Hugo Cuevas

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

A consensus linkage map for molecular markers and quantitative trait loci associated with economically important traits in melon (Cucumis melo L.).

BackgroundA number of molecular marker linkage maps have been developed for melon (Cucumis melo L.) over the last two decades. However, these maps were constructed using different marker sets, thus, making comparative analysis among maps difficult. In order to solve this problem, a consensus genetic map in melon was constructed using primarily highly transferable anchor markers that have broad potential use for mapping, synteny, and comparative quantitative trait loci (QTL) analysis, increasing breeding effectiveness and efficiency via marker-assisted selection (MAS).ResultsUnder the framework of the International Cucurbit Genomics Initiative (ICuGI, http://www.icugi.org), an integrated genetic map has been constructed by merging data from eight independent mapping experiments using a genetically diverse array of parental lines. The consensus map spans 1150 cM across the 12 melon linkage groups and is composed of 1592 markers (640 SSRs, 330 SNPs, 252 AFLPs, 239 RFLPs, 89 RAPDs, 15 IMAs, 16 indels and 11 morphological traits) with a mean marker density of 0.72 cM/marker. One hundred and ninety-six of these markers (157 SSRs, 32 SNPs, 6 indels and 1 RAPD) were newly developed, mapped or provided by industry representatives as released markers, including 27 SNPs and 5 indels from genes involved in the organic acid metabolism and transport, and 58 EST-SSRs. Additionally, 85 of 822 SSR markers contributed by Syngenta Seeds were included in the integrated map. In addition, 370 QTL controlling 62 traits from 18 previously reported mapping experiments using genetically diverse parental genotypes were also integrated into the consensus map. Some QTL associated with economically important traits detected in separate studies mapped to similar genomic positions. For example, independently identified QTL controlling fruit shape were mapped on similar genomic positions, suggesting that such QTL are possibly responsible for the phenotypic variability observed for this trait in a broad array of melon germplasm.ConclusionsEven though relatively unsaturated genetic maps in a diverse set of melon market types have been published, the integrated saturated map presented herein should be considered the initial reference map for melon. Most of the mapped markers contained in the reference map are polymorphic in diverse collection of germplasm, and thus are potentially transferrable to a broad array of genetic experimentation (e.g., integration of physical and genetic maps, colinearity analysis, map-based gene cloning, epistasis dissection, and marker-assisted selection).

Syntenic relationships between cucumber (Cucumis sativus L.) and melon (C. melo L.) chromosomes as revealed by comparative genetic mapping

BackgroundCucumber, Cucumis sativus L. (2n = 2 × = 14) and melon, C. melo L. (2n = 2 × = 24) are two important vegetable species in the genus Cucumis (family Cucurbitaceae). Both species have an Asian origin that diverged approximately nine million years ago. Cucumber is believed to have evolved from melon through chromosome fusion, but the details of this process are largely unknown. In this study, comparative genetic mapping between cucumber and melon was conducted to examine syntenic relationships of their chromosomes.ResultsUsing two melon mapping populations, 154 and 127 cucumber SSR markers were added onto previously reported F2- and RIL-based genetic maps, respectively. A consensus melon linkage map was developed through map integration, which contained 401 co-dominant markers in 12 linkage groups including 199 markers derived from the cucumber genome. Syntenic relationships between melon and cucumber chromosomes were inferred based on associations between markers on the consensus melon map and cucumber draft genome scaffolds. It was determined that cucumber Chromosome 7 was syntenic to melon Chromosome I. Cucumber Chromosomes 2 and 6 each contained genomic regions that were syntenic with melon chromosomes III+V+XI and III+VIII+XI, respectively. Likewise, cucumber Chromosomes 1, 3, 4, and 5 each was syntenic with genomic regions of two melon chromosomes previously designated as II+XII, IV+VI, VII+VIII, and IX+X, respectively. However, the marker orders in several syntenic blocks on these consensus linkage maps were not co-linear suggesting that more complicated structural changes beyond simple chromosome fusion events have occurred during the evolution of cucumber.ConclusionsComparative mapping conducted herein supported the hypothesis that cucumber chromosomes may be the result of chromosome fusion from a 24-chromosome progenitor species. Except for a possible inversion, cucumber Chromosome 7 has largely remained intact in the past nine million years since its divergence from melon. Meanwhile, many structural changes may have occurred during the evolution of the remaining six cucumber chromosomes. Further characterization of the genomic nature of Cucumis species closely related to cucumber and melon might provide a better understanding of the evolutionary history leading to modern cucumber.

Spanish melons ( Cucumis melo L.) of the Madrid provenance: a unique germplasm reservoir

Melon (Cucumis melo L.) landraces of the Madrid provenance, Spain, have received national distinction for their high fruit quality and sensorial attributes. More specifically, a unique array of Group Inodorus landraces have been continuously cultivated and conserved by farmers in the municipality of Villaconejos since the 19th century. Their genetic relationships to other Group Inodorus and Flexuous melon market classes is not known, and, thus, a study was designed to determine their genetic relationships using 52 simple sequence repeat (SSR) markers, and then make genetic comparisons between these accessions and a previously published “Standard Reference Germplasm Array” (RA) containing Group Inodorus (14 Spanish and one USA), Flexuosus (1 Spanish), and Cantalupensis ( 2 USA) melon accessions. This subset consisted of 15 Spanish Group Inodorus landraces that circumscribed the genetic variation of major Spanish melon market classes (Groups Inodorus and Flexuosus), and USA commercial varieties (Groups Cantalupensis and Inodorus). Based on genetic distances, Villaconejos (Madrid) genotypes differed substantially from RA subset accessions, thus defining their genetic uniqueness. Principal component analysis (PCA) partitioned the accessions examined into four distinct groups revealing that Villaconejos black epidermis melons (landraces ‘Largo’, ‘Largo Negro Escrito’ and ‘Puchero’) were distinctly different from all other accessions examined, as cluster analysis separated Rochet market type Villaconejos’ accessions (landraces ‘Mochuelo’, ‘Mochuelo Tradicional’ and ‘Melón de Villaconejos’) from RA of the same market type. Genetic assessment of principal Spanish market classes revealed comparatively low intra-market heterogeneity in Piel de Sapo type accessions and high heterogeneity in Black and Yellow market type accessions. While a relatively high level of genetic introgression was detected between Yellow and Green market types, black epidermis market types were genetically unique. Given the uniqueness and high genetic diversity resident in Villaconejos landraces, this germplasm pool should be considered as a genetic source for broadening the comparatively narrow genetic base of Group Cantalupensis and Inodorus melon market types, especially standard commercial Spanish Group Inodorus market types (e.g., Piel de Sapo, Rochet, and Canari).

Population structure of the NPGS Senegalese sorghum collection and its evaluation to identify new disease resistant genes

Sorghum germplasm from West and Central Africa is cultivated in rainy and high humidity regions and is an important source of resistance genes to fungal diseases. Mold and anthracnose are two important biotic constraints to sorghum production in wet areas worldwide. Here, 158 National Plant Germplasm System (NPGS) accessions from Senegal were evaluated for agronomic traits, anthracnose, and grain mold resistance at two locations, and genetically characterized according to 20 simple sequence repeat markers. A total of 221 alleles were amplified with an average of 11 alleles per locus. Each accession had a unique genetic profile (i.e., no duplicates), and the average genetic distance between accessions was 0.42. Population structure and cluster analysis separated the collection into four populations with pairwise FST values >0.15. Three of the populations were composed of Guinea-race sorghum germplasm, and one included multiple races. Anthracnose resistant accessions were present at high frequency and evenly distributed among the three Guinea-race populations. Fourteen accessions showed resistance to grain mold, and eight were resistant to both diseases. These results indicated that the NPGS of Senegal is a genetically diverse collection with a high frequency of disease resistant accessions. Nevertheless, its population structure suggests the presence of few sources of resistance to both grain mold and anthracnose, which are fixed in the germplasm. The phenotypic and genotypic information for these accessions provides a valuable resource for its correct use to broaden the genetic base of breeding programs.