Genome size The flow cytometry (FCM) estimate of the Matina 1-6 genome size is 445 Mbp; this value approximates the mean determined for 28 different T. cacao genotypes (see Additional file 1, Table S1), including representatives of all 10 structural diversity groups defined by Motamayor et al. [2]. When analyzed by structural group, the genome sizes fall into three statistically distinct sets (see Additional file 1, Table S2). Genotypes within the Nacional group have the largest genomes, whereas most of the other genotypes have genomes similar in size to that of Matina 1-6, a member of the Amelonado group. Cultivars within the Criollo group, including B97-61/B2 (409 Mbp) whose genome was recently published [11], have some of the smallest genomes within the species (see Additional file 1, Table S2).

Assignment of chromosomes to pseudomolecules using fluorescence in situhybridization 16 17 18 11 19 2 20 22 2 1 1 19 1 1 We performed fluorescence in situ hybridization (FISH)-based karyotyping of mitotic T. cacao chromosomes, using two types of probes: genomic repeats (centromeric repeats) for differential chromosome 'painting', and bacterial artificial chromosomes (BACs) for chromosome identification. Centromeric repeat-based FISH has been used to identify chromosomes in soybean [] and maize []. We identified sequences homologous to a previously identified candidate centromeric repeat [] by searching unassembled sequence reads acquired during generation of the Matina 1-6 physical map []. A ClustalW alignment (not shown) of the 236 sequences with the highest percentage of identity indicated a length for the Matina 1-6 Cent-Tc repeat of 171 bp (see Additional file, Figure S1 for consensus), comparable with the centromere monomer length in other plant species []. We designed specific oligonucleotide probes that target highly conserved or less well conserved centromeric repeat T. cacao (Cent-Tc) regions (see Additional file, Figure S1). Probe OLI-07 was present in the majority (74%) of the aligned Cent-Tc repeats, whereas probe OLI-13 was present in 12.5%. When used together in FISH experiments (see Materials and methods section for details) the two probes differentiated the mitotic chromosomes into several distinct color and intensity subgroups (Figure). To identify individual chromosomes, BAC probes (Materials and methods; also see Additional file, Table S3) derived from low-repeat content regions of each of the 10 pseudomolecules defined by Matina 1-6 physical mapping [] were individually mapped to Cent-Tc-labeled chromosomes (Figureand data not shown). We then developed a six-component (two Cent-Tc probes plus four BAC probes) FISH 'cocktail' that simultaneously identified every chromosome in a single mitotic chromosome spread (Figure). Because the Matina 1-6 physical map is anchored by high-density molecular markers that relate the map to the sequenced genome, pseudomolecule-derived BAC probes permit the association of molecular-linkage groups and the pseudomolecules with the chromosomes themselves, thereby enabling chromosome numbering based on pseudomolecule numbering, and generating a single, unified cytogenetic map for Matina 1-6.

Sequencing and assembly of the Matina 1-6 genome In total, 32,460,307 sequence reads (a combination of Sanger and Roche 454 pyrosequencing; see Additional file 1, Table S4), were assembled using a modified version of Arachne (version 20071016 [23]). The resulting assembly was integrated with multiple high-quality genetic maps (see Additional file 1, Table S5) to produce a chromosome-scale assembly. Sanger sequences were obtained from fosmids and from BAC ends selected using the minimum tiling path of a previously reported physical map [19] (see Materials and methods). The initial assembly generated 1,672 scaffold sequences with a scaffold N50 of 7.4 Mbp, and 91 scaffolds longer than 100 kbp (see Additional file 1, Table S6), giving a total scaffold length of 348.7 Mbp. To generate version 1.1 of the Matina 1-6 genome assembly, we removed contaminating sequences and scaffolds containing exclusively any of the following: unanchored repetitive sequences, unanchored ribosomal (r)DNA sequences, mitochondrial DNA, chloroplast DNA, and sequences less than 1 kbp in length. Next, we integrated the maps and constructed chromosome-scale pseudomolecules (see Materials and methods). Unanchored repetitive sequences were identified from the scaffolds remaining from the construction of pseudomolecules. These are scaffolds composed of more than 95% 24-mers occurring more than four times in scaffolds longer than 50 kb. Unanchored rDNA, mitochondrial, and chloroplast sequences were identified by aligning the remaining scaffolds against the nr/nt nucleotide collection at the National Center for Biotechnology Information (NCBI). 1 2 1 Scaffold total 711 Contig total 20,103 Scaffold sequence total 346.0 Mbp Contig sequence total 330.8 Mbp (4.4% gap) Scaffold N50, size (number) 34.4 Mbp (5) Contig N50, size (number) 84.4 Kbp (1,080) The chromosome-scale assembly contains 711 scaffolds that cover 346.0 Mbp of the genome, with a contig N50 of 84.4 kbp and a scaffold N50 of 34.4 Mb. The resulting final statistics are shown in Table. Plots of the marker placements were constructed for all three genetic maps used for the assembly, and for the synteny between exons in the T. cacao Matina 1-6 and Criollo genomes (see Additional file, Figure S2). The pseudomolecules comprise 99.2% (346.0 Mbp of 348.7 Mbp) of the assembly. For detailed comparison of the Criollo and Matina scaffold assemblies, see Additional file(Table S7). The genome assembly has been deposited in GenBank (accession number [ALXC01000000]).

Arachne assembly assessment An initial assessment of the completeness of the Arachne2 euchromatic genome assembly was estimated using 1,015,064 Roche/454 leaf transcript sequence reads [24]. The results of this analysis indicated that 98.9% of these (see Materials and methods) mapped to the 10 chromosomes. Other completeness assessments are described in the annotation results (see below). We also validated the Arachne2 sequence assembly against a 998 kbp reference region of Sanger-sequenced BAC clones from the Matina 1-6 library [25]. A dot-plot comparing the assembled genome and the BAC clone reference region was constructed (see Additional file 2, Figure S3). Overall, the analysis indicated high contiguity across the region; a detailed summary reporting error events and bps affected is presented (see Additional file 1, Table S8).

Evidence-based gene annotation 2 We prioritized the generation and use of RNA sequencing data from diverse experiments, and from available plant whole-gene sets, for evidence-based annotation. The overall results are shown schematically in Figure Longer transcript read sets (Roche/454, see Materials and methods) were first used to confirm the completeness of genome assembly version 1.1 as above. Seven million reads (see Additional file 1, Table S9) sequenced from leaf, bean, and floral RNA from different genetic backgrounds mapped at rates ranging from 88.25% to 99.25% in Matina 1-6 (version 1.1) and from 85.23% to 97.89% in Criollo (version.1 [11]) genome assemblies. Mapping sequence identities were similar, suggesting that no significant differences were derived from sampling (see Additional file 1, Table S9). The aforementioned reads were also used for gene model annotation together with 1.22 billion shorter paired-end reads from Illumina RNA sequencing (see Additional file 1, Table S10). We used coding and intron spans from protein alignments of eight annotated plant genomes for homology modeling (see Materials and methods; also see Additional file 1, section 3). The contributions of RNA and protein analyses are displayed independently with final models in Figure 2. Evidence-supported models represent 29,408 loci with 14,806 alternative transcripts determined from RNA assemblies (see Additional file 1, Table S11). Additionally, about 20,000 elements were annotated with little or no support. These included around 13,000 gene-like regions that overlapped substantially (≥ 33%) with transposon loci (described below) and showed little or no evidence of expression, as well as loci predicted ab initio only, partial gene models, possible non-coding RNAs, and pseudogenes. If the evidence-supported gene models alone are considered, around 15% of the Matina 1-6 genome (54 Mb) is expressed, and 36 Mbp of this represents protein-coding sequence. Intron size distribution was bimodal, as seen in other plant genes [26] (see Additional file 1, Table S12). The breadth of RNA data and the larger set of plant genome sequences available since our first cacao annotation release (version 0.9) contributed to a more substantiated annotation by every measure. The improved gene models are characterized by longer protein-coding sequence, full expression support through untranslated regions, and fewer fragments relative to both Matina (version 0.9 and Criollo (version 1) (see Additional file 1, Table S13).

Analysis of gene content and orthology Among the primary cacao protein structures, we found 18,457 unique alignments to UniProt references [27] and 4,903 that were without match in this broadly comprehensive database. Products of 3,426 gene models (5,819 transcripts) were associated with 140 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway maps [28]. This compares with 125, 124, 123, and 124 currently annotated metabolic pathways for Arabidopsis, poplar, grape, and castor bean, respectively [28]. Cacao genes were categorized using OrthoMCL [29] into 15,523 putative orthologous groups situated among those of 8 other plants used in the annotation, and of these groups, 13,700 contained a single representative. Remarkably, only 43 orthologous groups that were defined based on genes present in the other plant species had no cacao gene representative, the lowest level among the fully sequenced plant genomes analyzed (see Additional file 1, Table S14a). Orthology analyses performed in this study (see Materials and methods; see Additional file 1, section 3) generate group partitions that sometimes divide known phylogenetic clades. Ten (23%) of the groups not represented in cacao were defined by uncharacterized or hypothetical proteins in the other plants. The remainder of the unrepresented groups averaged only one or two copies in the other plants, and generally reflected sequence divergence within plant gene families whose paralogs were indeed present in cacao. For example, although one group in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily was not represented in cacao, a number of cacao genes falling into 59 orthologous groups and an additional 8 ungrouped genes were annotated as members of this superfamily. There were 16 calcium-dependent kinase (CDPK) groups and three ungrouped CDPKs found in cacao, although one group that was defined for the other plants was not represented in cacao by this analysis. The over-represented and under-represented gene families for cacao, including these examples are tabulated (see Additional file 1, Table S14b), and the results summarized in a Venn diagram form as shared gene families for five compared plants (see Additional file 1, Figure S4). The identified proteins were assigned to gene ontology (GO) categories, provided by The Arabidopsis Information Resource (TAIR), and the proportion of genes assigned to each category were found to be relatively close between poplar, cacao, and grape, but significantly different from Arabidopsis (χ2 = 2895.128, degrees of freedom (df) = 78, P < 2.2 ×10-16; see Additional file 1, Table S14c). This initial comparison suggested that cacao has the highest overall similarity to poplar (1.0 correlation, χ2 = 7.2923, df = 25, P= 0.9998), and that Arabidopsis drives most of the differences in gene content per GO category. Although further work will contribute to improve the functional annotation of the proteins identified in Matina 1-6, the initial assessment and functional characterization provided here considerably improves the genomic tools available for cacao, as we describe below for the mapping and identification of genes regulating phenotypic traits. Lineage-specific genes are undoubtedly among the 703 T. cacao gene clusters [29] that share no apparent orthology with genes in any of the other 8 plants used in this study (see Additional file 1, Table S14a). Ongoing analyses, which incorporate an annotated gene set for cotton, a second member of the Malvaceae family that was recently sequenced [30], will refine this picture, and will also help define genes that are associated with speciation. Hierarchical clustering of cacao gene families, among those of many other plants, is available in Phytozome (version 9 [31]). To further understand the results from the previously described analyses and to annotate seemingly divergent or uncharacterized unique genes in cacao, we performed additional hidden Markov model (HMM) searches [32]. We annotated 4244 transcripts from 4,085 genes (transposon overlaps excluded) based on expression, that is, association with RNA sequencing evidence alone, as these showed no homology to proteins in the other 8 plant genomes. We then surveyed potentially remote homology that would not have been identified within our annotation standards by searching these cacao proteins against the profile HMM databases Pfam and TIGRFAMS, using HMMER3 [32]. In total, 126 HMM families (E-value 0.005) were found within the no-homology set. We compared these with the set of HMM families obtained from an identical scan of cacao proteins with other plant homology (for groupings of these annotations and the proportion of HMM families between the two sets, see Additional file 1, Table S14d). Although the Pfam database is part of InterPro (used above), probabilistic scoring in the HMMER3 algorithm can exceed the accuracy of finding motifs by single optimal alignments [33, 34]. Although only four HMM families were unique to the no-homology set, these were members of clades that were represented in the set of annotated plant homologs (see Additional file 1, Table S14d). Overall, this analysis therefore added modest numbers of additional members to known structural categories, and approximately 3% more definition to the unknown proteins of cacao. Gene models of the Matina 1-6 (version 1.1) sequence have been deposited in NCBI as (GenBank accession number [ALXC00000000]).

Transposable elements A significant portion of eukaryotic genomes is composed of transposable elements (TEs) [35–37]. Using a structure-based and homology-based strategy, we identified 8,542 intact TEs in the T. cacao Matina 1-6 genome. Those elements, together with numerous truncated elements and other fragments, cover 41.53% of the assembled genome. The overall results are shown schematically in Figure 2. In a parallel comparative analysis using the same approach, we identified 5,089 intact TE copies in the T. cacao Criollo genome [11], and found only 35.40% of the Criollo genome assembly to be comprised of TEs (see Additional file 1, Table S15). Although this estimate represents a nearly 10% increase in TEs over a previous annotation of Criollo (version 1 [11]), the proportion of TEs in the assembled portion of the Criollo genome is notably less than that in the assembled portion of the Matina 1-6 (version 1.1) genome. Retrotransposons, especially long terminal repeat (LTR) retrotransposons, are the predominant class of TEs identified in the cacao genome. Two types of LTRs are found: intact and solo. Solo LTRs are thought to be formed by unequal intra-element homologous recombination (UR) between two closely identical intact LTRs [38]. The 7,545 LTR retrotransposons identified, comprising 5,345 solo LTRs and 2,200 intact LTRs, can be classified into 369 families as follows: 123 copia-like, 233 gypsy-like, and 13 unclassified families (see Additional file 1, Table S16). Of the 333 retrotransposon families identified in Criollo, 277 are common to both Criollo and Matina 1-6 genomes, 56 families are specific to Criollo, and Matina 1-6 contains 92 LTR retrotransposons families not identified in Criollo (see Additional file 1, Table S16). While 96.02% (Criollo) and 98.10% (Matina 1-6) of the intact copies of these retrotransposons belong to these 277 shared families, the copy numbers of elements in each family differ substantially. For example, for Tcr53, which is the largest family in both genomes, 1,124 copies were identified in the Matina 1-6 genome, whereas only 301 copies were detected in the Criollo genome. It should be noted that, because the portions of either the Matina 1-6 or the Criollo sequences that were not assembled into the current genome pseudeomolecules remain unknown, the contribution of TEs to the difference in the contents and sizes of the two genomes could not be assessed more precisely. Nevertheless, four times more copies of Tcr53 elements were detected in Matina 1-6 than in Criollo, which may be explained mainly by the different levels of independent amplification of this family in the two genomes after their divergence. Most of the families that are unique to either genome contained only single copies. DNA transposons represent 8.87% of the Matina 1-6 genome and 7.00% of the Criollo genome. The gypsy-like retrotransposons comprise 79.93% (4.90% out of 6.13%) of the difference in TE coverage between Matina 1-6 and Criollo. These results suggest that retrotransposons may have undergone a recent amplification in the Matina 1-6 genome.

Synteny between Matina 1-6 and Criollo We further compared the whole-genome sequences of Matina 1-6 (version 1.1) and the Criollo-type B97-61/B2 (version 1 [11]). The results, depicted using Circos [39], indicate very well conserved synteny, as expected, with 271 orthologous regions (ORs) identified (see Additional file 2, Figure S5). The longest OR is a 7.7 Mbp region in chromosome 9 in both Criollo and Matina 1-6, which contains 2,580 matching exons. The mean number of matching exons in each OR is 150, and the mean lengths of the ORs are 681 kbp in Criollo and 717.5 kbp in Matina 1-6. Of the total number annotated in each genome, 87.23 % (28,666 of 32,862) of the Criollo genes and 76.08 % (22,374 of 29,408) of the Matina 1-6 genes are located in the ORs that we identified. The orthology is well conserved at the chromosomal level, but fewer and smaller ORs between non-orthologous chromosomes were also detected (see Additional file 2, Figure S5). Regions on chromosome 2 of Matina 1-6 are orthologous to regions on chromosome 9 of Criollo, for example, and regions on chromosome 3 of Matina 1-6 are orthologous to regions on chromosome 10 of Criollo. In total, 12 ORs between non-orthologous chromosomes were detected and the mean size of the ORs between non-orthologous chromosomes was much smaller than that of the ORs between orthologous chromosomes (see Additional file 1, Table S18). We analyzed the distribution of the genes associated with transposons in ORs between non-orthologous chromosomes and those between orthologous chromosomes (see Additional file 1, Table S18). We examined the mean span (kb) between genes and between transposons in each region of the two cultivars. In both cultivars, the mean span between transposons was shorter in ORs between non-orthologous chromosomes, even though the mean span between genes was larger in ORs between non-orthologous chromosomes. These results suggest that a much higher proportion of the genes in ORs between non-orthologous regions are associated with transposon activity than those in ORs between orthologous regions. The higher frequency of transposon-related genes in the ORs between non-orthologous chromosomes, along with the smaller size of the ORs between the non-orthologous chromosomes, suggests that those blocks were translocated to non-orthologous regions by transposon activity. Although more detailed studies are required to discern if the observed differences are inherent to the different assemblies and annotation approaches, we determined whether any disease resistance-related genes (within the leucine-rich repeats: LRR-RLK class) or genes potentially involved with cacao-bean quality [11] reside in ORs (see Additional file 1, Table S19). The genes that reside outside ORs or in ORs between non-orthologous chromosomes could be responsible for the differences in crop quality and disease resistance between the two cultivars. It has been reported that genes encoding proteins involved in plant interaction with biotic and abiotic extrinsic factors are far more likely to have been transposed than those that are involved in relatively stable processes [40]. Many of the LRR-RLK genes, flavonoid biosynthetic pathway genes, lipid biosynthesis genes, and terpenoid synthesis genes identified in T. cacao by Argout et al. [11] are outside ORs (see Additional file 1, Table S19; for the specific gene models involved in this analysis, see Additional file 1, Table S20a). However, this analysis was based on the Criollo annotation [11] and did not use the genes identified through our evidence-based annotation pipeline in Matina 1-6 for these pathways, many of which are also in non-orthologous regions. For example, the LRR-RLK gene motif is found in twice as many genes in non-orthologous regions in the Matina 1-6 assembly as in the Criollo assembly (see Additional file 1, Table S20b).

Mapping pod color In both T4 mapping populations (see Materials and methods), the progeny segregate in a 1:1 ratio for pod color, suggesting the involvement of a single gene. The common parent in both of these populations, Pound 7, has green pods. The other parents of these crosses, UF 273 Type 1 and Type 2, have red pods (see Additional file 2, Figure S6). In the MP01 mapping population, pod color segregates in a 3:1 red:green ratio, and we infer that both parents of this population, TSH 1188 and CCN 51, are heterozygous for the dominant red allele (see Additional file 2, Figure S6). Linkage-mapping analyses previously suggested that pod color is regulated by gene(s) located on chromosome 4 [14]. To corroborate this result, we performed Fisher's exact test for each single-nucleotide polymorphism (SNP), using a contingency table of genotype counts and phenotype scores (that is, red and green) and the marker data from the ten linkage groups on the three mapping populations (T4 Type 1, T4 Type 2, and MP01). In all three mapping populations, a large region on chromosome 4 showed significant (based on the Bonferroni correction at α = 0.05) association with pod color (see Additional file 2, Figure S7). 41 42 43 3 Using haplotypes can be more powerful than using single-marker methods to detect phenotypic effects of low-frequency variants in genome-wide association studies []. Haplotype-based methods are also useful for inferring the underlying causal genetic basis for various traits in linkage-mapping populations because, as shown here, it is possible to evaluate the parental inheritance of the associated haplotype more efficiently. We therefore determined parental haplotypes for chromosome 4 in individuals from the three mapping populations studied to help us identify candidate genes that are associated with this trait. We resolved the genotypes of the progeny in each of the mapping populations for chromosome 4 into the two corresponding parental haplotypes, using the output of HAPI-UR [] (see Materials and methods). To investigate the effect from each parental haplotype separately on pod color, we performed Fisher's exact tests separately for each parent, on individuals from each mapping population, using a 2 × 2 contingency table of parental haplotypes for each marker and red and green pod color. This analysis identified a difference between the T4 and MP01 populations; in both T4 populations, haplotypes belonging to one parent, Pound 7, did not affect the color phenotype; whereas in MP01, haplotypes from both parents did affect pod color (Figure). The observation that a single haplotype from each parent associates with red pod color in MP01, but not in T4, is consistent with the mendelian model outlined above, and suggests the existence of a potential common allele in the two T4 mapping populations, even though the parents are different. Our results also suggest that red pod color is dominant over green and that both UF 273 parents are heterozygous for the red allele, whereas the common parent, Pound 7, is homozygous for the recessive green allele. We then focused on the small region that we had identified on chromosome 4 (Figure 3) as that most significantly associated with the trait. The locations of the most significant SNP markers vary only slightly between the mapping populations, ranging from 20,987,989 bp to 21,726,996 bp. The haplotype combinations, taking into account SNP 21,126,449 bp, explain the phenotypes in the data with at least 97% accuracy in each population (see Additional file 1, Table S21; see Materials and methods for more details on the computation with Fisher's exact test). 4a,b 4a,b To fine-tune the mapping of the region of interest, we examined trees exhibiting a recombination of the haplotypes within the associated region, and added flanking markers to extend the region to 20.35 to 22.05 Mbp. In each T4 population, there were 8 to 15 trees with recombination events in at least one parental haplotype (Figure). The T4 recombinants refined the location of the causal locus to the region between 20,562,635 and 21,726,996 bp on chromosome 4 (Figure). Using MP01 trees that exhibit recombination in this region, the location of the causal locus was further refined (Figure 4c) to the region between 20,562,635 and 21,126,449 bp on chromosome 4. Indeed, the alleles in TcAPO4 and TcMYB6 associated with red pod color in the CCN 51 and TSH 1188 parents were now also associated with green pod color in the recombinants. This result further supports the hypothesis of a single allele underlying pod color (although there is the possibility that different mutations in the same gene could be responsible).

Candidate genes for pod color The availability of the high-quality Matina 1-6 reference genome permits straightforward identification of candidate genes within the restricted chromosome region identified. Recombination events identified in the T4 Type 1 and Type 2 mapping populations delimit the region regulating pod-color variation to between 20,562,635 and 21,726,996 bp on chromosome 4 (Figure 4a,b). In this region, three gene models are of particular interest: TCM_019192, homologous to Arabidopsis MYB113; TCM_019219, homologous to the Vitis vinifera APO4 gene; and TCM_019261, homologous to Arabidopsis MYB6. Both MYB genes encode transcription factors of the R2R3 domain class, which are known to be diverse in plants [44]; whereas APO proteins promote photosystem 1 complex stability and associated chlorophyll accumulation [45]. Cacao pod color is determined by the degree of red pigment that is superimposed over the base green color of the pod during development. The green color itself ranges widely, from light to dark green [7]. Because the intensity of pod pigment is influenced by the status of the base green color, we considered the gene homolog to APO4 to be a candidate gene for pod-color determination. It has been hypothesized that pod color could be regulated by one locus with alleles that determine the intensity of the green color acting in concert with a second locus that determines pigmentation [7]. Two of the 18 MYB genes on chromosome 4 of the T. cacao Matina 1-6 genome version 1.1_ (see Additional file 1, Table S22), which are localized within the 20,562,635 and 21,726,996 bp interval, are particularly promising candidate genes for pod-color variation in cacao because variants in MYB transcription factors are known to cause variation in berry pigmentation in grapes [46, 47]. While MYB6 is less characterized, MYB113 is in a small clade known to act in complexes with basic helix-loop-helix (bHLH) proteins to activate genes encoding enzymes acting in late stages of flavonol biosynthesis. MYB variants cause pigmentation variation in potato, tomato, and pepper [48], as well as in kale [49]. Pigmentation in apple skin and flesh is also regulated by MYB transcription factors [50]. Moreover, when genes encoding the R2R3 MYB transcription factors (cloned from apple) are transformed into tobacco, they induce the anthocyanin pathway, when co-expressed with bHLH proteins [50]. MYB gene evolution is thought to involve duplication events [51]; a single gene cluster of three MYB genes is involved in berry pigmentation in grape [46], and three genes of this type (including MYB113) are tandemly arrayed in Arabidopsis. The gene cluster in grape that is involved in berry-color variation includes the VvMYBA1 gene (homologous to TCM_019192), VvMYBA4 (homologous to TCM_019261), and VvMybA2. Mutations within VvMybA1 or VvMybA2 cause berries to be pigmented white/green instead of the wild-type purple color [52]. As described above, the MP01 recombinants narrow the region of interest to genes located between 20,562,635 and 21,126,449 bp on chromosome 4 (Figure 4c). Within this region, only one of the two MYB transcription factors is present: TCM_019192, which is homologous to VvMybA1. We refer to this gene as TcMYB113, and it encodes a protein with 275 amino acids sharing 61% identity with grape VvMYBA1 (see Additional file 2, Figure S8 for protein-sequence comparison). In the Criollo genome [11], TCM_019192 is annotated as two genes: Tc04_t014240 and Tc04_t014250. This difference may be attributed to the strict evidence-based Matina 1-6 version 1.1 annotation procedures that we used (see above).

Resequencing of additional genotypes and haplotype phasing of resequenced data The availability of a high-quality reference genome greatly facilitates resequencing data analyses of additional genotypes. Five parents of the mapping populations (MP01: CCN 51 and TSH 1188,; T4 Type 1: Pound 7 and UF 273 Type 1; T4 Type 2: Pound 7 and UF 273 Type 2), and eight additional genomes (KA 2 101, K 82, LCTEEN 141, NA 331, PA 51, mvP 30, mvT 85, and Criollo 13) were resequenced (see Materials and methods). Reads were phased for the region surrounding the pod-color mapping interval (between 20,561,460 and 21,386,830 bp) on chromosome 4 (see Materials and methods). We identified 21,225 SNPs and 1,879 putative insertions/deletions (indels) within this region. Phasing of the reads permitted generation of haplotype sequences and, consequently, identification of the alleles associated with pod color, specifically for the three candidate genes. The phased resequencing data from 13 genotypes plus the Matina 1-6 (version 1.1) sequence were used to generate clusters based on the haplotypes for the two MYB genes and APO4 (see Additional file 2, Figure S9). The haplotypes for TcMYB113 appear to cluster in agreement with the haplotype they induce or the haplotype of the phenotype of the clones they represent; this is not the case for TcMYB6 or APO4, in which green and red haplotypes are positioned within the same cluster (see Additional file 1, Figure S9). These results corroborate the association between TcMYB113 and pod-color variation across multiple resequenced unrelated samples with diverse genetic backgrounds. The phylogenetic relationships between the haplotypes also indicate that the haplotypes of the Criollo 13 green-pod genotype are closest to the cluster of red-pod-associated haplotypes (see Additional file 2, Figure S9). This suggests that the TcMYB113 alleles that are associated with red pod color in this study originated from the Criollo genetic group. In fact, only a single allele was found for MYB6 and APO4 in the red pod-associated haplotypes from the mapping population parents (see Additional file 2, Figure S9), but their sequence is identical to the one from the resequenced green-pod Criollo 13. This indicates that the red-pod parents of the mapping populations may have inherited a large segment of chromosome 4 from a Criollo genotype, and in fact, the Criollo genetic group was previously identified as the most likely source of the red-pod gene [7].

Sanger sequencing, association mapping, and putative functional effects of polymorphisms found within TcMYB113 1 1 2 1 1 1 53 54 5 2 55 56 57 54 5 5 54 The resequencing data shows specific SNPs in TcMYB113 within coding regions in the alleles associated with red pod color, which are at positions 20,878,747, 20,878,891, 20,878,957, 20,879,122, and 20,879,148. We corroborated this result by Sanger sequencing of the three candidate genes studied and by association mapping (see Additional file, Table S23). The association-mapping analysis was performed using 73 SNPs generated via Sanger sequencing (see Materials and methods) and 95 other SNPs from chromosomes 1 to 10 in 54 genotypes with green pods and 17 genotypes with red pods from diverse genetic backgrounds. Without accounting for genetic structure, the most significant P-values from the Fisher test were for the following SNPs (from highest to lowest significance): 20,878,891; 20,875,691, and 20,879,148 (see Additional file, Table S23). SNPs at these positions permitted differentiation of alleles that are associated with the green-pod Criollo genotypes from those associated with the red-pod trees of Criollo origin (see Additional file, Figure S10). The least significant of these (position 20,879,148; see Additional file, Table S23) results in an amino-acid change from serine to asparagine at codon 221 of the TcMYB113 protein in the alleles associated with red pod color. This residue occurs outside of the R2R3 DNA binding domain, but within the C-terminal region that varies substantially between MYB family members, making the structural consequence of this substitution difficult to predict. When genetic structure was taken into account in the association-mapping analysis, the significance of the association between this SNP and pod color was considerably lower (see Additional file, Table S23). The second most significant SNP (position 20,875,691) was detected via Sanger sequencing in the TcMYB113 5' untranslated region (UTR), located 25 bases upstream of the ATG start site (see Additional file, Table S23). The function of this SNP is difficult to infer; however, mutations occurring near translation start sites are known to affect protein-translation rates []. The SNP that was most significantly associated with pod color (position 20,878,891) is a synonymous mutation found within the coding region of TcMYB113. Intriguingly, this SNP is positioned within a target site for a dicot trans-acting small interfering RNA (tasiRNA) derived from TAS4 (TRANS-ACTING siRNA 4) TAS4-siR81(-) as identified by Luo et al. [] (Figure; see Additional file, Figure S8). TAS-derived siRNAs post-transcriptionally downregulate protein-coding transcripts in a manner similar to microRNA (miRNA)-directed repression []. MYB113 regulation in Arabidopsis additionally involves miR828, which acts both to cleave TAS4 to generate the interfering small RNA TAS4-siR81(-) [] and also, independently, to silence MYB113 []. We identified not only the TAS4-siR81 site, but also a conserved miR828 target sequence within TcMYB113 (Figure), suggesting that these regulatory mechanisms are highly conserved in cacao. However, we detected no polymorphism within the miR828 target sequencewhich overlaps with the highly conserved R3 domain (Figure). The activities of both TAS4 and miR828 ultimately regulate anthocyanin biosynthesis through MYBs in Arabidopsis []. Conservation of and natural variation in this regulatory loop is yet to be explored in other plants. Resequencing data of the region 4 kbp upstream of TcMYB113 also revealed SNPs associated with the red pod-inducing haplotypes we had identified (see Additional file 2, Figure S11). However, because these SNPs are also present in the resequenced green-pod genotype Criollo 13, these mutations in the putative region containing the promoter of TcMYB113 are unlikely to be functionally associated with the red-pod phenotype.