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. 2010 Jul 15;28(2):241–254. doi: 10.1007/s11032-010-9477-2

Mining and validating grape (Vitis L.) ESTs to develop EST-SSR markers for genotyping and mapping

Hong Huang 1,2, Jiang Lu 1,✉, Zhongbo Ren 1, Wayne Hunter 3, Scot E Dowd 4, Phat Dang 5
PMCID: PMC3132434  PMID: 21841909

Abstract

Grape expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping. An integrated pipeline including several computational tools for SSR identification and functional annotation was developed to identify 6,447 EST-SSR sequences from a total collection of 215,609 grape ESTs retrieved from NCBI. The 6,447 EST-SSRs were further reduced to 1,701 non-redundant sequences via clustering analysis, and 1,037 of them were successfully designed with primer pairs flanking the SSR motifs. From them, 150 pairs of primers were randomly selected for PCR amplification, polymorphism and heterozygosity analysis in V. vinifera cvs. Riesling and Cabernet Sauvignon, and V. rotundifolia (muscadine grape) cvs. Summit and Noble, and 145 pairs of these primers yielded PCR products. Pairwise comparisons of loci between the parents Riesling and Cabernet Sauvignon showed that 72 were homozygous in both cultivars, while 70 loci were heterozygous in at least one cultivar of the two. Muscadine parents Noble and Summit had 90 homozygous SSR loci in both parents and contained 50 heterozygous loci in at least one of the two. These EST-SSR functional markers are a useful addition for grape genotyping and genome mapping.

Electronic supplementary material

The online version of this article (doi:10.1007/s11032-010-9477-2) contains supplementary material, which is available to authorized users.

Keywords: SSR, EST, Marker, Genotyping, Grape

Introduction

Microsatellites, or simple sequence repeats (SSRs), are short (1–6 bp) repeat DNA motifs that are usually single locus markers with characteristics of hypervariability, abundance and reproducibility. The variation of the SSR repeat units can be easily differentiated by PCR products amplified with primers flanking the SSR motif. SSRs have been widely used for bacteria screening (Lin et al. 2005), plant genotyping (Chen et al. 2006), linkage mapping (Zhang et al. 2002), gene tagging (Roy et al. 2002), and map-based gene cloning (Tekeoglu et al. 2002).

The availability of ESTs greatly accelerates the systematic identification of SSRs and corresponding marker development based on computer analytical approaches (Varshney et al. 2002; Gao et al. 2003; Thiel et al. 2003; Chen et al. 2006). EST-derived SSRs have been well documented in some plant species including Arabidopsis (Depeiges et al. 1995), sugarcane (Cordeiro et al. 2001), cereal species (Kantety et al. 2002), cacao (Lima et al. 2008), and rubber tree (Feng et al. 2009). Using homology searches, putative functions can be deduced for the SSRs and thereby provide a new resource that can further aid in genetic and evolutionary studies (Cho et al. 2000; De Keyser et al. 2009).

EST-SSR and genomic SSR markers should be considered as complementary to plant genome mapping, with EST-SSR being less polymorphic but concentrated in the gene-rich regions (Varshney et al. 2006). With hundreds of thousands of ESTs available in the public domain, the process of developing EST-SSR markers has been greatly accelerated by using optimized computational pipelines and high-throughput genotyping techniques.

SSR markers have been widely used in grape genotyping. The high polymorphism of Vitis-derived microsatellite loci has been reported extensively in the literature and used for fingerprinting (Thomas and Scott 1993; Bowers et al. 1996, 1999a, b; Sefc et al. 1998; Arroyo García et al. 2002; Di Gaspero et al. 2005, 2007; Merdinoglu et al. 2005; Lamoureux et al. 2006; Costantini et al. 2007; De Mattia et al. 2007; Cipriani et al. 2008; Bocharova et al. 2009; Riaz et al. 2009). Several publications have also demonstrated transferability of SSR markers across the Vitis genus (Lin and Walker 1998; Tessier et al. 1999; Di Gaspero et al. 2000; Fernández et al. 2008).

SSR markers have been used for construction of grape genetic maps (Dalbò et al. 2000; Doligez et al. 2002; Grando et al. 2003; Adam-Blondon et al. 2004; Doucleff et al. 2004; Riaz et al. 2004, 2006; Lowe and Walker 2006; Di Gaspero et al. 2007; Vezzulli et al. 2008). While the majority of the loci in grape linkage maps are microsatellite markers developed from genomic DNA libraries, the availability of EST-SSRs will serve as new genetic markers to be included into the linkage map (Decroocq et al. 2003; Akkak et al. 2006; Salmaso et al. 2008). EST-SSRs have been reported to be less polymorphic but to have higher transferability than genomic SSRs in grape and other plants because of greater DNA sequence conservation in transcribed regions (Scott et al. 2000; Cho et al. 2000; Chabane et al. 2005).

Traditionally, SSR PCR products are separated by polyacrylamide gels (Thiel et al. 2003) or Metaphor Agarose gels (Chani et al. 2002). The electrophoresis-based technology is low-throughput and comes with imprecise sizing at times. Automatic capillary sequencing using fluorescently-labeled primers (Eujayl et al. 2002) provides more accurate and high-throughput genotyping results but the cost of dye-labeling each forward primer is high. Using M13 universal labeled primers with automatic capillary sequencing can not only reduce the cost but also provide fast and precise genotyping results (Oetting et al. 1995, Chen et al. 2006).

Here we report the identification and characterization of 1,701 unique grape EST-SSRs derived from a total of 215,609 grape ESTs. A set of SSR markers was developed from this analysis and validated by using M13 universal primers and an automatic capillary sequencing system.

Materials and methods

Plant materials

For PCR amplification, genotyping and polymorphism analysis, we selected four genotypes which are parents of two mapping populations: V. vinifera cvs. Cabernet Sauvignon and Riesling, and V. rotundifolia cvs. Noble and Summit. Genomic DNA was extracted from young leaves/shoot tips of these grape cultivars using a modified CTAB protocol (Qu et al. 1996).

Grape EST and genomic sequences retrieval from NCBI

All grape EST sequences available in the NCBI database on 10 February 2006 were retrieved. Among the total of 215,609 ESTs, 194,200 were from V. vinifera, 10,704 were V. shuttleworthii, 2,177 were V. aestivalis, 1,995 were V. riparia, and 6,533 were V. hybrids (V. rupestris A. de Serres × V. spp. b42-26).

A total of 31,910 genomics sequences were also retrieved from NCBI on 12 June 06. Among them, 30,832 were from a BAC library of V. vinifera cv. Pinot Noir, and 1,078 from V. vinifera cvs. Syrah and Maxxa.

Computer programs for mining SSRs from ESTs

A Perl script program named Microsatellite (MISA) developed by Thiel et al. (2003, http://pgrc.ipk-gatersleben.de/misa) was used to identify EST-SSRs. The SSRs are between 2 and 6 nucleotides in size. The minimal length of SSR repeats was defined as 2 × 9 = 18 bp for dinucleotides, 3 × 6 = 18 bp for trinucleotides, 4 × 5 = 20 bp for tetranucleotides, 5 × 4 = 20 bp for pentanucleotides, and 6 × 4 = 24 bp for hexanucleotides. ESTs containing SSRs were assembled in Sequencher® version 4.2 (Genecodes, Ann Arbor, Michigan, USA) under criteria of 40% minimum overlap and 90% minimum match percentage. A flow chart for mining and developing the grape EST-SSR markers is provided in Fig. 1.

Fig. 1.

Fig. 1

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Flow chart of Vitis EST-SSR identification and validation

Functional annotation of EST-SSRs

HTGOFAT, a data mining and annotation tool kit developed in Microsoft NET 2003, was utilized to functionally annotate the assembled EST-SSRs sequences (Dowd and Zaragoza 2005). The putative functional genes were classified using the Munich Information Centre for Protein Sequences (MIPS) Arabidopsis thaliana functional catalogue (MATDB, http://mips.gsf.de).

PCR and fragment analysis

EST sequences flanking the microsatellite motifs were used to design PCR primers using the program Primer3®. A total of 150 primer pairs (Table 3) were screened for assessment of polymorphisms among the four parents using a CEQ Genetic Analyzer (Beckman Coulter, California, USA).

Table 3.

Genotyping and allelic details of the 145 EST-SSRs in four grape cultivars

Marker ID Accession no. Repeat type Forward primer Reverse primer Expected size (bp) Riesling Cabernet Summit Noble R × C Allele S × N Allele
FAM01 CA817092 (ga)9 TTACCCGACACTGGACAC ACTTACCACCGAGATGAGG 310 967/983 967/983 989 989 ab × ab
FAM02 CB973719 (tc)10 GCCTTGGACCGAACTATC CTAAGAAACACCATTCATCAG 199 218/236 208/216 218 218 ab × cd
FAM03 CF371950 (tca)6 CACCGAAAGAGCACAAGA CACCGAAAGAGCACAAGA 232 239/266 243/251 262 252/280 ab × cd aa × bc
FAM04 CF405747 (ct)10 GTGACTTACAATCCTTCCAAA AGGGAGAGAGAGAGAGAGAGA 180 198 196 189/191 189/191 aa × bb ab × ab
FAM05 CB035928 (tat)6 ATTTCACCACCTGTCAATAAA CCACTTCCATACACACATACA 331 470 470/476 458 454 aa × ab aa × bb
FAM06 CB915165 (aag)11 AGATAATGACCGCTATGTGAA CAACAATCCCTACCCAAAC 296 313 313 304/335 316/322 ab × cd
FAM07 CV092730 (ctt)6 ACTTGTCTCCAAATCATCACA CATCAGCAGGGTAGAAATAGA 236 360/369 369 362 362/365 ab × aa aa × ab
FAM08 CV093018 (tcg)6 TCATCATCCACCACAACAC AGTCTCTTCGCATTAGGGA 235 248 248 254 254
FAM09 BQ106736 (ga)9 TGAGGCCTACATCTTGTTCT TCTGTGTGTCTCTCTGGTGA 277 282/310 286/294 306 296/324 ab × cd aa × ab
FAM10 CA814604 (aat)13 TGAAGCACTGATGCTTATTG ACAATGTCACACACAAGGTG 126 117 120/147 147 120/147 aa × bc aa × bc
FAM11 CB348477 (cgat)11 TACTCTAGGTCCATTGTGGG CGAATAACAATCTGGCTACC 367 568 566 570 570 aa × bb
FAM12 CB346585 (cct)6 GAGAGAAGAGTGGTGGTGAA CTCCGTGTAGCACCTTAAAT 352 379 NA 356/371 356/368 ab × ac
FAM13 CB974681 (cag)6 CTCTTCAGGAAACACTGGAG CCTGGAGTTCCTGGTAGATT 195 214 214 185/211 187/211 ab × ac
FAM14 CF208236 (ctt)8 AGACCACCATGGATCACTT CTTGATATTCTTAATGGGCG 196 212 212/215 198/201 198/201 aa × ab ab × ab
FAM15 CF208572 (tc)13 TCATCCTTTCCATACAGACC CTCCATTGGAAGACACTCAT 113 123 125/131 133/139 NA aa × bc
FAM16 CF214062 (tgc)6 GTTATGAAGCTGGAGGTGAG AAACTGGAGGACATTGCTAA 325 333/342 342 330 330 ab × aa
FAM17 CF214143 (ttc)7 TTTGCTTCTCCATTTGATCT CTTCAATCCTTGACAGGAAG 286 NA NA 517 517
FAM18 CF211871 (aga)7 AGAGAGCAAAGGAACATGAA ACAAACCCTAACCCTAGCTC 204 220 220 220/225 229 ab × cc
FAM19 CF211908 (caa)6 TTATCAGAGACGAGTCCACC TAAGTTATGGACTTGGACGG 302 314 314/316 314 314 aa × ab
FAM21 CF403813 (ag)9 TTCCAGAGACCTGTTTGTTT TGGAGGAGTAGGATGAGCTA 309 332 326 316/320 320 aa × bb ab × aa
FAM22 CF415755 (ttc)11 CTTGCTTCTCATACTCGTCC GAATCACCCATGGTTTCTAA 274 280/290 284/288 276 276 ab × cd
FAM23 DT026156 (aaaat)5 AGATCCTCCGAAACAAACTT CAAGATCAAGGAGAAACTGC 371 388/392 NA 276 276
FAM24 CF512631 (tatg)7 TCCATCTTCTTCTCGTGTTT TTTGAAGAAACAGGGACTTG 271 281/288 281/288 302 298/300 ab × ab aa × bb
FAM25 CV095500 (acc)9 CCACTATCACCACTACCACC CTTGTTCTTGGTCTGAGAGG 333 348 348 348/356 348 ab × aa
FAM26 CF515612 (gttt)5 CTCTCCACATTACGTCTTCC ATCAGGGCAAGTCTCTTGTA 290 310/318 310/318 316 316 ab × ab
FAM28 CF516198 (tc)21 TGGCCTTATATGCAGTTTCT AGGCTCAATTCCAACTGTTA 371 392 392 356 356/392 aa × ab
FAM29 BM437681 (att)6 TATAGTGGTCAATGCAACCA GGTGAGTCCACATGGTAAAT 117 135 135 133 133
FAM30 CB003274 (tc)9 ACTCAGCCAAACCAAGTAAA TTAGATCAAGCCCAGTCATT 277 294/302 298 298 298 ab × cc
FAM31 CD012233 (ta)10 CTTGGTGTCCTAAGGTTTCA AGAATTGCTGTCAGCTTCAT 231 240 248 241 241 aa × bb
FAM32 BM437023 (cac)7 AAACTGGACTCCACTGTCTG GTGGAGATGGCACTAATAGC 211 227 221/227 213 213 aa × ab
FAM33 CB912861 (ct)14 TACAGAAACCGAGTCACACA AATTCAAACTTGCAATCCAT 142 148/152 148/150 166/168 NA ab × ab
FAM34 CB914464 (cca)7 GTATGGGTTTGAGCAGAAAG GTTGTGGTGGTCGTGAAG 145 153 153 153 153/163 aa × ab
FAM35 CB005343 (cag)7 CACTCTCCAACTCCAGATGT ATGTTTCCCATATTCACAGC 156 160/182 172/182 180 180 ab × ac
FAM36 CB920562 (tct)6 TATCATTGTTTCCCTTCTGG GAAGAATTCAAGAGTGTCCG 359 380/386 396 381 381 ab × aa
FAM37 CB915120 (ct)16 CTTTGATTTGGGATGTGTCT TGGAAGCTCTTGATGAAGTT 121 139 139 138 138
FAM38 CB005457 (ct)10 GCTTCCATACGAGAAACTCA TAGGGTAATCCACAGTTTGC 225 235/238 238/242 231/235 231/235 ab × cd ab × ab
FAM39 CB915484 (ag)53 TACGTTCTGTTATCCAGGCT CAATATTTCAGTAGGGCCAG 388 388/414 NA 388 388
FAM40 CB920839 (ctt)7 AAGGTACTCAGCTTCCCTTC CACCATCTCTTCTCCACAAT 320 328/334 328/338 326 326 ab × ac
FAM41 CB922482 (agc)6 CAGAAGTTGAGAAGTCAGGG ACTTTGGCATTCCTAACTGA 175 185/191 185/191 185 185/200 ab × ab aa × ab
FAM42 CB916884 (gat)7 AAATTGATTTCATCAGTGCC CTATCATTGTCGCTTTCCTC 292 412 397/412 402 402 aa × ab
FAM43 CB911681 (cag)6 AATAGGGAAAGAGAACAGGC TCAATGTATGCACCCAAGTA 340 489 489 491 491
FAM44 CB922984 (aag)6 GAGGAGGTGGAAGGAGAA TTTGATAAGGTTGATGGTCC 113 129/135 135 135/141 132/141 ab × aa ab × ac
FAM45 CD801743 (ga)10 ATATAAGCCAAAGGTTCACA CAAAGGATGGAAAGCATAAG 379 395 395 381/398 383 ab × cc
FAM46 CD801804 (aaagg)4 TAACCTCACATCACATCCCT TATTAGGGTCTGCTGCAAAT 144 154/160 154/160 172/182 172/182 ab × ab ab × ab
FAM47 CD798867 (aata)5 GGGATGATGCTACCAGTCT CAAGTATAACAGGGTCCCAA 399 417 417 417 417
FAM48 CD798949 (cac)6 CTCAACAACGAATACCCACT AAGCATCGTTTCAAGTGTTT 241 519 519 523 523
FAM49 CD799025 (aaaac)5 AGCCTGAACACAATTTCTTT CAGCAAGAACTGAAGTGTGA 217 204/230 204/230 196 196 ab × ab
FAM50 BQ794329 (ag)14 CACAAAGCATGTCCATAAAC GGCTTATGCATTACTGGACT 178 185 185 197 185 aa × bb
FAM51 BQ798187 (ct)9 CTTGAGTCCTCACTCCAAAG TGTGACCATGGTGGACTT 101 129/130 129/130 126 126 ab × ab
FAM52 BQ800590 (ta)21 AAATCACACCCTACCATATACT AGGTGCACTAGCTTGAGTTC 142 133 NA 134 NA
FAM53 CF603660 (aacaag)4 AACCTCTGCCACCACAAC CCACACCTCATCGAATATCT 385 394/400 394/400 390 390 ab × ab
FAM54 CF605507 (cca)7 ATCTCAAGCCTCTTCTTTCC ATCAAGAATATCATCCACGC 319 326/335 326/335 326 326 ab × ab
FAM55 CF605791 (acc)6 GCACCCACTCACAATGTT AGGGAAGGAGTAGTAGGTGG 271 284/290 290 293 287/293 ab × aa aa × ab
FAM56 DT019642 (ttc)6 GCAGAACCCAAGTCTCATAC CAGGTATGAGAGGACTGAGC 356 368 368 368 368
FAM57 CB968692 (ct)16 CCATCTACCATCACCTTTGT GGAGAAGTGGTATTTGGTGA 152 155 173 158/163 171 aa × bb ab × cc
FAM58 CF403802 (at)18 TAGACGTTTGCCCTATTTGT CCTCTAACATGTCCCATTTC 158 154 154 NA 154
FAM59 CB917857 (gca)7 GATGGTATACGACGGAGAAA AGAGTACGACCCTTCGATCT 175 186/192 192 180 180 ab × aa
FAM60 CB916170 (caa)6 CCTCATCTGGCTTTCATAAC CTGGACAGAACTTGGATCAT 143 152/158 132/152 155 155 ab × cd
FAM61 CF608950 (ct)9 GCTACTTCTGGGAATGTTCA AGTCCTCATAAATTCTCAAACA 375 395 395 406 406
FAM62 CB342303 (gca)6 CTAATCTCCAGCGAAACAAC GTCAGCAATGTTGTCATTTG 274 257/289 NA 253/286 286 ab × aa
FAM63 CB346557 (tca)6 AAATGCACTCGTCTCTTCAT CGCTTGACCTTACATACTCC 334 350 350 348/360 348/360 ab × ab
FAM64 CN007369 (aag)8 TATACTTCACCGCAATTCCT TGATCAGCTCCTCGATATTT 261 277 274/286 291 NA aa × bc
FAM65 DT019756 (gca)6 CCCTATCCAGCAGACACTAC TTCATCTGGCTATACATCCC 129 145/148 145 142 142 ab × aa
FAM66 BQ794995 (at)10 TTTATCTCAAACCTTCACATCT GTTGTTAGGAGTGACTTCCG 200 210 210 210 207/212 aa × bc
FAM67 CB977433 (atttt)4 GGACTTCATCCTGGAGTACA CTTGCAGGACACCTAAATTC 376 395 395 387 387
FAM68 CF607255 (aga)6 AAACCCTACCGAAGTCTCTC CTTCTTCTTCGCCTCTGTTA 307 321 321 321 321
FAM69 CF608166 (gaa)6 GATACATAAGATGCCAAGGG CATCCTCGTCTTCAATCTTC 335 352 352 352 352
FAM70 CN545596 (ga)10 TGGCAATAGAAGAGGAGTGT AACAACACTTCCAGTATGGG 281 389/392 NA 380/397 397/403 ab × ac
FAM71 CN548152 (at)9 AGTCTCTTCAAGTGCCTCAG CTGCATAGACTGACGAAACA 180 202 195 195 197 aa × bb aa × bb
FAM72 CN549034 (ct)14 TCAGTCCAGATTTACCTTGC TCATGTGGTTCTGCAATAGA 171 172/188 172/188 180 170/180 ab × ab aa × ab
FAM73 CO818811 (tttc)5 GGCATATGGAAAGGGATAA ATTTGGTCAGATGGATCAAG 359 390/399 377/399 369 369 ab × ac
FAM74 CO819364 (ag)8 GGCCTCCAGATCAACTAGTAA GCGCCTCTGTCATAGAATAC 289 307 307 234/318 309 ab × cc
FAM75 DT004860 (ctt)11 CCTGTAAACGCTTCAAATCT ATGGCTGAGTCATAGAGAGG 169 184/187 187 172/175 172/175 ab × aa ab × ab
FAM76 DT009858 (cag)6 ATTAACGAGGATGTGTTTGG AAGGATCCATTTCACATACG 394 409 409 427 433 aa × bb
FAM78 DT010742 (tgaag)5 TTCATGACAATTGTGTTTGG CGGACTCATCAGAGAAGAAG 303 322 322 339 339
FAM79 DT011109 (ag)12 GCAGAAGCAAGAAGTGAAGT AGATTCAAAGCCACTGAAGA 147 162/172 162/172 153/157 153/157 ab × ab ab × ab
FAM80 DT011686 (ct)9 AACTCATTCAGACAGACCCA ATGATTTCCTCAGCTTTCAA 316 424 424 428 428
FAM81 DT011972 (gcc)8 TTCTCTCAACATACATGGCA GCACTGAATACACTTGGGTT 203 218 218 226 226
FAM82 DT012098 (tc)8 AGAAGCACTCCATCTGAGAA GAAGGCATAATCATCCTGAA 344 357/361 361 348 348 ab × aa
FAM83 DT012268 (gagagg)4 CTCCGTGAGAGAAGGTTATG CATTCCTGACAACCATGC 249 251/275 255 244/255 244/255 ab × cc ab × ab
FAM84 DT014885 (ga)18 CCCATATTCTCAACCAAG TCCCAATATGTAGAACCTGG 177 195 195/210 184 184 aa × ab
FAM85 DT015345 (ta)10 GAATTCAAGGAGAGGACACA TATATATTGCGAGGCAACAA 283 300 300/304 308 308 aa × ab
FAM86 DT016122 (at)11 AGAAACCAGCTGCCAATA GGAGGAGACCATAGACATGA 267 276 284 277 NA aa × bb
FAM87 DT026426 (gat)6 TCCGAAGAAGAAGAAGAAGA CTGGCCATACTGTTTAAAGG 375 388/400 388 421/436 421/433 ab × aa ab × ac
FAM88 DV220116 (at)14 AATGTCAAAGATTCACCAGG CAGTTGCAGCTCATAGAACA 237 230/242 254/256 223 226 ab × cd aa × bb
FAM89 DV220950 (ag)9 CCTTGTTTGGACTTTGGAG CTAATGGCTTCTGATATGGC 193 211 211 211 211
FAM90 DV221456 (ct)10 GATCAAAGATTATTGCGAGG AACAAGCAAACAGAGGGTTA 368 358/387 387 392/400 400 ab × cd
FAM91 DV222762 (tct)8 ATTATCGCAACCAAGATGTC ATCAGCCTCTGTAACTGGAA 159 176 173/176 161 161 aa × bc
FAM92 DV939679 (ta)9 TTGTACTTTGGTGCACCTTT ACCTTTGATAACCATTGGG 388 405 405 417 417
FAM93 DV940288 (ag)12 ATTACATCTCATCCCGGTAA ATGCTCTCAGAGGAGTCTCA 242 258 244/258 238/243 238/243 aa × ab ab × ab
FAM94 CF206303 (ttc)6 GGCAATGCAAGGCTATTT ATCTTCATATGCAGCACCTT 190 277 277 277 277
FAM95 CF205720 (ga)10 ATCATCTTCTGCCTCGAATA TGAAACTGTGCATTCATCAT 176 191 191 191 191
FAM96 CF205720 (ga)10 ATCATCTTCTGCCTCGAATA TGGTGAAGGTTAGTGTGATCT 302 300 300 304 304
FAM97 CF205251 (aat)6 AGGTCTCCAGCTTGTACTCA ATATGTAGCCAGACGTGTCC 301 326 318/326 320 320/324 aa × ab aa × ab
FAM98 CF205081 (acc)6 AAAGGGTCTTCTGAACTTCC TCCTAACTGAAACGAAAGGA 382 395 395 395 395
FAM99 CF204388 (aaaat)6 ATTCCAAACAAAGCAGGTAA GGATTGTGAATAAGCCCATA 247 261/266 261 NA NA ab × aa
FAM100 CF203674 (tc)13 CATTTCACGAGCTCTAAACC GGATGAGACCAAATTCAAGA 185 186 186 182/191 182 ab × aa
FAM101 CF202410 (tatg)5 TGTGACTATGTTTCTTTGTATGT CAAATCTGATTGTTCCAGGT 251 283/286 283 292/294 292/298 ab × aa ab × ac
FAM102 CF201608 (tatg)5 ACCCATGTTCTCTTCAACAC CGAGAGATTGGAGAGTATCG 147 145 145 125 125
FAM103 CV092439 (gga)7 GGAGCTTCTTGACATCATTC CGAATTTCATCATTCTCACA 245 347 347 350 344/350 aa × ab
FAM104 CV092870 (agc)7 TGGATCCTATTCTCTCCTCA AAATATTTCCTAATACCCGACT 113 132 132 132 132
FAM105 CV092969 (atc)8 CCCTCTCACTCTTTGAAATC ATGATTGGATGGTCATGTCT 279 291 291 285 285
FAM106 CV093018 (tcg)6 TCATCAACATCATCATCCAC GCACTCTTCTCACCTTTGTT 202 216 216 222 219/225 aa × bc
FAM107 CV094376 (cag)11 ATCAGGTCGAAATAATGGTG GACCATTGTTAACCGTAGGA 302 301 301 304 304
FAM108 CV094448 (ctt)8 CTCTTCTCAAACTCCAATGC AGGAGTCACCAATGATGAAG 151 156 156 156 156
FAM109 CV094765 (at)9 GTTAACATCTAGGCGGTTTG GCTTGCACATGTTAACAGAA 321 333 333 333 333
FAM110 CV095258 (gct)6 GGCTATTGATTCAGCTCCTA TACAAGCCGTTCTATCCATT 192 287/303 303 290 290 ab × aa
FAM112 CV097295 (gaa)8 AGTTTCGTATTCGAAGTCCC TCTTGAAATCGACTGAGGTT 205 206 206 199 199
FAM113 CV098232 (gga)6 ACTTCCATCTACCGTCCTCT GACTTCCTTCCAGTCCTTCT 246 275 275/291 260 260 aa × ab
FAM115 CV098402 (agg)6 AACTAACTCAGCCAAGGACA CACAGCCTTGTACATTATGC 318 345 345 333 330
FAM116 CV099053 (ag)16 CTTCTATTTCTGGCACCCTT CTTCTGTGGAGGAAGAGTTG 330 333 333/341 333 333 aa × ab
FAM117 CV099069 (tga)7 TAGTGGAATACCAGAGTGGG AGTCGTTCAGATTGATCACC 225 229 229 229 229
FAM118 CV100438 (aag)11 AAAGCTTAAGCAACACCTTG AACAAATCACACGTTATCCC 301 300 305/325 312 NA aa × bc
FAM119 CA810326 (cca)7 GCAAATGAGTTACCAGAAAG GTAGAAAGGAGGAAGGACCA 311 428/444 424/444 420 420 ab × ac
FAM120 CA810919 (agg)7 CGCATCAGAAGTCATCAAC ACCCTCACTCTCACACTCAC 324 430 426/430 432 432 aa × ab
FAM121 CA816978 (gat)6 CCCTTCCATACTCCAACATAC CCTCAATCTTAGTCGCTCC 348 348 348 348/351 348/351 ab × ab
FAM122 CB343602 (ct)9 AGAGGAAGAAGCACAAATCTC AAAGAGTGGAGGAATCGG 354 370 370 385 385/391 aa × ab
FAM123 CB343426 (ga)9 GTAGCCAACAGAACCAGAGA CAAACACATCCTCACCCTT 516 534 530/534 492 NA aa × ab
FAM124 CB349648 (tga)6 TAAGGAAGCATTAGAAACAAG AACCAAGAAGGAAGAAGAGAA 312 308/315 315 313 313 ab × aa
FAM125 CB969938 (cag)6 TCTTGTCATCTACCTCATCTTG CACAGTCCCTCCTCCTCT 215 238 238 238 238
FAM126 CB973643 (ag)10 CGACCTAAGAAACACCATTC CCTTGGACCGAACTATCTG 202 220/236 208/218 218 218 ab × cd
FAM127 CB982007 (gga)6 ACGGAAGAAGAGAAGAAAGAG ATCCACCGAAACAAACTTAC 197 216/222 216 NA NA ab × aa
FAM128 CF214574 (act)6 TACAAGAGCCAAGAGGGATT GGATAACGAAGGAGACAGAGT 315 784 786 782 782 aa × bb
FAM129 CF212154 (aaagg)4 ACATCCCTTTGTTGTCTTCTT ATTTGTGCTGTTGTCTGTTGT 119 130 130 141 141
FAM130 CF405979 (ctc)6 ACAAAGCAGGTAAGTAGCAAA AAGACGGAAGAAGAGAAGAAA 272 286 286 378 378
FAM131 CF514744 (atg)6 TGACTGGCATACTGATTTACC CCCAATGAACTACCTTTACCT 259 272/275 275 272/275 272/275 ab × aa ab × ab
FAM132 CF518394 (tca)6 ACCCAATGAACTACCTTTACC AGGAACAAGACAAACAATACACT 133 149 149 146/149 146/149 ab × ab
FAM133 CA812979 (ga)13 GGGAGATTGAAAGGAAGTG GGAGACCGACGAGGATAA 373 385 385 388 388
FAM134 CA813367 (cct)7 GTAGCCAACAGAACCAGAGA AAACACATCCTCACCTTCC 514 533 529/533 493 493 aa × ab
FAM135 CB004296 (cat)6 AGGGTTGTGTCTCTTCTCAA GATACTTCATCTGTTGCTTCTG 400 413 417/419 413 413 aa × bc
FAM136 CB919516 (ctt)14 AGGGAGATGACAAAGATGAAG CCAAACACCGTAGGAGAGA 245 249/264 NA 261/264 234/261 ab × ac
FAm137 CB920177 (tc)9 CAAACTGTCCAATCCTCATAGT AGTAGAGCCAAGTGTCAAACC 146 157 155 157 157
FAM138 CB005751 (gca)9 CGAGTGGTAGAGAGGAGAGAG GTTGAGGGTGATGGTAAGG 208 223 223/237 237 237 aa × ab
FAM139 CD013208 (atttt)4 GGCAGAAAGGCATAAATAGTC TGGGCACTCTCCAACCTG 377 395 395 395 395
FAM140 CB916384 (tttc)6 AAGGGAAGAGAGGTATCGG CCATAAACGAGAAGAAACAAA 253 274 265/279 269 269 aa × bc
FAM141 CD711290 (ttc)6 GAACCATAGACAAGACAAACAA AAGAGAGAAGCAACGAAGAAC 182 192 186/198 192 192 aa × bc
FAM142 CN603827 (aga)5 AAGACCGAAGAAGAGAAGAAA TAATACCGTGGAAATCACAAA 246 271 267 263 263/267 aa × bb aa × ab
FAM143 CV099313 (ctc)6 CTCTTTGACCGTTTCCAG CCCACTACCTCTTACCTTCTT 199 217 217 217 217
FAM144 CV092545 (acc)9 CACCACTATCACCACTACCAC AGGAGGCGAATGAAGGTC 193 211 NA 211/220 211/220 ab × ab
FAM145 CV093192 (caa)7 TCCAACAACAACAACTACTAC AGGAATCTCGTGTCGCTC 215 238/247 238/241 230/236 230/236 ab × ac ab × ab
FAM146 CB920177 (tc)9 CAAACTGTCCAATCCTCATAGT AGTAGAGCCAAGTGTCAAACC 146 157/160 155 157 157 ab × aa
FAM147 CV092326 (gat)6 TACAACCACATAGAGGCACTT TCTTCTTCAGTTTCTTCACCA 185 606/612 606 602 606 ab × aa aa × bb
FAM148 CV099379 (caa)7 TCCTCCTTGTTATCCTCTTCT TAGTAGTTGTTTCGGTTGGAC 400 413 413 410 410
FAM149 CV094327 (ctt)9 TAGACCTCCACCACTCTCTC GTCATCAGCGAAAGCATC 343 362/365 359/365 335/353 332/335 ab × ac ab × ac
FAM150 CF519163 (gct)6 ATCTGACAAAGGAAAGGAGAA GTAACATACCGAGGAAGGCA 235 244 NA 237 237/244   aa × ab
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NA not available

To save cost, a 20-bp long universal M13 forward primer sequence GTT GTAAAA CGA CGG CCA GT (Oetting et al. 1995) was added as a common tail to the 5′ end of all 180 SSR forward primers. All SSR primers, including regular and M13-tailed forward primers, were synthesized by Operon Technologies (Huntsville, Alabama, USA). The universal M13 primers were labeled by Sigma-Genosys (USA) and used for CEQ Genetic Analyzer Fragment Analysis.

PCR reactions were performed in a 20-μl reaction mix including 30 ng of genomic DNA, 10 × PCR buffer (Promega), 2 μl of 2 mM dNTP (Promega), 1.0 U Taq DNA polymerase, 2.8 μl of 25 mM MgCl2 (Promega), and 0.3 μM primers. The PCR reactions were carried out in a PTC-200 thermal cycler (MJ Research) with the following thermal profile: 3 min at 94°C followed by 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 48 to 58°C (based on the T m of the different primer sets), and 2 min extension at 72°C, followed by a final step of 6 min extension at 72°C. The same conditions were also used for labeling the primers.

For fragment analysis using the CEQ Genetic Analyzer, 0.25 μl of each M13 labeled PCR product was mixed with 40 μl Sample Loading Solution (Beckman Coulter 608087) with 0.2 μl 400-bp DNA size standard (Beckman Coulter 608098) and overlaid with one drop of light mineral oil, then loaded into the 96-well sample microtiter plates (Beckman Coulter 609801). CEQ Sequencing Separation Buffer (Beckman Coulter 608012) were also loaded into the 96-well separation plate (Beckman Coulter 609844). Dye-labeled amplicons were automatically sized by running on “Frag-3” separation and the GenomeLab software (Beckman Coulter) and then visually examined.

Results and discussion

Identification and characterization of grape EST-SSRs

A total of 6,447 out of 215,609 (3%) grape ESTs retrieved from NCBI on 1 February 2006 contained SSRs (Table 1). With some of them having multiple SSR sites, a total of 6,815 SSR motifs were identified among these 6,447 EST sequences. The percentage of EST-SSRs varied slightly among different Vitis species, ranging from 2.98% for V. vinifera (5,782 of 194,200), 3.50% for V. aestivalis (74 of 2,116), 3.55% for V. shuttleworthii (389 of 10,933), to 5.43% for V. riparia (59 out of 1,087). The EST-SSRs accounted for 2.71% for a Vitis hybrid of (V. rupestris A. de Serres × V. spp. b42-26) (177 of 6,533; Electronic Supplementary Material 1).

Table 1.

Characterization of grape redundant and non-redundant EST and genomic SSRs

  Redundant SSR-ESTs Non-redundant SSR-ESTs Genomic SSR sequences
Total % 6,447 1,701 1,346
 Di- 1,835(28.5%) 664(39.0%) 699(51.9%)
 Tri- 3,235(50.2%) 710(41.7%) 341(25.3%)
 Tetra- 391(6.1%) 125(7.3%) 154(25.2%)
 Penta- 618(9.6%) 134(7.9%) 99 (7.4%)
 Hexa- 736(11.4%) 179(10.5%) 53 (3.9%)
Abundant type
 Di- AG/CT(18.9%) AG/CT(26.9%) AT/AT (33.0%)
 Tri- AGG/CCT(14.8%) AAG/CTT (11.3%) AAT/ATT (18.6%)
 Tetra- AAGG/CCTT(1,7%) AAAT/ATTT(2.4%) AAAT/ATTT(7.0%)
 Penta- AAAAT/ATTTT(2.8%) AAAAT/ATTTT(2.1%) AAAAT/ATTTT(3.8%)
 Hexa- AGGGTC/AGTCCC(2.6%) ACCCTG/ACTGGG(1.1%) AAAAAT/TAAAAA(1.7%)
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Among the redundant EST-derived SSR repeats, tri-nucleotide, which accounted for 50.2% of total SSRs, was the most abundant repeat unit followed by di (28.5%), hexa (11.4%), penta (9.6%), and tetranucleotide (6.1%; Table 1). These findings are in agreement with previous observations on abundance of SSR repeat units in barley, maize, rice, sorghum, and wheat (Kantety et al. 2002). The dominance of trinucleotide SSRs was viewed as the result of a frame shift in size of one amino acid read, or three nucleotides, a selection against possible frame shift mutations (Metzgar et al. 2000; Toth et al. 2000; Wren et al. 2000; Cordeiro et al. 2001). For the same reason, a higher percentage was also observed in hexanucleotide SSRs than tetra- and penta-repeats. In both non-redundant and redundant EST-SSRs (Table 1), di- and tri-repeats were accounted for about 80% of the total EST-SSRs for each group (redundant: di-28.5%, tri-50.2%; non-redundant: di-39%, tri-41.7%). Interestingly, the proportion of tri repeats dropped from 50.2% in redundant to 41.7% in non-redundant ESTs while di repeats increased from 28.5 to 39.0% after eliminating the redundancy by contig assembling (Table 1). The result was interpreted to suggest that tri-repeat SSRs were mainly found in coding regions (Yu et al. 2004) and many of these redundant EST-SSRs were eliminated because these sequences contain tri-repeats representing putative amino acid runs (Li et al. 2004) as overexpressed ESTs representing the same set of genes. Another explanation is the effect of gene duplication and paralogy. Depending on the parameters used for clustering, untranslated regions of paralogous genes, which are more divergent and contain all types of SSR, might have remained separated, while ESTs covering exons of paralogous genes, which are more conserved and highly enriched in tri-nucleotide SSR, might have collapsed more frequently into a “single” redundant EST.

Comparison between genomic and EST derived SSRs

Unlike the tri-nucleotide repeats as the dominant type in SSR-ESTs, the number of genome sequence-derived SSRs were dominated by di-nucleotide repeats that accounted for 51.9% of total genomic SSRs, followed by tri- (25.3%), tetra- (25.2%), penta- (7.4%), and hexa-SSRs (3.9%; Table 1). Similar patterns for EST-SSRs having a higher proportion in tri-repeats than genomic SSRs were reported in the literacture (Cardle et al. 2000). Among the top 20 SSRs in ranking, the most abundant di-nucleotide repeat in non-redundant ESTs was AG/CT which accounted for 17.9% of total EST-SSRs, followed by AT/AT (8.4%; Table 2), while the most abundant di-nucleotide repeat in genomic sequences was AT/AT which accounted for 33.0%, followed by AG/CT (15.5%) and AC/GT (3.5%). The most common EST-derived tri-nucleotide repeat was AAG/CTT (14.0%), while AAT/ATT (18.6%) was the most abundant tri-nucleotide SSRs derived from genomic sequences. Among grape genomic sequences, around 67.1% of the SSRs belonged to three types of repeats: AT/AT (33.0%), AAT/ATT (18.6%), and AG/CT (15.5%; Tables 2 and 3).

Table 2.

Top 20 SSR motifs in grape ESTs and genomic sequences

EST-SSR repeats Total % Genomic SSR repeats Total %
AG/CT 1,217 17.9 AT/AT 444 33.0
AGG/CCT 957 14.0 AAT/ATT 251 18.6
AT/AT 575 8.4 AG/CT 208 15.5
AAG/CTT 547 8.0 AAAT/ATTT 94 7.0
AAT/ATT 403 5.9 AAAAT/ATTTT 51 3.8
ACC/GGT 326 4.8 AAG/CTT 48 3.6
AGC/CGT 237 3.5 AC/GT 47 3.5
ACG/CTG 201 2.9 AAAAAT/ATTTTT 21 1.6
AGT/ATC 201 2.9 AAAAG/CTTTT 17 1.3
AAAAT/ATTTT 195 2.9 AAAAAG/CTTTTT 15 1.1
ACT/ATG 168 2.5 AATT/AATT 14 1.0
AAC/GTT 163 2.4 AAAG/CTTT 13 1.0
AGGGTC/AGTCCC 144 2.1 AGT/ATC 12 0.9
AAAAG/CTTTT 98 1.4 ACAT/ATGT 10 0.7
AAAAC/GTTTT 97 1.4 ACT/ATG 9 0.7
AAAT/ATTT 89 1.3 AGAT/ATCT 8 0.6
AAGG/CCTT 88 1.3 AAC/GTT 7 0.5
AAAGG/CCTTT 88 1.3 AAAAC/GTTTT 6 0.4
ACCCTG/ACTGGG 81 1.2 AAATT/AATTT 6 0.4
AAAAAG/CTTTTT 78 1.1 AGG/CCT 5 0.4
Other motifs 862 12.6 Other motifs 60 4.5
Total 6,815 100.0   1,346 100.0
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Functional analysis of EST-SSR sequences

The 1,701 assembled non-redundant EST-SSRs were functionally annotated using the HTGOFAT program (Dowd and Zaragoza 2005). Fifty-eight percent (994 out of 1,701) of the EST-SSRs were annotated and grouped by the Biological Process Classification using the MIPS MATDB Arabidopsis Scheme. The most abundant EST-SSRs belonged to the categories of protein-binding (22%) and subcellular localization (18%

;

Fig. 2), which demonstrated a similar pattern to wheat, rice, maize and barley (Tang et al. 2006). The 150 validated markers were further functionally annotated (Electronic Supplementary Material 2) and estimation of their genomic/chromosome locations by comparison to grapevine genome assembly (Jaillon et al. 2007; Velasco et al. 2007) is given in Electronic Supplementary Material 3.

Fig. 2.

Fig. 2

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Functional prediction of 994 grape SSR-EST based on the MIP MATDB classification scheme

SSR marker development and validation

The Beckman CEQ8800 Genetic Analyzer was used for the SSR validation and analysis. This system can detect DNA fragment length polymorphism in a “single base pair”. A set of 150 primer pairs was initially screened for SSR marker development and validation. Parents of two mapping populations, V. vinifera Riesling × Cabernet Sauvignon (Riaz et al. 2004) and V. rotundifolia Summit × Noble (Ren et al. 2000) were used for the screening. Results showed that 145 out of 150 primers had well-amplified fragments among the four cultivars (Table 3). Some of the fragment sizes exceeded expected sizes possibly due to their having introns within the flanking regions or the length of the repeat being shorter than the source species, and less prone to polymerase slippage. Polymorphisms were found in 66 primer pairs between Riesling and Cabernet Sauvignon, and 40 between Summit and Noble. Only 16 of the polymorphic primers shared the same polymorphic lengths between these two parent pairs, reflecting the fact that the alleles between V. vinifera and V. rotundifolia grape are distinct (Riaz et al. 2008).

The homo and heterozygosity of these 145 loci were screened in the four testing cultivars; 92 of 144 were identified as homozygous and 52 were heterozygous loci in Riesling, while 86 of 136 were homozygous and 49 were heterozygous loci in Cabernet Sauvignon. Among the 92 Riesling and 86 Cabernet Sauvignon homozygous loci, 68 are common in both parents (Table 4). As for the muscadine grapes Noble and Summit, Noble showed 97 homozygous and 39 heterozygous loci and the respective number for Summit was 108 and 34 (Table 4). Some of those microsatellite loci were selected for having long stretches in V. vinifera grapes, and thus may show more polymorphisms than in muscadines. From this screening of 145 loci, muscadine grapes demonstrated a higher homozygosity compared to V. vinifera grapes.

Table 4.

Level of heterozygosity in two Vitis vinifera and two Muscadinia rotundifolia genotypes for a set of 145 SSR markers

Genotype Riesling Cabernet Sauvignon Summit Noble Riesling vs Cabernet Sauvignon Summit vs Noble
Homozygous 92 86 108 96 66(9)a 86(6)b
Heterozygous 52 49 34 40
Failed 1 10 3 9
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a9 primers are homozygous in both Riesling and Cabernet Sauvignon, but are polymorphic between two cultivars

b6 primers are homozygous in both Submit and Noble, but are polymorphic between two cultivars

Pairwise comparison between Riesling and Cabernet Sauvignon showed that 72 loci were monomorphic with either one allele (60) or two (12). Seventy loci were heterozygous in at least one cultivar with either two (53), three (10), or four alleles (7; Table 5). The muscadine Noble and Summit showed 50 heterozygous loci in at least one parent with either two (31), three (17), or four alleles (2). Ninety homozygous loci were found in both cultivars with either one (84) or two alleles (6; Table 5). According to the results from these 145 EST-SSR loci, the percentage of polymorphisms is about 49% between Riesling and Cabernet Sauvignon, and 29% between Summit and Noble. However, those polymorphic SSRs that are homozygous (e.g. aa × bb) in both parents cannot be mapped in F1 populations although they are useful for mapping in F2 or backcross populations (Chen et al. 2006). The heterozygous monomorphic SSRs (e.g. ab × ab) can be used for mapping in F1 populations (Table 5). As a result, the estimated number of SSRs that can be mapped in the F1 populations between Riesling and Cabernet Sauvignon is about 46%, which means that out of the total 1,037 SSRs with successful primers designed, around 477 EST-SSR putative markers can be mapped in the F1 population, and about 33% of the total SSRs (342 EST-SSR loci) can be mapped in the F1 of Summit × Noble.

Table 5.

Distribution of the segregation types expected for the two mapping populations

Alleles R × Ca Number S × Nb Number Mappable in F1
1 aa × aa 57 aa × aa 80 No
2 aa × bb 9 aa × bb 6 No
2 aa × ab 15 aa × ab 13 Yes
2 ab × aa 18 ab × aa 4 Yes
2 ab × ab 12 ab × ab 13 Yes
3 aa × bc 8 aa × bc 4 Yes
3 ab × cc 2 ab × cc 4 Yes
3 ab × ac 6 ab × ac 8 Yes
4 ab × cd 9 ab × cd 1 Yes
Total mappable   70 47  
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aR × C: Riesling × Cabernet Sauvignon

bS × N: Summit × Noble

EST-SSR marker transferability was evaluated and the current research showed a high transferability across species. All but two of the 145 EST-SSR markers in Vitis vinifera appeared in the muscadine as well. This result indicated that development of EST-SSR markers is a cost-effective method for obtaining additional markers for grape genome typing and gene mapping.

EST-SSRs provided sources of additional markers for marker development. Compared to genomic-derived markers, EST-SSRs are highly transferable for detecting the gene-rich areas within the genome. We can utilize these markers to evaluate marker transferability across taxa, and conduct analysis in comparative mapping and gene functional diversity analysis, in addition to genotyping. The functional EST-SSR markers should be even more useful for developing a linkage map or tagging a viticulturally important trait. In addition, the polymorphic EST-SSR markers are much needed for genotyping, cultivar identification and development of a linkage map in muscadine grapes since they are genetically much less diversified than Vitis species.

Electronic supplementary material

Below is the link to the electronic supplementary material.

(XLS 77 kb) (77KB, xls)
(XLS 44 kb) (44.5KB, xls)
(XLS 42 kb) (42KB, xls)

Acknowledgments

This work was supported by USDA Capacity Building Grant (#0205031) and FAMU-ARS Science Center for Excellence. The authors thank two anonymous reviewers for invaluable comments on the manuscript. Special thanks go to Ms. Elisa Scott for helping us to prepare the DNA and PCR samples.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Contributor Information

Hong Huang, Email: huanghon2003@gmail.com.

Jiang Lu, Email: jiang.lu@famu.edu.

Zhongbo Ren, Email: zhongbo.ren@famu.edu.

Wayne Hunter, Email: whunter@ushrl.ars.usda.gov.

Scot E. Dowd, Email: sdowd@pathogenresearch.org

Phat Dang, Email: pdang@nprl.usda.gov.

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