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. 2009 Sep 28;10:454. doi: 10.1186/1471-2164-10-454

Ontology and diversity of transcript-associated microsatellites mined from a globe artichoke EST database

Davide Scaglione 1, Alberto Acquadro 1, Ezio Portis 1, Christopher A Taylor 2, Sergio Lanteri 1,✉, Steven J Knapp 2
PMCID: PMC2760586  PMID: 19785740

Abstract

Background

The globe artichoke (Cynara cardunculus var. scolymus L.) is a significant crop in the Mediterranean basin. Despite its commercial importance and its both dietary and pharmaceutical value, knowledge of its genetics and genomics remains scant. Microsatellite markers have become a key tool in genetic and genomic analysis, and we have exploited recently acquired EST (expressed sequence tag) sequence data (Composite Genome Project - CGP) to develop an extensive set of microsatellite markers.

Results

A unigene assembly was created from over 36,000 globe artichoke EST sequences, containing 6,621 contigs and 12,434 singletons. Over 12,000 of these unigenes were functionally assigned on the basis of homology with Arabidopsis thaliana reference proteins. A total of 4,219 perfect repeats, located within 3,308 unigenes was identified and the gene ontology (GO) analysis highlighted some GO term's enrichments among different classes of microsatellites with respect to their position. Sufficient flanking sequence was available to enable the design of primers to amplify 2,311 of these microsatellites, and a set of 300 was tested against a DNA panel derived from 28 C. cardunculus genotypes. Consistent amplification and polymorphism was obtained from 236 of these assays. Their polymorphic information content (PIC) ranged from 0.04 to 0.90 (mean 0.66). Between 176 and 198 of the assays were informative in at least one of the three available mapping populations.

Conclusion

EST-based microsatellites have provided a large set of de novo genetic markers, which show significant amounts of polymorphism both between and within the three taxa of C. cardunculus. They are thus well suited as assays for phylogenetic analysis, the construction of genetic maps, marker-assisted breeding, transcript mapping and other genomic applications in the species.

Background

The globe artichoke Cynara cardunculus is a member of the Asteraceae (Compositae) family, and originates from the Mediterranean basin [1]. The species is subdivided into three taxa - the globe artichoke (var. scolymus L.), the cultivated cardoon (var. altilis DC), and their progenitor, the wild cardoon [var. sylvestris (Lamk) Fiori]. The edible part of the globe artichoke plant is provided by its immature inflorescence, referred as a capitulum or head [2], and represents a significant component of the Mediterranean diet. The cultivated cardoon is grown for its fleshy stems, which are used in traditional cuisine. Leaf extracts of the species contain a number of bioactive compounds (e.g., quercetin, rutin, luteolin, gallic acid, di-caffeoylchinic acid, and sesquiterpene lactones) which have been shown to have anti-oxidative and anti-carcinogenic activity, to inhibit cholesterol biosynthesis, and to enhance lipid metabolism [3-8]. The antioxidant content per serving of globe artichoke ranks very highly among vegetables [9], while the roots provide a source of inulin, a proven enhancer of human intestinal flora [10,11]. In spite of its economic importance, however, little breeding effort has been applied to date in the globe artichoke.

Progress has been made in the development of DNA marker based genetic maps in globe artichoke [12-14]. The most saturated map has been recently developed from a set of F1 progeny of a cross between a globe artichoke and a cultivated cardoon genotypes [14]. This map consisted of 20 linkage groups comprising 326 loci and spanned ~1500 cM with a mean inter-marker distance of ~4.5 cM. A set of loci common to this map and a previously developed one [12] allowed for map alignment and the definition of 17 homologous linkage groups, corresponding to the haploid chromosome number of the species.

It was long assumed that SSRs were primarily associated with non-coding DNA, but it has now become clear that they are more frequent in transcribed than non-transcribed sequences and equally frequent in the transcriptomes of plants with dramatically different nuclear DNA contents [15]. EST databases therefore represent a convenient resource for the identification of microsatellites, some of which, as a result of their presence within coding DNA, have the potential to deliver informative within gene markers, exploitable as COS (conserved orthologous set) for genomic comparative analysis.

Here, we report: i) the unigene assembly based on the globe artichoke EST database deposited in GenBank by the Compositae Genome Project (CGP), ii) the identification of a wide set of EST-based microsatellite markers and iii) the evaluation of the informativeness of a subset of these markers using a panel of C. cardunculus genotypes. Furthermore, we performed a comprehensive functional annotation, inferred from sequence alignment (ISA), as well as a gene ontology categorisation inferred from sequence orthology (ISO) of the SSR-containing unigenes. At last we assessed whether motif type and relative position within CDSs (Coding DNA Sequences)/UTRs (Untraslated Regions) may be preferentially associated with a particular gene ontology term.

Results and Discussion

EST microsatellite discovery, frequency and primer design

Globe artichoke ESTs were trimmed, assembled, and annotated using a customized bioinformatic pipeline (Figure 1) into 19,055 unigenes (6,621 contigs and 12,434 singletons) spanning 15 Mbp. The transcript assembly and unigene consensus sequences are supplied as electronic supplementary materials (See Additional file 1, 2, 3: EST assembly, 19,055 unigenes, ACE assembly file). The unigenes had a mean length of 786 ± 1.7 bp, with a mean GC content of 43.9 ± 0.03% (range 24.7 - 67.9%, Figure 2) and a mean ambiguity code ratio of 0.51 ± 0.01. Within the unigene set, 3,308 contained 4,219 uninterrupted tracts of (perfect) di-, tri-, tetra-, penta-, and hexa-nucleotide repeats, delivering a mean microsatellite density of one per 3.6 kb. Comparable microsatellite frequencies and densities have been discovered in the transcriptomes of other Compositae species [16-18]. Only perfect repeats were selected, as these appear to be the more prone to strand slippage and, consequently, tend to be more polymorphic than interrupted ones [19]. Sufficient flanking sequence (in terms of both length and read quality) was present in 1,974 of the unigenes, containing 2,311 perfect microsatellites. The resulting PCR primers designed for these loci are given in Additional File 4 (primer pairs designed).

Figure 1.

Figure 1

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The schema used for EST assembly, annotation, primer design and amplicon screening.

Figure 2.

Figure 2

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Output of the EST assembly. Distribution of unigene (A) length, and (B) GC-content.

Allelic diversity with the EST microsatellites

A subset of 300 microsatellites (ranging in length from 10 to 84 bp, and representative of a broad spectrum of the repeat types) was surveyed for their informativeness. The targeted amplicon length ranged from 98 to 456 bp and the set was selected to optimize the possibility of multiplexing on the capillary genotyping platform employed. The test germplasm panel consisted of twelve genotypes of globe artichoke, nine of cultivated cardoon, and seven of wild cardoon (Table 1). In all, 238 (79.3%) of the assays were successful; of these, 236 were informative among the taxa, while 215, 216 and 223 were polymorphic within, respectively, globe artichoke, cultivated cardoon and wild cardoon (Table 2 and Additional file 5: full statistics on 300 SSR-containing loci). A total of 1,546 alleles was generated from the 238 successful assays, giving a mean of 3.8 (range 1-15) alleles per locus. The largest range in amplicon length observed was 196 - 252 bp, observed for a TCAn microsatellite (CyEM-171). In 85% of the loci, the assay generated the predicted length of amplicon, in 12.2% the amplicon was longer than expected, and in 2.8% it was shorter. The allelic range (in terms of amplicon length) was greater for the wild cardoon (17.1 ± 1.0 bp) than for globe artichoke (13.6 ± 0.8 bp) or cultivated cardoon (13.7 ± 0.7 bp).

Table 1.

Genotypes set.

C. cardunculus taxa Genotypes Cluster1
scolymus Violet de Provence A1
AVM 7 A1
Blanco A1
Pasquaiolo A2
Pietralcina A2
Romanesco C3 A2
Green Globe A2
Sakiz B1
Spinoso di Palermo B1
Spinoso violetto di Liguria B2
Empolese B2
Violetto di Toscana B2
altilis Blanco de Peralta A1
Lleno de España A1
Rojo de Corella A2
Valencia A3
Gigante di Romagna B1
Bianco Avorio B2
Gobbo di Nizza B2
Bianco Pieno Migliorato C
Altilis 41 -
sylvestris Bronte Sicily
Roccella Sicily
Palazzolo Sicily
Sassari Sardinia
Oristano Sardinia
Nuoro Sardinia
Creta 4 -
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The 28 C. cardunculus accessions assayed for genotypic variation. Respectively 12 globe artichokes, 9 cultivated cardoons and 7 wild cardoons were analysed.1Globe artichoke, cultivated cardoon and wild cardoon clusters defined in [20,21,26], respectively.

Table 2.

CyEM loci statistics.

Locus Accession N°/unigene name Exp. Size Motif N° of repeats Allele size range na ne Ho PIC GxG GxC GxW
CyEM-001 GE602982 148 ATAC 5 141-150 3 2,018 0,679 0,504 + + +
CyEM-002 GE604144 150 TTTG 6 139-143 2 1,733 0,464 0,423 + + +
CyEM-003 GE604267 144 CAATGG 9 110-141 5 3,324 0,440 0,699 - + -
CyEM-004 GE604528 175 ATC 20 125-138 3 1,075 0,071 0,070 - - -
CyEM-005 GE605519 154 TC 9 137-170 9 2,446 0,571 0,591 + - +
CyEM-006 GE606838 152 ATC 13 135-162 8 3,269 0,556 0,694 - - +
CyEM-007 GE609770 165 TTC 17 121-165 8 3,735 0,577 0,732 + + +
CyEM-008 GE610358 149 CAT 19 106-156 9 3,347 0,654 0,701 + + +
CyEM-009 GE610418 143 ATAC 6 118-146 10 6,693 0,500 0,851 + + +
CyEM-010 GE612656 180 TGTA 7 152-194 12 7,259 0,643 0,862 + + +
CyEM-011 GE613193 209 TTGGTT 10 170-214 5 3,057 0,393 0,673 - + +
CyEM-012 GE578689 153 GAA 8 146-169 8 4,036 0,538 0,752 + + +
CyEM-013 GE588213 149 GAT 10 124-151 8 3,416 0,429 0,707 + + +
CyEM-014 GE595596 170 ATG 11 153-172 7 4,308 0,643 0,768 + + +
CyEM-015 CL2994Contig1 92 GTTT 5 84-97 4 2,592 0,750 0,614 + + +
CyEM-016 GE587666 127 TATG 5 117-129 4 2,922 0,481 0,658 + + +
CyEM-017 CL469Contig1 187 TTGGT 6 173-205 7 3,588 0,429 0,721 - - +
CyEM-018 CL6299Contig1 156 GGTCT 6 139-156 2 1,813 0,321 0,448 - - -
CyEM-019* CL2425Contig1 179 TGGTA 6 - \ \ \ \ + + +
CyEM-020 GE612053 107 TCATCT 6 67-104 7 3,724 0,321 0,732 + + +
CyEM-021 CL8Contig1 231 CGC 5 234-234 1 1,000 0,000 0,000 - - -
CyEM-022 CL167Contig1 222 ATG 5 224-224 1 1,000 0,000 0,000 - - -
CyEM-023 CL290Contig1 234 CT 5 214-233 4 1,675 0,429 0,403 - + -
CyEM-024 CL432Contig1 125 ATC 7 113-126 4 3,655 0,464 0,726 + + +
CyEM-025 CL489Contig1 229 CTA 6 229-239 3 1,878 0,321 0,467 + + +
CyEM-027 CL768Contig1 228 ACC 6 224-227 2 1,036 0,036 0,035 - - -
CyEM-028 CL1480Contig1 228 CGATTA 7 530-544 4 1,845 0,095 0,458 + - +
CyEM-029 CL2522Contig1 219 CTTC 7 202-223 5 3,315 0,607 0,698 + + +
CyEM-030 CL2739Contig1 240 TC 19 212-245 11 7,245 0,667 0,862 - + +
CyEM-031 CL2833Contig1 223 CT 8 117-226 7 3,682 0,333 0,728 + - +
CyEM-032 CL5674Contig1 229 CAT 8 214-236 5 3,294 0,500 0,696 + + +
CyEM-033 CL6305Contig1 227 CTT 12 205-231 8 3,213 0,571 0,689 + - +
CyEM-034 CL6392Contig1 212 CT 17 185-224 9 6,078 0,750 0,835 + + +
CyEM-035 CL136Contig1 231 GGTTA 5 212-239 6 3,435 0,739 0,709 + + +
CyEM-036 CL840Contig1 231 GAATT 5 222-237 4 2,379 0,500 0,580 + + +
CyEM-037 CL1651Contig1 220 CT 16 191-225 12 6,759 0,679 0,852 + + +
CyEM-038 CL3137Contig1 220 AAGTG 5 216-226 3 2,316 0,536 0,568 + + +
CyEM-042 CL4773Contig1 168 AG 14 148-176 12 6,438 0,577 0,845 + + +
CyEM-043 CL5064Contig1 292 ACA 10 272-297 7 4,021 0,500 0,751 + + +
CyEM-045 CL5134Contig1 301 CAATC 5 290-299 3 1,651 0,280 0,394 + + +
CyEM-046 CL5445Contig1 278 CTTTGC 5 273-278 2 1,415 0,071 0,293 - - +
CyEM-047 CL6123Contig1 286 CATCTT 5 274-303 5 3,226 0,571 0,690 + + +
CyEM-048 CL703Contig1 292 CAATCC 5 270-294 6 3,378 0,560 0,704 + + +
CyEM-049 CL1527Contig1 305 CAGAAG 6 284-307 5 3,246 0,393 0,692 + - -
CyEM-050 CL1584Contig1 311 TTGGT 5 269-315 8 1,617 0,240 0,382 - - +
CyEM-051 CL1735Contig1 162 TGGCAA 5 129-167 5 1,551 0,074 0,355 - - -
CyEM-052 CL1878Contig1 301 CT 15 277-303 11 6,788 0,964 0,853 + + +
CyEM-053 CL2037Contig1 327 CT 20 297-330 13 8,667 0,885 0,885 + + +
CyEM-054 CL4038Contig1 307 ATGTGG 6 268-305 11 9,191 0,600 0,891 + + +
CyEM-055 CL4185Contig1 297 CAACAG 7 271-293 5 3,995 0,630 0,750 + + +
CyEM-056 CL5289Contig1 301 GA 21 283-319 9 4,404 0,385 0,773 + + +
CyEM-057 CL6231Contig1 301 AT 6 300-323 6 2,620 0,308 0,618 + + +
CyEM-058 CL815Contig1 299 CA 7 291-298 2 1,642 0,000 0,391 - + +
CyEM-059 CL1157Contig1 289 GGT 9 280-297 7 2,116 0,429 0,527 + + +
CyEM-060 CL1449Contig1 306 GATTC 5 294-307 4 2,605 0,429 0,616 + + +
CyEM-063 GE590526 375 GATGG 5 432-457 8 3,673 0,481 0,728 + + +
CyEM-064 GE590983 122 CT 15 96-126 12 8,145 0,741 0,877 + + +
CyEM-066 GE591829 368 GAT 15 372-405 10 6,231 0,704 0,840 + + +
CyEM-069 GE594774 386 AGGA 5 370-392 6 3,556 0,519 0,719 - + +
CyEM-070 GE594818 272 TGCA 5 309-380 5 3,769 0,393 0,735 + + +
CyEM-071 GE595888 376 GTTTG 5 438-468 6 2,713 0,357 0,631 - - -
CyEM-072 GE595959 376 AAGCA 5 367-386 4 3,308 0,536 0,698 + + +
CyEM-073 GE596794 376 AGCC 6 457-465 4 2,088 0,286 0,521 + + -
CyEM-075 GE597515 390 TC 16 363-393 7 3,458 0,462 0,711 - + +
CyEM-076 GE597588 378 AACCA 14 436-449 6 3,627 0,556 0,724 + + +
CyEM-077 GE598177 378 CCAT 6 370-380 5 3,492 0,429 0,714 + + +
CyEM-079 GE601502 375 AATG 6 463-488 8 5,333 0,625 0,813 + + +
CyEM-080 GE602408 382 TTCACG 14 652-694 4 3,273 0,714 0,695 + + +
CyEM-083 CL5605Contig1 465 AG 13 448-475 7 3,689 0,643 0,729 + - -
CyEM-084 CL5717Contig1 128 AATCA 5 108-123 3 2,018 0,429 0,504 + + +
CyEM-086 GE577139 430 ATGTAA 6 410-460 6 3,447 0,389 0,710 + + +
CyEM-087 GE578205 450 CCAAC 5 443-457 5 1,488 0,125 0,328 - - -
CyEM-088 GE578232 451 GAGGAA 7 436-459 5 2,045 0,222 0,511 - - +
CyEM-090 GE580735 201 ATAC 6 190-218 5 1,615 0,222 0,381 - + -
CyEM-091 GE581152 452 GGTAT 5 657-669 4 2,493 0,464 0,599 + + +
CyEM-092 GE581504 106 TTGC 7 83-104 6 4,683 0,750 0,786 - - -
CyEM-093 GE581834 435 GA 18 410-453 12 7,362 0,857 0,864 + + +
CyEM-094 GE581842 450 TCA 14 417-454 5 2,068 0,333 0,516 + + +
CyEM-096 GE586326 451 CTCTAT 6 426-465 9 3,144 0,346 0,682 + - -
CyEM-097 GE587846 446 GT 12 437-449 5 3,415 0,556 0,707 + + +
CyEM-098 GE588210 262 AAGAG 5 620-650 4 3,068 0,357 0,674 - - -
CyEM-099 GE588482 448 AAGTG 5 536-547 3 2,594 0,593 0,615 + + +
CyEM-100 GE589916 434 AT 11 522-554 10 6,857 0,375 0,854 - - +
CyEM-102 GE590134 100 ACC 7 87-105 5 1,871 0,250 0,466 - + +
CyEM-103 GE592369 100 AGC 7 93-105 4 1,821 0,571 0,451 + - +
CyEM-104 GE595980 100 CAG 7 94-117 8 4,226 0,679 0,763 + + +
CyEM-105 GE588534 101 AAG 7 85-107 6 3,355 0,654 0,702 + + +
CyEM-106 GE588636 101 CAG 7 84-99 6 3,908 0,692 0,744 + + +
CyEM-107 GE591921 101 GAA 7 91-115 6 3,391 0,704 0,705 + + +
CyEM-108 GE590638 102 ACA 7 293-356 12 5,481 0,778 0,818 + + +
CyEM-109 GE586147 103 GGA 7 50-102 7 3,980 0,357 0,749 - - +
CyEM-110 GE586350 103 GTT 7 98-105 3 1,618 0,393 0,382 - + -
CyEM-111 GE587414 165 CGG 7 105-124 6 4,467 0,464 0,776 + + +
CyEM-112 GE593991 104 TCA 7 99-117 7 4,519 0,679 0,779 + + +
CyEM-113 GE584535 107 CAC 7 97-102 3 1,332 0,286 0,249 + + +
CyEM-115 GE582326 109 CTG 7 94-120 6 2,190 0,179 0,543 - - -
CyEM-117 GE596127 110 GAT 7 103-109 3 1,742 0,429 0,426 + + +
CyEM-118 GE597580 111 GCT 7 96-117 6 3,197 0,593 0,687 + + +
CyEM-120 GE597566 113 ATT 7 99-120 6 3,350 0,464 0,702 + + +
CyEM-121 GE590328 114 ACA 7 265-287 7 3,574 0,519 0,720 + + +
CyEM-122 GE583054 115 CTG 7 118-133 5 2,149 0,385 0,535 - + +
CyEM-123 GE592105 115 GTG 7 107-120 3 1,640 0,500 0,390 + + +
CyEM-124 GE597437 117 GT 12 112-126 8 4,598 0,571 0,783 + + +
CyEM-126 GE601086 119 AGC 8 111-158 7 2,834 0,731 0,647 - + +
CyEM-127 GE586328 110 CCA 8 99-116 7 3,960 0,857 0,747 + + +
CyEM-128 CL4629Contig1 120 AG 12 103-131 10 2,830 0,500 0,647 + + +
CyEM-129 GE594087 123 AGT 8 105-124 5 2,649 0,250 0,622 - - -
CyEM-130 GE610344 123 GAT 8 112-131 6 3,168 0,393 0,684 + + +
CyEM-131 GE580155 258 TC 12 260-296 9 4,989 0,538 0,800 - - -
CyEM-132 GE589900 126 GTG 8 112-127 5 2,851 0,286 0,649 - - +
CyEM-133 GE582083 128 CAT 8 121-155 10 3,806 0,464 0,737 - + +
CyEM-134 GE587520 128 TGA 8 123-129 3 2,402 0,407 0,584 + + +
CyEM-135 GE580164 129 TC 12 125-151 10 6,426 0,893 0,844 + + +
CyEM-136 GE599224 129 GA 12 118-145 10 5,502 0,679 0,818 + + +
CyEM-138 CL2919Contig1 130 TC 12 112-153 13 6,788 0,714 0,853 + + +
CyEM-139 CL5080Contig1 130 CAA 8 122-139 6 2,835 0,500 0,647 + - +
CyEM-141 GE588755 133 ATC 8 119-134 5 3,019 0,593 0,669 + + +
CyEM-142 GE581587 134 CAT 8 119-140 5 2,246 0,231 0,555 - - -
CyEM-143 GE577330 135 AG 12 125-145 8 3,540 0,357 0,717 - - +
CyEM-144 GE602230 136 TGA 8 128-158 6 3,853 0,679 0,740 + + +
CyEM-145 GE594958 139 GAT 8 133-181 8 3,807 0,519 0,737 + + +
CyEM-146 CL4926Contig1 164 CCA 6 226-241 7 2,259 0,143 0,557 + + -
CyEM-147 CL2920Contig1 165 CTC 7 140-164 7 2,864 0,286 0,651 + + +
CyEM-148 GE608083 167 GAT 6 161-179 5 2,420 0,250 0,587 - + +
CyEM-149 GE605451 168 CA 10 115-124 3 2,378 0,222 0,580 - - -
CyEM-150 GE588087 169 AG 10 138-171 9 4,681 0,286 0,786 + + +
CyEM-151 CL1781Contig1 169 CAC 7 150-169 6 3,136 0,429 0,681 + - +
CyEM-152 GE579243 170 GA 10 165-187 8 4,976 0,407 0,799 + + +
CyEM-153 GE603802 171 AAC 6 165-183 6 2,450 0,393 0,592 - + +
CyEM-154 GE581295 102 TC 10 94-106 7 1,653 0,423 0,395 + - +
CyEM-155 GE608129 173 CAG 6 165-174 4 2,877 0,571 0,652 + + +
CyEM-156 GE596512 174 TC 10 171-185 6 4,094 0,464 0,756 + + +
CyEM-157 CL5016Contig1 174 TGA 6 154-197 8 6,698 0,458 0,851 + + +
CyEM-158 GE599088 200 TCA 6 193-199 2 1,899 0,308 0,473 + + +
CyEM-159 GE612769 176 TC 10 167-187 9 3,260 0,571 0,693 + - +
CyEM-160 CL3274Contig1 176 CTT 7 163-185 6 1,589 0,286 0,371 - - -
CyEM-162 CL1575Contig1 258 TAG 7 253-261 5 2,914 0,500 0,657 + + +
CyEM-163 GE595958 179 AC 10 174-190 8 4,442 0,607 0,775 + + +
CyEM-164 GE583509 182 AC 10 380-401 5 3,406 0,440 0,706 + + +
CyEM-165 GE581808 188 TC 10 182-194 4 3,004 0,393 0,667 - - -
CyEM-166* GE605263 189 CT 10 - \ \ \ + + +
CyEM-167 CL1848Contig1 189 AG 11 173-212 13 7,095 0,643 0,859 + + +
CyEM-169 GE610017 190 CTT 7 176-191 4 1,958 0,393 0,489 + + +
CyEM-170 GE598301 192 TGG 7 186-198 4 1,292 0,179 0,226 - - +
CyEM-171 GE610233 194 TCA 7 196-252 8 3,136 0,429 0,681 - - +
CyEM-172 CL5891Contig1 195 CT 11 394-432 13 4,532 0,393 0,779 + + +
CyEM-173 GE607339 196 GAC 7 187-204 6 2,777 0,593 0,640 + + +
CyEM-174 GE609927 196 CCA 7 109-209 7 3,503 0,538 0,714 + + +
CyEM-175 CL6045Contig1 197 CT 11 190-211 6 5,058 0,643 0,802 + + +
CyEM-176 GE604158 198 ACA 7 191-203 4 3,588 0,393 0,721 + + +
CyEM-178 GE612882 200 CAG 7 188-202 5 2,883 0,385 0,653 + + +
CyEM-179 GE601991 201 ATG 7 194-203 3 1,338 0,000 0,253 - - -
CyEM-180 CL431Contig1 201 CT 11 194-202 5 2,920 0,536 0,658 - - +
CyEM-181 GE577085 108 GT 11 96-111 8 4,599 0,593 0,783 + + +
CyEM-182 GE607197 202 TCA 7 192-225 9 6,453 0,393 0,845 + - +
CyEM-183 GE597664 98 CAA 7 91-103 5 3,174 0,786 0,685 + + +
CyEM-185 GE610261 208 GAA 7 183-296 12 6,258 0,741 0,840 + + +
CyEM-186 GE607652 209 CTC 7 198-216 7 4,695 0,536 0,787 - - -
CyEM-187 GE602677 210 AGC 7 255-261 3 1,075 0,071 0,070 - - -
CyEM-188 GE613233 210 GCA 7 194-225 10 6,288 0,538 0,841 + + +
CyEM-189 GE605002 215 ATC 7 211-220 4 2,877 0,571 0,652 + + +
CyEM-190 CL1046Contig1 242 CAG 7 235-250 6 3,530 0,630 0,717 + + +
CyEM-193 CL1609Contig2 246 ATC 7 254-268 6 2,256 0,571 0,557 + + +
CyEM-195 CL6448Contig1 249 ACA 7 397-420 7 3,778 0,357 0,735 + + +
CyEM-196 GE580410 250 CAT 7 238-267 8 3,919 0,231 0,745 + + +
CyEM-197 CL3269Contig1 250 TGA 7 245-274 7 3,496 0,370 0,714 - + -
CyEM-199 CL3496Contig1 251 ATC 7 246-260 8 6,426 0,679 0,844 + + +
CyEM-200 CL4126Contig1 237 CAT 7 217-259 6 3,471 0,519 0,712 + + +
CyEM-201 CL6303Contig1 252 GAA 7 225-251 6 2,521 0,214 0,603 + + -
CyEM-202 CL2754Contig1 105 CAG 7 92-106 5 4,084 0,630 0,755 + + +
CyEM-203 CL2776Contig1 253 ACC 7 325-330 3 2,074 0,333 0,518 - - +
CyEM-204 CL5986Contig1 253 TCT 7 242-263 7 3,769 0,321 0,735 - + -
CyEM-205 CL3033Contig1 237 CAC 7 230-239 4 3,073 0,462 0,675 + + +
CyEM-207 CL4470Contig1 257 CAC 7 240-268 10 6,231 0,741 0,840 + + +
CyEM-208 CL5699Contig1 257 AAG 7 254-256 2 1,080 0,000 0,074 - - -
CyEM-209 CL1652Contig1 188 ATC 7 180-194 5 1,576 0,143 0,365 + + +
CyEM-210 GE611460 260 TGA 8 253-267 6 3,446 0,478 0,710 + + +
CyEM-211 CL6394Contig1 120 GCA 7 114-123 4 2,116 0,370 0,527 + + +
CyEM-212 CL2349Contig1 262 ACC 7 262-268 3 1,124 0,038 0,110 - - -
CyEM-213 GE603351 263 TC 13 235-283 12 7,801 0,821 0,872 + - +
CyEM-214 CL1016Contig1 263 CAT 9 253-293 12 5,045 0,731 0,802 + + +
CyEM-215 CL6059Contig1 263 CAC 9 258-274 4 2,379 0,214 0,580 - - +
CyEM-216 GE578065 264 CAT 9 229-265 9 3,707 0,321 0,730 - - +
CyEM-218 GE611429 265 TC 13 350-370 9 5,580 0,536 0,821 + + +
CyEM-219 GE611316 267 CAC 8 256-278 6 2,925 0,464 0,658 + + -
CyEM-220 CL4549Contig1 274 CAT 9 261-276 6 4,326 0,704 0,769 + + +
CyEM-221 GE611385 275 TG 12 254-282 7 5,042 0,679 0,802 + + +
CyEM-223 CL5961Contig1 276 CAC 9 263-275 5 2,296 0,500 0,564 + + +
CyEM-225 GE580984 278 GAA 9 274-286 5 3,081 0,571 0,675 + + +
CyEM-226 GE611110 280 AGA 8 517-529 4 3,817 0,370 0,738 + + +
CyEM-227 CL5817Contig1 280 AAG 9 241-281 6 3,142 0,667 0,682 + + -
CyEM-228 CL4460Contig1 283 GAT 9 348-357 4 1,566 0,321 0,362 + - +
CyEM-229 GE577281 285 CT 13 270-294 10 6,222 0,786 0,839 + + +
CyEM-230 CL1174Contig1 285 CCA 9 271-293 6 3,159 0,462 0,683 + + +
CyEM-231* CL548Contig1 286 AGA 9 - \ \ \ \ + + +
CyEM-232 CL4621Contig1 287 CAG 9 273-287 6 4,915 0,679 0,797 + + +
CyEM-233 GE583211 288 AC 13 444-471 11 3,672 0,679 0,728 + + +
CyEM-234* GE602543 310 TC 11 - \ \ \ \ + + +
CyEM-236 CL923Contig1 315 CTT 8 401-433 6 3,729 0,667 0,732 + + +
CyEM-237 GE589921 316 CT 11 310-331 10 6,202 0,885 0,839 + + +
CyEM-238 CL1788Contig1 321 TC 12 304-334 9 2,904 0,296 0,656 - - -
CyEM-240 CL2307Contig1 324 AGC 8 303-333 8 5,063 0,630 0,802 + + +
CyEM-241 CL2526Contig1 325 CT 12 311-325 3 2,263 0,179 0,558 - - -
CyEM-243 CL5805Contig1 325 CT 12 309-338 10 4,472 0,667 0,776 + + +
CyEM-244 GE609380 326 TC 11 318-337 9 5,080 0,519 0,803 + + +
CyEM-246 GE604318 327 TG 11 320-354 8 3,117 0,520 0,679 + + +
CyEM-247 CL2951Contig1 327 CTG 8 316-339 7 3,815 0,500 0,738 + + +
CyEM-248 GE581850 330 AG 11 325-331 4 2,379 0,714 0,580 + + +
CyEM-250 CL3943Contig1 331 AG 10 321-334 5 3,516 0,321 0,716 + + +
CyEM-253 CL3338Contig1 142 CT 15 125-159 9 4,653 0,571 0,785 + - +
CyEM-254 CL3757Contig1 125 TC 12 108-141 15 7,682 0,654 0,870 + + +
CyEM-256 CL6387Contig1 338 TCA 8 323-350 7 4,148 0,464 0,759 - + -
CyEM-259 CL5381Contig1 197 CA 15 176-220 12 6,826 0,696 0,853 + + +
CyEM-260 CL2855Contig1 151 CCA 10 131-151 7 4,780 0,630 0,791 + + +
CyEM-261 GE613227 185 GGT 9 168-202 9 5,765 0,643 0,827 + + +
CyEM-264 CL3958Contig1 350 GAT 10 611-625 5 3,636 0,593 0,725 + + +
CyEM-266 GE598991 352 AG 14 343-359 8 5,985 0,571 0,833 + + +
CyEM-272 GE599540 356 TAC 9 348-357 4 3,333 0,600 0,700 + + +
CyEM-273* GE599578 356 AAC 9 - \ \ \ + + +
CyEM-277 CL2561Contig1 138 TC 14 116-157 13 5,911 0,783 0,831 + + +
CyEM-278 GE579023 391 CAT 10 376-399 5 3,057 0,259 0,673 + - +
CyEM-279 CL4781Contig1 106 TC 17 90-105 7 5,507 0,720 0,818 + + +
CyEM-280 GE591354 394 ATG 11 375-432 12 7,010 0,852 0,857 + + +
CyEM-281 GE610121 395 TC 15 372-412 9 5,302 0,778 0,811 + - +
CyEM-282 GE604802 399 GAT 11 380-401 5 4,085 0,423 0,755 + + +
CyEM-284 CL2318Contig1 111 AG 17 88-114 9 5,209 0,679 0,808 + + +
CyEM-285 CL4633Contig1 109 CT 16 91-129 13 6,284 0,667 0,841 + - +
CyEM-286 GE602088 405 TC 17 386-410 9 5,074 0,679 0,803 + + +
CyEM-288 GE595961 410 ATG 10 388-425 7 3,072 0,542 0,674 + + +
CyEM-289 GE580749 411 CTT 12 461-501 4 1,247 0,214 0,198 + + +
CyEM-290 GE583378 160 TC 15 151-174 9 5,383 0,500 0,814 + - -
CyEM-291 CL1901Contig1 147 TG 18 120-164 9 3,540 0,607 0,717 + + +
CyEM-293 CL3287Contig1 413 TC 20 384-410 9 4,717 0,440 0,788 + + +
CyEM-294 GE593962 231 GAT 11 216-232 7 4,556 0,407 0,781 + + +
CyEM-295 CL2047Contig1 414 AG 16 480-517 9 3,282 0,500 0,695 + + +
CyEM-296 GE602341 415 GAT 11 397-428 8 5,629 0,593 0,822 + + +
CyEM-299 CL6551Contig1 424 CAC 12 469-520 9 2,254 0,333 0,556 + - +
CyEM-300 GE610516 425 TC 15 405-432 7 3,885 0,308 0,743 + + +
Average 6,6 3,677 0,484 0,660
s.e. 0,2 0,107 0,013 0,012
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Main information reported: locus name, unigene (for contigs-derived loci)/Accession number (for singletons-derived loci), expected size, perfect microsatellite motif, number of repeats, observed alleles range, number of observed alleles (no), effective alleles (ne), observed heterozygosity (Ho), polymorphic information content (PIC) and mapping utility for the three progenies ["Romanesco C3" × "Spinoso di Palermo" (GxG), "Romanesco C3" × "altilis41" (GxC) and "Romanesco C3" × "Creta-4" (GxW)]. Additional information are available in the electronic supplementary material. *Data showing a multi-locus amplification were excluded from the analysis.

Allelic diversity was, as expected given the breeding history of these taxa [2,20,21], greater in the wild than in the two cultivated forms (Figure 3A). The frequency of taxon-specific alleles was two fold more in the wild cardoon, and the polymorphic information content (PIC) was higher in the wild cardoon (0.576 ± 0.015) than in either the globe artichoke (0.484 ± 0.013) or the cultivated cardoon (0.466 ± 0.015). The observed heterozygosity level (Ho) was significantly less in the cultivated cardoon than in globe artichoke, presumably because the globe artichoke is primarily a vegetatively propagated plant, and thus able to maintain a high level of heterozygosity over time [20,21]; whereas cultivated cardoon is seed propagated and has been subjected to purifying selection aimed at increasing genetic uniformity for stabilizing its production. We previously developed three mapping population for the development of C. cardunculus genetic maps by crossing one globe artichoke non spiny genotype (common female parent) with a spiny genotype of globe artichoke or cultivated cardoon or wild cardoon. When the parents of the three mapping populations were tested with the set of microsatellites, 214 were informative in at least one combination, while 153 across all the three combinations, thus supplying landmarks for comparative mapping of phenotypic and quantitative trait loci (QTLs). As expected, the most polymorphic combination (198 microsatellites) was the one involving the cross between the most genetically divergent taxa: globe artichoke and wild cardoon (Figure 3B).

Figure 3.

Figure 3

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Allelic diversity revealed by the set of EST microsatellite markers. (A) Allelic patterns and the level of heterozygosity within each taxon. Observed (no), effective (ne) and the number of taxon-specific alleles per marker are represented by bars. PIC and Ho are indicated by points. (B) Markers showing segregation within the three mapping populations. GxG: within scolymus, GxC: scolymus × altilis, GxW: scolymus × sylvestris.

Diversity analysis

The informativeness of the newly developed EST microsatellites was comparable with that described for microsatellite markers in globe artichoke [13,22], sunflower [23,24] and lettuce [25]. A set of five EST microsatellites was sufficient to discriminate between each of the 28 members of the germplasm panel (e.g. CyEM-10, -37, -54, -105, -254). The inferred genetic relationships were in good concordance with those derived from AFLP profiling [20,21,26,27]. Thus, the globe artichoke accessions clustered with one another (Figure 4A, cluster A), but two sub-clusters, corresponding to the contrasting capitulum types (i.e. non spiny vs. violet, spiny types), could be recognised. The clade most closely related to the globe artichokes contained the cultivated cardoons (Figure 4A, cluster B), and among these, the most well differentiated accession was 'Bianco Pieno Migliorato', as previously observed [21]. The Spanish cultivated cardoon accessions were genetically very similar to one another. The wild cardoon accessions formed a discrete, but rather loose group (Figure 4A, cluster C). A principal co-ordinate analysis further illustrated the genetic relationships between members of the germplasm panel (Figure 4B). Axes 1 and 2 accounted for ~ 73% of the genetic variation, the former contributing ~ 47%, and the latter ~ 26%. Axis 1 distinguished the globe artichokes from the cultivated and wild cardoons, while Axis 2 separated the two cardoon taxa. As expected, F1 progenies mapped to intermediate positions with respect to those of their parents (Figure 4B).

Figure 4.

Figure 4

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Diversity analysis. (A) A UPGMA dendogram based on 1,546 EST microsatellite alleles. The parentheses indicate the globe artichoke, cultivated cardoon and wild cardoon clusters defined by [20,21,26]. (B) Principal co-ordinate analysis based on the genetic distance matrix of 46 individuals, including the parents (red circles) and progeny of the three mapping populations GxG: within scolymus, GxC: scolymus × altilis, GxW: scolymus × sylvestris.

Distribution of microsatellite

Of the 4,219 microsatellites, trinucleotide motifs accounted for 49%, dinucleotides for 33%, hexanucleotides for 13%, tetranucleotides for 3% and pentanucleotides for 2% (Figure 5). Only ESTs (2,498 of the 3,308) having a non-ambiguous ortholog in Arabidopsis thaliana were taken forward for the purpose of annotation. The position of the microsatellite tract (5'-UTR or 3'-UTR or CDS) was derived from the BlastX result in conjunction with the ORF (Open Reading Frame)-Predictor algorithm [28]. About 33% of the microsatellites were present in the 5'-UTRs, ~ 20% in the 3'-UTRs and ~ 47% in the CDSs (Figure 5), similar to the relative frequencies in both the A. thaliana and rice genomes [15]. Most of the CDS microsatellites consisted of trinucleotides, while dinucleotides were the most abundant in the 5'-UTRs, and di- and trinucleotides were equally represented in the 3'-UTRs. Tetra- and pentanucleotide motifs were more frequent in the 3'-UTRs than in either the CDSs or the 5'-UTRs (data not shown). Trinucleotide (and hexanucleotide) motifs are expected to predominate in the population of CDS microsatellites, as variation in their repeat number is not associated with a frame shift event [29]. The most abundant dinucleotide repeat was AG/CT, followed by AC/GT, although AT/TA predominated in the 3'-UTRs. Among the trinucleotides, the most frequent was AAG/CTT, followed by ATC/GAT and CAC/GTG (Figure 6). This distribution is consistent with the situation in A. thaliana and Brassica spp. orthologs, in which a preference for AG/CT and AAG/CTT motifs has been identified in the 5'-UTRs, thought to be associated with the cis-acting regulation of transcription [30]. In the globe artichoke 5' UTRs, dinucleotide motifs were over-represented, with AG/CT being the most abundant (Figure 6), similar to the situation in the 5'-UTRs of many plant (both mono- and dicotyledonous species) genes [31,32], which has been reported to play a role in post-transcriptional gene regulation at the RNA level [33,34]. Dinucleotide motifs were also frequent in the 3'-UTRs, possibly because AT-rich elements are able to act as cis mediators of mRNA turnover [33]. Overall, present data confirm that homopurine/homopyrimidine repeats contribute markedly in 5'-UTR and CDS, as previously reported by Morgante et al [15].

Figure 5.

Figure 5

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Position and motifs of EST microsatellites. (A) Distribution within specific regions of the unigenes. (B) Frequencies of repeat motifs within the unigene set.

Figure 6.

Figure 6

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Distribution of microsatellite classes. Di- and trinucleotide classes belonging to each unigene region (5'-UTR, coding sequence, 3-UTR).

The function of genes containing microsatellites

Microsatellites within coding sequences can have a major effect on gene activity, since the expansion/contraction of triplets within the coding sequence alters the gene product, thereby sometimes causing a significant phenotypic change. In humans, the effects on phenotype due to the presence of SSRs in coding regions of genes playing key roles in neuronal disorders and cancer have been extensively studied [35]. Among the microsatellites in the globe artichoke transcriptome, the six most frequent amino acid stretches present in the CDS were poly-serine (94 unigenes), poly-aspartic acid (75 unigenes), poly-glutamic acid (57 unigenes), poly-lysine (46 unigenes), poly-glycine (45 unigenes) and poly-threonine (35 unigenes). It has been reported that particular amino acid repeats tend to be associated with specific classes of proteins [36]. Acidic and polar amino acid repeats have generally been associated with transcription factors and protein kinases, whereas serine repeats are common within membrane transporter proteins [37]. In the globe artichoke, poly-serine and poly-glycine stretches are particularly frequent in the CDS. Poly-serine linkers are common in eukaryotic genomes, and are thought to provide a flexible interdomain. They are frequently associated with modular proteins, and are involved in complex carbohydrate degradation [38] and the binding of proteins with extracellular matrix components, such as the laminin binding protein. Poly-glycine (also poly-asparagine and poly-proline) microsatellites may provide a domain for DNA binding or protein-protein interactions, and has been found to be necessary for chloroplast envelope targeting. Poly-glutamic and poly-aspartic acid tracts feature in many NLS (nuclear localisation signal) proteins [39], and it has been suggested that both basic karyophilic and acidic clusters can enhance their selective binding to transport machinery components [40]. Poly-glutamic acid stretches have also been implicated in transcription activation/de-activation [41-45], and microsatellite allelic variants of these genes have been identified as the genetic basis of a number of human diseases [46].

To support the occurrence of certain repeated motifs in the assembled unigenes we have exploited sequence alignment and gene ontology in order to annotate their functions and assess whether their motif type and position are preferentially associated with a particular gene ontology term.

In preparation, the set of globe artichoke unigenes was filtered to include only those with a BlastX E-value of < 1e-29 when matched with the A. thaliana reference protein set. In all, 12,783 queries satisfied this criterion (Additional file 6: 12,783 globe artichoke unigenes annotation). The A. thaliana gene accession numbers were used to categorize the unigenes using TAIR gene ontology (data not shown). The GoStat2 web interface was then used to identify gene ontology categories which were over-represented. By comparing either the set of microsatellite-containing unigenes, or subsets of it (e.g.: genes including di- or trinucleotide motifs in their CDS or UTRs) with the complete set of annotated unigenes, it was possible to identify over-representation in gene ontology (GO) categories (Table 3). Microsatellites appeared to be over-represented in loci involved in certain biological processes and functions, while no significant association was found with GO cell components (data not shown).

Table 3.

Functional enrichments.

CDS 5'-UTR
ID Name unigenes All SSRs All SSRs TRI DI AAG_CTT ATC_GAT AG_CT All SSRs DI TRI AG_CT
Biological process GO:0006139 nucleic acid metabolic process 7,33 9,58 10,90 10,88 - - - - - - - -
GO:0019219 regulation nucleic acid metab. process 3,30 5,85 7,56 8,21 - - 9,09 - - - - 7,86
GO:0006350 transcription 3,56 5,85 7,70 8,21 - - 9,09 - - - - 7,42
GO:0006351 transcription, DNA-dependent 1,99 - 4,65 4,96 - - - - - - - -
GO:0006355 regulation of transcript., DNA-depend. 1,92 - 4,51 4,96 - - - - - - - -
GO:0010467 gene expression 8,12 - 11,19 11,64 - - - - - - - -
GO:0010468 regulation of gene expression 3,50 6,07 7,70 8,40 - - 9,09 - - - - 7,42
GO:0016070 RNA metabolic process 3,51 - 5,96 6,11 - - - - - - - -
GO:0019222 regulation of metabolic process 3,62 6,36 7,85 8,59 - - 9,09 - - 6,83 - 7,86
GO:0031323 regulation of cellular metabolic process 3,45 6,14 7,70 8,40 - - 9,09 - - - - 7,86
GO:0032774 RNA biosynthetic process 1,99 - 4,65 4,96 - - - - - - - -
GO:0045449 regulation of transcription 3,29 5,77 7,56 8,21 - - 9,09 - - - - 7,42
GO:0050789 regulation of biological process 4,17 6,94 8,58 9,16 - - - - - - - -
GO:0050794 regulation of cellular process 3,82 6,65 8,43 8,97 - - 9,09 - - - - 7,86
GO:0065007 biological regulation 5,07 7,68 9,16 9,73 - - 10,74 - - - - -
GO:0006281 DNA repair 0,48 - - - - - - 3,61 - - - -
GO:0002376 immune system process 0,32 - - - 2,73 - - - - - - -
GO:0050896 response to stimulus 5,60 - - - 11,82 - - 13,25 - - - -
GO:0006950 response to stress 2,71 - - - - - - 8,43 - - - -
GO:0006974 response to DNA damage stimulus 0,48 - - - - - - 3,61 - - - -
GO:0006855 multidrug transport 0,14 - - - - - - 2,41 - - - -
GO:0009266 response to temperature stimulus 0,63 1,46 - - - - - - 1,90 - - -
GO:0009409 response to cold 0,38 - - - - - - - 1,14 - - -
GO:0009628 response to abiotic stimulus 1,46 - - - - - - - 2,91 - - -
GO:0009733 response to auxin stimulus 0,26 - - - - - - - - - 2,10 -
GO:0009737 response to abscisic acid stimulus 0,27 - - - - - - - - 1,71 - 1,75
GO:0009738 abscisic acid mediated signaling 0,11 - - - - - - - - 1,02 - 1,31
GO:0065004 protein-DNA complex assembly 0,19 - - - - 2,17 - - - - - -
GO:0046483 heterocycle metabolic process 0,75 - - - - 3,62 - - - - - -
Molecular function GO:0003676 nucleic acid binding 8,89 13,67 17,01 - - 18,12 - 18,07 - - - -
GO:0003677 DNA binding 5,05 8,92 11,34 - - - 13,22 - - - - 10,04
GO:0003700 transcription factor activity 3,34 6,51 8,14 - - - 9,09 - - 6,48 - 8,30
GO:0008270 zinc ion binding 3,18 4,39 - - - - - - - - - -
GO:0043169 cation binding 4,89 6,65 - - - - - - - - - -
GO:0016667 oxidoreduct. activ. on SH group of donors 0,13 - - - - - - 2,41 - - - -
GO:0015297 antiporter activity 0,45 - - - - - - 3,61 - - - -
GO:0051219 phosphoprotein binding 0,11 - - - - - - - 0,63 1,71 - 1,75
GO:0004721 phosphoprotein phosphatase activity 0,93 - - - - - - - 2,02 - - -
GO:0005544 Ca-dependent phospholipid binding 0,03 - - - - - - - - - 1,40 -
GO:0015071 protein serine/threonine phosphatase activity 0,45 - - - - - - - - - - 2,18
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GO terms statistically enriched (showed in percentage) for specific SSRs subsets. Fisher's exact test was performed between each SSRs subset versus the whole unigenes categorization; only significant over-represented subset are reported (p < 0,01). Analysis is displayed referring to "biological process" and "molecular function" classifications; "cellular component" is not reported due to the absence of particular enriched subsets.

Most of the unigenes containing trinucleotide motifs in their CDS were associated with nucleic acid metabolic processes (GO:0006139), transcription (GO:0006350) and the regulation of transcription (GO:0006355), consistent with the encoding by the GAT trinucleotide of aspartic acid, since stretches of this residue are characteristic of 'karyophilic' acidic clusters in NLS (nuclear localization signal) proteins. Similarly, the AAG/TTC motif, which occurred frequently in the CDS, encodes poly-glutamate, which is thought to be involved in both protein-DNA complex assembly (GO:0065004) and heterocyclic metabolic processes (GO:0046483). Unigenes carrying dinucleotide motifs in their CDS were found to be specifically associated with the response to stimulus (GO:0050896). The AG/CT repeats in the CDSs were over-represented among genes responding to stress (GO:0006950), involved in DNA repair (GO:0006281) and in nucleic acid binding (GO:0003676). This is consistent with the presence of domains involved in protein-RNA/protein-protein sticky interactions.

The commonest microsatellite motifs occurring in 5'-UTR of unigenes were dinucleotide repeats (mostly AG/CT). These unigenes were associated with nucleic acid metabolism (GO:0006139), the regulation of gene expression (GO:0010468), transcription (GO:0006350) and the regulation of transcription (GO:0006355). AG/CT repeats were also over-represented in genes involved in the response to ABA (GO:0009737 and GO:0009738). Moreover, trans-acting elements (GO:0003700: "transcription factor activity"), which show an over-representation of trinucleotidic (ATC/GAT) in their CDSs, were also frequently enriched in their 5'UTRs by AG/CT motifs, suggesting a cascade of signal transmission. Trinucleotide motifs were not common in the 5'-UTRs, except in genes involved in the response to auxin stimulus (GO:0009733).

Conclusion

We have demonstrated here the utility of a set of de novo globe artichoke EST-based microsatellite markers for the definition of genetic diversity, phylogeny and genetic mapping. Since EST microsatellites lie within expressed sequences, they have the potential to represent perfect markers for genes underlying phenotypic variation. Most of these assays are fully transferable to other C. cardunculus taxa, providing anchor points for the integration of taxon-specific genetic maps. The functional annotation of these EST sequences increases their utility as a source of gene-based markers for the study of synteny and other applications.

Methods

EST microsatellites discovery and primer design

A collection of 36,321 EST, generated from the 'Green Globe' variety of C. cardunculus var. scolymus, as part of the output of the Compositae Genome Project http://compgenomics.ucdavis.edu, was downloaded from the NCBI database http://www.ncbi.nlm.nih.gov. To generate a set of unique assemblies, the sequences were first trimmed to remove any remaining vector fragments and polyA tails, using the perl script SeqCleaner, and assembled adopting a second perl script, TGICL, employing the following parameters: p = 95 (identity percentage), l = 40 (minimum overlap length), v = 10 (maximum length of unmatched overhangs); the maximum mismatch overhang was set to 10 bp, since the sequences had already been purged of vector stretches and polyA tails. The two scripts are available at http://compbio.dfci.harvard.edu/tgi/software. The unigene set was then searched for perfect microsatellite sequences using a modified SSRIT perl script [47], with the minimum number of dinucleotides set as five, of tri-, tetra- and penta-nucleotides set as four, and of hexanucleotides as three. A sample of 300 non redundant microsatellite-containing sequences, selected to include the longer microsatellite motifs, was taken forward for PCR screening. Primer design was carried out using BatchPrimer3 http://probes.pw.usda.gov/batchprimer with an optimal GC content of 50%, a maximum melting temperature difference of 3°C, variable amplicon size (to allow multiplexing), and all other parameters set to default values. The de novo microsatellite markers were prefixed with 'CyEM' (Cynara Expressed Microsatellite) and numbered sequentially.

Plant materials and genomic DNA isolation

DNA was extracted from young leaves following a modified CTAB method [48]. The primers were used to amplify genomic DNA template extracted from a germplasm panel consisting of twelve globe artichoke genotypes, representative of crops grown in the Mediterranean Basin [20]; nine cultivated cardoon genotypes, representative of both the Spanish and Italian gene pools [21]; and seven wild cardoon genotypes sampled from both Sicily and Sardinia [26]. Full genotypes details are reported in Table 1. The set also included DNA of the four parents of three established mapping populations, i.e. two globe artichoke accessions ['Romanesco C3' (C3) and 'Spinoso di Palermo' (SP)], one cultivated cardoon ('Altilis 41') and one wild cardoon ('Creta 4'); furthermore six F1 individuals from each of the segregating populations (C3 × SP, C3 × Altilis 41 and C3 × Creta 4) were included in the analyses.

Genotyping and diversity analysis

Primer pairs CyEM-001 to CyEM-300 (Additional file 4: primer pairs designed) were tested for their informativeness on the germplasm panel. Amplification was carried out in 10 μl reactions containing 7 ng template DNA, 1× PCR Buffer (Qiagen Inc., Venlo, Netherlands), 1.0 mM MgCl2, 0.5 U Taq DNA polymerase (Qiagen), 40 nM 5'-labelled (FAM, HEX or TAMRA) forward primer, 40 nM unlabelled reverse primer and 0.2 mM dNTPs. A touchdown cycling regime was applied, consisting of 1 cycle at 94°C for 150 sec, 9 cycles at 94°C for 30 sec, 63°C for 30 sec (-0,7°C/cycle) and 72°C for 60 sec, then 30 cycles at 94°C for 30 sec, 57°C for 30 sec and 72°C for 60 sec, followed by a final extension at 72°C for 5 min.

Weakly amplified reactions were re-run using 1.5 mM MgCl2 and applying a final annealing temperature of 55°C. Amplicons were separated on an ABI3730 capillary DNA sequencer (Applied Biosystem Inc., Foster City, CA, USA). Internal ROX-labelled GS500 size standards were included in each capillary. Fragment data were analysed using GeneMapper v3.5 software (Applied Biosystems). The genotypic data were analysed using the GenAlex Excel package [49]. Genetic diversity was calculated separately for the globe artichoke, cultivated cardoon and wild cardoon genotypes on the basis of (1) the mean number of alleles observed per locus (no), (2) the effective number of alleles per locus (ne) as predicted by 1/Σpi2 where pi is the frequency of the ith allele at the locus, (3) the mean observed heterozygosity (Ho), and (4) the polymorphic information content (PIC), estimated following [49]. A co-phenetic distance matrix for co-dominant markers was generated as described by Smouse and Peakall [50] and used to construct a UPGMA-based dendrogram [51] by means of NTSYS software package v2.10 [52]. Principal co-ordinate analysis was based on the distance matrix, with data standardization provided by the GenAlex package.

Annotation of the unigene set

The unigene set was aligned by a BlastX [53] search against the A. thaliana reference proteins database (NCBI), applying an E-value threshold of e-29. The location within the gene sequence of the microsatellite (5'-UTR, CDS or 3'-UTR) was inferred from this alignment, while ORF-Predictor [28] was used to predict the position of the stop codon. The frequencies of peptide repeat tracts within the CDS were used to identify any over-representation of particular triplets. For this purpose, the unigenes were divided into ten subgroups on the basis of the identity of the trinucleotide motif present in the CDS. Each subgroup was then subjected to an analysis based on the ORF-Predictor algorithm, considering only the positive reading frames (+1, +2, +3), since the sequenced transcripts were originally directionally cloned. All the unigenes were assigned a function based on the GeneOntology tool http://www.arabidopsis.org, using the A. thaliana orthologs (from BlastX output) as input (using AGI codes from TAIR). Enrichment within the GO hierarchical levels by mean of different subset of unigenes was estimated using the GoStat2 interface http://gostat.wehi.edu.au/cgi-bin/goStat2.pl based on Fisher's exact test [54], adopting a threshold p-value of 0.01 and considering terms starting from the 3rd hierarchical level of the DAG (directed acyclic graph; Table 3).

Authors' contributions

SL and SK planned and supervised the work. DS were responsible for the in silico analysis of the EST sequence data, primer design and amplification; AA and EP selected the constitution of the C. cardunculus gerplasm panel; DS, AA, EP and CT analysed the data. All the authors contributed to the final version of the manuscript.

Supplementary Material

Additional file 1

EST assembly. The data provided represent the list of 6,621 assembled globe artichoke contigs derived from 23,871 ESTs.

Click here for file (1MB, XLS)
Additional file 2

19,055 unigenes. The data provided represent the fasta sequences of the assembled unigenes (contigs and singletons).

Click here for file (15.2MB, TXT)
Additional file 3

ACE assembly file (EagleView available at http://bioinformatics.bc.edu/marthlab/EagleView. Contig representation file parsed from TGICL output file by a customised PERL script.

Click here for file (12MB, ZIP)
Additional file 4

primer pairs designed. The data provided represent the list of the 2,311 designed primer pairs and loci/SSRs description.

Click here for file (497KB, XLS)
Additional file 5

full statistics on 300 SSR-containing loci. The data provided represent the list of 300 selected SSR-containing loci, their allele statistics, polymorphism information, repetitive motif position and gene annotation.

Click here for file (642.5KB, XLS)
Additional file 6

12,783 globe artichoke unigenes annotation. The data provided represent the list of 12,783 globe artichoke unigenes annotation using BlastX (e-value threshold < 1e-29) on Arabidopsis reference protein database (June, 2008).

Click here for file (3.3MB, XLS)

Acknowledgments

Acknowledgements

Globe artichoke ESTs were produced by the Compositae Genome Project http://compgenomics.ucdavis.edu/. This research was supported by grants from: (i) the National Science Foundation Plant Genome Research Program (No. 0421630), (ii) the Georgia Research Alliance, (iii) the University of Georgia Research Foundation, and (iv) by MIPAAF (Ministero delle Politiche Agricole, Alimentari e Forestali - Italy) through the CAR-VARVI ("Valorizzazione di germoplasma di carciofo attraverso la costituzione varietale ed il risanamento da virus") project.

We are grateful to Prof. Giovanni Mauromicale and Dr. Rosario Mauro (D.A.C.P.A. Agronomical Sciences, University of Catania) for providing the C. cardunculus parental accessions and the F1 individuals.

Contributor Information

Davide Scaglione, Email: davide.scaglione@unito.it.

Alberto Acquadro, Email: alberto.acquadro@unito.it.

Ezio Portis, Email: ezio.portis@unito.it.

Christopher A Taylor, Email: taylor75@uga.edu.

Sergio Lanteri, Email: sergio.lanteri@unito.it.

Steven J Knapp, Email: sjknapp@uga.edu.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1

EST assembly. The data provided represent the list of 6,621 assembled globe artichoke contigs derived from 23,871 ESTs.

Click here for file (1MB, XLS)
Additional file 2

19,055 unigenes. The data provided represent the fasta sequences of the assembled unigenes (contigs and singletons).

Click here for file (15.2MB, TXT)
Additional file 3

ACE assembly file (EagleView available at http://bioinformatics.bc.edu/marthlab/EagleView. Contig representation file parsed from TGICL output file by a customised PERL script.

Click here for file (12MB, ZIP)
Additional file 4

primer pairs designed. The data provided represent the list of the 2,311 designed primer pairs and loci/SSRs description.

Click here for file (497KB, XLS)
Additional file 5

full statistics on 300 SSR-containing loci. The data provided represent the list of 300 selected SSR-containing loci, their allele statistics, polymorphism information, repetitive motif position and gene annotation.

Click here for file (642.5KB, XLS)
Additional file 6

12,783 globe artichoke unigenes annotation. The data provided represent the list of 12,783 globe artichoke unigenes annotation using BlastX (e-value threshold < 1e-29) on Arabidopsis reference protein database (June, 2008).

Click here for file (3.3MB, XLS)

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