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

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.

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

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

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%)

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

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

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

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

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.

Below is the link to the electronic supplementary material.

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