Joanne A. Labate

Tomato SNP discovery by EST mining and resequencing

Many economically important crop species are relatively depauparate in genetic diversity (e.g., soybean, peanut, tomato). DNA polymorphism within cultivated tomato has been estimated to be low based on molecular markers. Through mining of more than 148,000 public tomato expressed sequence tags (ESTs) and full-length cDNAs, we identified 764 EST clusters with potential single nucleotide polymorphisms (SNPs) among more than 15 tomato lines. By sequencing regions from 53 of these clusters in two to three lines, we discovered a wealth of nucleotide polymorphism (62 SNPs and 12 indels in 21 Unigenes), resulting in a verification rate of 27.2% (28 of 103 SNPs predicted in EST clusters were verified). We hypothesize that five regions with 1.6–13-fold more diversity relative to other tested regions are associated with introgressions from wild relatives. Identifying polymorphic, expressed genes in the tomato genome will be useful for both tomato improvement and germplasm conservation.

Evidence of cryptic introgression in tomato (Solanum lycopersicum L.) based on wild tomato species alleles

BackgroundMany highly beneficial traits (e.g. disease or abiotic stress resistance) have been transferred into crops through crosses with their wild relatives. The 13 recognized species of tomato (Solanum section Lycopersicon) are closely related to each other and wild species genes have been extensively used for improvement of the crop, Solanum lycopersicum L. In addition, the lack of geographical barriers has permitted natural hybridization between S. lycopersicum and its closest wild relative Solanum pimpinellifolium in Ecuador, Peru and northern Chile. In order to better understand patterns of S. lycopersicum diversity, we sequenced 47 markers ranging in length from 130 to 1200 bp (total of 24 kb) in genotypes of S. lycopersicum and wild tomato species S. pimpinellifolium, Solanum arcanum, Solanum peruvianum, Solanum pennellii and Solanum habrochaites. Between six and twelve genotypes were comparatively analyzed per marker. Several of the markers had previously been hypothesized as carrying wild species alleles within S. lycopersicum, i.e., cryptic introgressions.ResultsEach marker was mapped with high confidence (e<1 x 10-30) to a single genomic location using BLASTN against tomato whole genome shotgun chromosomes (SL2.40) database. Neighbor-joining trees showed high mean bootstrap support (86.8 ± 2.34%) for distinguishing red-fruited from green-fruited taxa for 38 of the markers. Hybridization and parsimony splits networks, genomic map positions of markers relative to documented introgressions, and historical origins of accessions were used to interpret evolutionary patterns at nine markers with putatively introgressed alleles.ConclusionOf the 47 genetic markers surveyed in this study, four were involved in linkage drag on chromosome 9 during introgression breeding, while alleles at five markers apparently originated from natural hybridization with S. pimpinellifolium and were associated with primitive genotypes of S. lycopersicum. The positive identification of introgressed genes within crop species such as S. lycopersicum will help inform conservation and utilization of crop germplasm diversity, for example, facilitating the purging of undesirable linkage drag or the exploitation of novel, favorable alleles.

Genetic structure of the four wild tomato species in the Solanum peruvianum s.l. species complex

The most diverse wild tomato species Solanum peruvianum sensu lato (s.l.) has been reclassified into four separate species: Solanum peruvianum sensu stricto (s.s.), Solanum corneliomuelleri, Solanum huaylasense, and Solanum arcanum. However, reproductive barriers among the species are incomplete and this can lead to discrepancies regarding genetic identity of germplasm. We used genotyping by sequencing (GBS) of S. peruvianum s.l., Solanum neorickii, and Solanum chmielewskii to develop tens of thousands of mapped single nucleotide polymorphisms (SNPs) to analyze genetic relationships within and among species. The data set was condensed to 14,043 SNPs with no missing data across 46 sampled plants. Origins of accessions were mapped using geographical information systems (GIS). Isolation by distance, pairwise genetic distances, and number of clusters were estimated using population genetics approaches. Isolation by distance was strongly supported, especially between interspecific pairs. Eriopersicon (S. peruvianum s.s., S. corneliomuelleri, S. huaylasense) and Arcanum (S. arcanum, S. neorickii, S. chmielewskii) species groups were genetically distinct, except for S. huaylasense which showed 50% membership proportions in each group. Solanum peruvianum and S. corneliomuelleri were not significantly differentiated from each other. Many thousands of SNP markers were identified that could potentially be used to distinguish pairs of species, including S. peruvianum versus S. corneliomuelleri, if they are verified on larger numbers of samples. Diagnostic markers will be valuable for delimiting morphologically similar and interfertile species in germplasm management. Approximately 12% of the SNPs rejected a genome-wide test of selective neutrality based on differentiation among species of S. peruvianum s.l. These are candidates for more comprehensive studies of microevolutionary processes within this species complex.

AlleleCoder: a PERL script for coding co-dominant polymorphism data for PCA

A useful biological interpretation of diploid heterozygotes is in terms of the dose of the common allele (0, 1 or 2 copies). We have developed a PERL script that converts FASTA files into coded spreadsheets suitable for principal component analysis. In combination with R and R Commander, twoand three-dimensional plots can be generated for visualizing genetic relationships. Such plots are useful for characterizing plant genetic resources. This method nicely illustrated the spectrum of genetic diversity in tomato landraces and the varieties categorized according to human-mediated dispersal.

Nucleotide diversity estimates of tomatillo (Physalis philadelphica) accessions including nine new inbred lines

To help support utilization of germplasm resources for tomatillo (Physalis philadelphica) crop improvement, we characterized genetic diversity in the National Plant Germplasm System collection. Genotyping by sequencing, a method of high-throughput DNA sequencing of reduced representation genomic libraries, was performed on 190 plant samples. This yielded 77,340 high-quality filtered single nucleotide polymorphisms from 179 plants sampled from 125 accessions. Geographical information systems data on geospatial references were verified using web- and PC-based software tools. We found that multiple plants sampled per accession were closely related to each other, but there was no apparent pattern related to original sampling location with respect to state in Mexico. There was no evidence for isolation by distance in a 15-accession, 53 plant geodiversity panel. Average proportion of heterozygous sites was halved in samples from nine inbred lines relative to samples from open-pollinated accessions (0.04 vs. 0.08). The genetic characterization of these accessions can help end users choose germplasm to support increased production of fresh and processed tomatillo products for expanding niche markets.