTT 161 ACTCGTCTCACTGCAATACTG 164 GTTAAACGAAAGGGAAATGGC 128 AATAACAAGCGCAACTATAT 160 GAACAATAAA CAAACAAGTTCAGAAGCTC 165

W, wheat; R, rice; M, maize; S, sorghum; SG, sorghum genomic SSR. SGs are only primers from genomic sequence. * Markers were tested on more bermudagrass accessions; ?, size of the original amplicon was unknown.

DNA bands on the agarose gels were counted but DNA bands amplified in species from which primers were designed were excluded.

Results Transfer rate of SSR markers across the grass family The average transfer rate of SSR markers from four major cereal crops to other grass species was 57% (Table 3). Among the four major cereal crops, the average transfer rate of SSR markers from maize to other grass species (48%) was less than the average transfer rate of the four major cereal crops. This might relate to maize SSR primer design and average size of maize original amplicons. On the other hand, the transfer rate of SSR markers across species or genera was closely related to their phylogentic relationship. For example, hexaploid

common wheat (2n ¼ 6x ¼ 42, AABBDD) and tetraploid durum wheat (2n ¼ 4x ¼ 28, AABB) belong to the same Triticum genus and thus the transfer rate of SSR markers from common wheat to durum wheat was high (80%, the highest observed). Maize and durum wheat belong to different subfamilies and the transfer rate of SSR markers from maize to durum wheat was low (only 44%). Sorghum, maize and paspalum belong to the same subfamily (Panicoideae; Fig. 1). The transfer rate of SSR markers from sorghum to paspalum and from sorghum to maize was 68% and 61%, respectively. The difference between these two transfer rates was not significant (t , a ¼ 0.05). The number of cross-species or cross-genus amplicons generated from transferable SSRs could not be predicted from the number of original amplicons. Sorghum genomic SSR marker Xtxp168 (Fig. 2A), for example, generated a single amplicon in sorghum, but multiple cross-genus amplicons in most tested species. Sorghum genomic SSR marker Xtxp21 (Fig. 2B) generated a

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M. L. Wang et al.

Table 3. Summary of cross-species or cross-genus amplification Species Wheat Rice Maize Sorghum Average

No. of markers

WT

RT

MT

ST

DT

FT

CT

PT

Average

50 EST-SSRs 50 EST-SSRs 50 EST-SSRs 60 SSRs: 30 EST-SSRs 30 genomic SSRs

x 39.00 40.00 52.43 43.33 61.53 43.91

52.08 x 40.00 57.43 53.33 61.53 50.00

48.96 45.00 x 61.03 56.67 65.39 51.95

66.67 69.00 59.00 x x x 64.87

79.69 50.00 44.00 63.40 57.57 69.23 59.07

65.63 53.00 57.00 57.65 54.17 60.58 58.09

58.33 53.50 41.50 62.21 60.00 64.42 54.04

65.10 59.00 50.50 68.43 66.67 70.19 60.91

64.11 53.09 47.72 61.24 57.27 65.21 56.53

Values are percentages. T is transferability; x transfers to itself. W, R, M, S, D, F, C and P are initials for common wheat, rice, maize, sorghum, durum wheat, finger millet, Cynodon and Paspalum, respectively.

single amplicon in sorghum but it did not generate crossgenus amplicons for most tested species. Sorghum ESTSSR marker CNL164 (Fig. 2C) generated two amplicons in sorghum, however it generated a single, double or multiple cross-genus amplicons in most tested species. Sorghum EST-SSR marker CNL170 (Fig. 2D) generated a single amplicon in sorghum and also generated single cross-genus amplicons for most tested species. The average transfer rate from sorghum EST-SSR markers and from sorghum genomic SSR markers was 57% and 65%, respectively. The difference between these two transfer rates was significant (a ¼ 0.05 , t , a ¼ 0.01).

Polymorphism level and size of cross-species and cross-genus amplicons The polymorphism level of 412 cross-species and crossgenus amplicons is summarized in Table 4. Transferable SSR markers were more polymorphic among species (67%) than within species (34%). The polymorphism level was also related to the degree of out-crossing for each species. The level of polymorphism detected within species was 57% from self-incompatible species (averaged from bermudagrass and seashore paspalum), 39% from out-crossing species (maize) and 20% from self-pollinated species (averaged across accessions of wheat, rice, sorghum, durum and finger millet). Sorghum was included in self-pollinated species because it only outcrosses 2–5% in nature. The level of polymorphism detected from sorghum (27%) was higher than the level of polymorphism detected from wheat (15%), finger millet (21%), rice (21%) and durum wheat (23%). Sorghum genomic SSR markers detected a significantly higher level of polymorphism (69%) than sorghum ESTSSR markers (33%) within sorghum. The level of polymorphism detected from transferred sorghum genomic SSR markers was 74% among species and 36% within species, while the level of polymorphism detected from transferred sorghum EST-SSR markers was 70% among species and 37% within species (differences between types of SSR markers was not significant).

The level of polymorphism was not related to the transfer rate. For example, the average transfer rate of SSR markers to durum wheat (59%; Table 3) was slightly higher than the average transfer rate to all species (57%) whereas the average level of polymorphism detected within species for durum wheat (23%; Table 4) was much lower than the average level of polymorphism detected within species for all species (34%). The size of cross-species or cross-genus amplicons could be similar to or different from the size of original amplicons (from primer-designed species). For example, the size of most cross-genus amplicons from the sorghum SSR marker CNL170 was very similar to the size of the original amplicons (Fig. 2D) whereas the size of most cross-genus amplicons from the sorghum SSR marker CNL164 (Fig. 2C) was very different from the size of the original amplicons. Although the sequence content of cross-genus amplicons could be different, the level of polymorphism detected may be related to the size variation of cross-species or cross-genus amplicons.

Use of transferred polymorphic SSR markers for germplasm characterization To demonstrate whether these cross-species or crossgenus amplicons can be used as DNA markers for germplasm characterization and evaluation, 40 pairs of SSR primers (labelled with an asterisk in Table 2), which generated clear bands by cross-species or crossgenus amplification, were tested on accessions from bermudagrass. The genomic SSR marker Xtxp303 from sorghum for example generated polymorphic amplicons that could distinguish three bermudagrass accessions within the species. Ninety-six accessions from bermudagrass were also tested and characterized by this transferred SSR marker (Fig. 3). These accessions were simply classified into four tentative groups on a 3% agarose gel: (1) accessions with bands a, b and d; (2) accessions with bands a, c and d; (3) accessions with bands b and d; and (4) accessions with only band d.

Transfer of simple sequence repeat markers

53

Fig. 2. Amplicons generated by PCR and separated by electrophoresis. Each well contains either 15 ml of molecular marker (150 ng) or 12.5 ml of PCR products. PCR products were amplified with sorghum primers: genomic Xtxp168 (A) and Xtxp21 (B), EST-derived CNL164 (C) and CNL170 (D). The first lane of each row was the molecular marker, a 50-bp ladder, followed by W1, W2, D1, D2, D3, D4, R1, R2, M1, M2, S1, S2, F1, F2, F3, F4, C1, C2, C3, C4, P1, P2, P3 and P4 in each panel (see Table 2 for primer information).

Discussion Transferability of SSR markers across the grass family The transferability of SSR markers depends on the genetic relatedness among species examined (including difference in DNA sequence, genome size and evolution rate) as well as PCR conditions used (including the amount of template DNA, annealing temperature, number of cycles and ion concentration). The transfer

rate (57%) of SSR markers across the grass family was very similar to the rate (55%) from wheat to barley (Gupta et al., 2003), the rate (57%) from tall fescue to several grass species (including wheat and rice) (Saha et al., 2004), lower than the rate (75%) from maize to Miscanthus (Herna´ndez et al., 2001) but much higher than the rates (8%; 31%) for SSR markers across the legume family (Peakall et al., 1998; Wang et al., 2004). Annealing temperature is critical for PCR and will affect the transferability of SSR markers. In comparison of the transferability across the grass family (this report) and

Values are percentages. For calculation of the average, data in bold were excluded. The first initial letter W, R, M, S, D, F, C and P is for common wheat, rice, maize, sorghum, durum wheat, finger millet, Cynodon and Paspalum, respectively. The second initial letter P is for polymorphism. Pw and Pa stand for the polymorphism of accessions within and among species, respectively. E, EST-SSR; G, genomic SSR.

40.18 39.71 19.71 37.14 35.71 34.03 75.00 78.00 58.00 83.33 80.77 73.53 62.50 78.00 40.00 56.67 57.69 59.31 70.83 70.00 52.00 70.00 80.77 67.16 70.83 70.00 30.00 56.67 73.01 58.82 68.75 64.00 54.00 56.67 61.54 61.28 20.83 36.00 18.00 20.00 15.39 23.04 35.42 30.00 16.00 33.33 69.23 27.02 16.67 18.00 6.00 16.67 23.08 14.74 Wheat Rice Maize Sorghum (E) Sorghum (G) Average

25.00 50.00 12.00 26.67 26.92 21.43

50.00 24.00 38.00 50.00 34.62 38.96

16.67 22.00 16.00 33.33 19.23 20.59

Average Pw PPa PPw CPa CPw FPa FPw DP SP MP RP WP Species

Table 4. Summary of polymorphism detected within species and among species

71.53 70.67 54.67 70.00 74.36 67.32

M. L. Wang et al.

Average Pa

54

across the legume family (Wang et al., 2004), the same PCR conditions were employed for these two separate studies. The most likely explanation for the difference in transferability (57% versus 31%) is that the grass species investigated are more closely related to each other than the legume species. For example, among the grass species in this study, common wheat is closely related to durum wheat, and sorghum is closely related to maize (Kellogg, 1998). However, among the legume species investigated by Wang et al. (2004), Medicago truncatula was not closely related to peanut and mung bean (Doyle and Luckow, 2003; Choi et al., 2004). The transfer rate (48%) of SSR markers from maize to other grass species was much lower than the average transfer rate (57%) of SSR markers from the four major cereal crops. The average size (143 bp) of the original amplicons from maize SSR markers was much smaller than the average size of the original amplicons from wheat (194 bp), rice (232 bp) and sorghum (270 bp). The size of an original amplicon generated from a SSR marker is the sum of the length of simple sequence repeats and the lengths of flanking sequences. If the size of the original amplicon is small and the maize genome has been expanded, the region corresponding to the amplicon in other species might be too small to be detected. This might partly explain the low transfer rate of SSR markers from maize to other grass species. On the other hand, the average length of primers from wheat, rice and sorghum was very similar (20-mer) but the average length of primers from maize was much longer (23-mer). A longer primer length needs a higher stringency for a specific amplification. This might be another reason to explain the low transfer rate of SSR markers from maize to other species.

Polymorphism level of cross-species or cross-genus amplicons The level of polymorphism detected across species or genera mainly depended on the genetic divergence of species tested and primers used. As expected, a higher level of polymorphism from cross-species or crossgenus amplicons was detected among species (67%) than within species (34%). The level of polymorphism detected was also related to the reproductive system for each species indicating that out-crossing contributes to more rapid genome evolution. A higher level of polymorphism was detected in sorghum from sorghum genomic SSR markers than from sorghum EST-SSR markers. This result was consistent with a higher level of polymorphism detected from genomic SSRs than EST-derived SSRs in rice and durum wheat (Cho et al., 2000; Eujayl et al., 2002). However, this phenomenon was not

Transfer of simple sequence repeat markers

55

Fig. 3. Characterization of plant germplasm by a transferred SSR marker. Amplicons generated from 96 accessions of Cynodon dactylon by genomic SSR marker (Xtxp303) from sorghum were separated on a 3% agarose gel. Distinguished bands were labelled as a, b, c and d. Faint bands on this gel were not scored. The first lane in each row contained a 50-bp DNA ladder followed by accessions from Cynodon dactylon.

observed with transferred polymorphic SSR markers. The polymorphism level of cross-species or cross-genus amplicons detected within species from transferable sorghum genomic SSR markers and EST-SSR markers was 36% and 37%, respectively. There was no significant difference between them. Cross-species and cross-genus amplicons generated from rice SSR markers were sequenced (Chen et al., 2002) indicating that not all cross-species or cross-genus amplicons contained simple sequence repeats. Because the sequence content of cross-species or cross-genus amplicons could be very different from original amplicons, it is inadequate to compare the level of polymorphisms detected from transferred genomic SSRs and EST-SSRs in the results.

Application of cross-species or cross-genus amplicons as DNA markers for germplasm characterization and evaluation The goal of this study was to identify SSR markers that can be transferred at a high rate to closely related species or genera and also detect a high level of polymorphism within species. As a pilot experiment, amplicons from 96 bermudagrass accessions generated by a sorghum SSR marker (Xtxp303) were simply classified into four tentative groups (Fig. 3). The preliminary results indicated that SSR markers developed in major cereal crops can be used as DNA markers for characterization and evaluation of germplasm from minor grass species.

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However, more transferred polymorphic SSR markers from major cereal crops need to be tested on more accessions of different minor grass species. As expected, the transferable SSR markers detected a much higher polymorphism among species than among accessions within species. There are some alternative approaches that can be used to overcome the low level of polymorphism. The separation sensitivity of cross-species or crossgenus amplicons on an agarose gel is limited to . 10 bp, however the sensitivity can be increased to . 2 bp by the separation of amplicons on a polyacrylamide gel. If necessary, these amplicons could also be sequenced to confirm whether they contain SSRs, insertions and deletions (InDels) or single nucleotide polymorphisms (SNPs). For the last decade, plant genome research has been focused on the major crops and model species such as Arabidopsis thaliana and Medicago truncatula. A vast amount of genomic information has been accumulated and will be exploited for improvement of the major cereal crops in the future. This will provide an exciting opportunity for plant germplasm scientists to use the major cereal crops as sources of information and markers to assess genetic diversity and phylogenetic relationships of thousands of minor grass species. In return, plant germplasm research will provide information about useful traits and sources of superior alleles for improving cultivars for agriculture production and will help to develop strategies for sustaining genetic diversity for future generations.

Disclaimer Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

Acknowledgements The authors gratefully thank Drs Harold Bockelman, Robert Stebbins, Jerry Johnson, Zhenbang Chen, Shiaoman Chao and Ms Lee Ann Chalkley for providing us with rice, maize, common wheat, durum wheat and sorghum seeds; Drs Tracie Jenkins, Jerry Johnson and Yong Seo, and two anonymous reviewers, for useful comments to improve the manuscript; Drs Roy Pittman and Jerry Davis for help preparing the phylogeny figure and statistics analysis; and Ms Meredith Reed for her excellent assistance.

M. L. Wang et al.

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