High-throughput marker discovery in melon using a self-designed oligo microarray

<p>Abstract</p> <p>Background</p> <p>Genetic maps constitute the basis of breeding programs for many agricultural organisms. The creation of these maps is dependent on marker discovery. Melon, among other crops, is still lagging in genomic resources, limiting the ability to discover new markers in a high-throughput fashion. One of the methods used to search for molecular markers is DNA hybridization to microarrays. Microarray hybridization of DNA from different accessions can reveal differences between them--single-feature polymorphisms (SFPs). These SFPs can be used as markers for breeding purposes, or they can be converted to conventional markers by sequencing. This method has been utilized in a few different plants to discover genetic variation, using Affymetrix arrays that exist for only a few organisms. We applied this approach with some modifications for marker discovery in melon.</p> <p>Results</p> <p>Using a custom-designed oligonucleotide microarray based on a partial EST collection of melon, we discovered 6184 putative SFPs between the parents of our mapping population. Validation by sequencing of 245 SFPs from the two parents showed a sensitivity of around 79%. Most SFPs (81%) contained single-nucleotide polymorphisms. Testing the SFPs on another mapping population of melon confirmed that many of them are conserved.</p> <p>Conclusion</p> <p>Thousands of new SFPs that can be used for genetic mapping and molecular-assisted breeding in melon were discovered using a custom-designed oligo microarray. A portion of these SFPs are conserved and can be used in different breeding populations. Although improvement of the discovery rate is still needed, this approach is applicable to many agricultural systems with limited genomic resources.</p

A consensus linkage map for molecular markers and Quantitative Trait Loci associated with economically important traits in melon (Cucumis melo L.)

Background A 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). Results Under the framework of the International Cucurbit Genomics Initiative (ICuGI, http://www.icugi.org webcite), 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. Conclusions Even 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).This work was supported in part by SNC Laboratoire ASL, Ruiter Seeds B.V., Enza Zaden B.V., Gautier Semences S.A., Nunhems B.V., Rijk Zwaan B.V., Sakata Seed Inc, Semillas Fito S. A., Seminis Vegetable Seeds Inc, Syngenta Seeds B. V., Takii and Company Ltd, Vilmorin & Cie S. A., and Zeraim Gedera Ltd (all of them as part of the support to the ICuGI); the grants AGL2009-12698-C02-02 from the Spanish "Ministerio de Ciencia e Innovacion" to AJM. NK lab was supported in part by Research Grant Award No. IS-4223-09C from BARD, the United States - Israel Binational Agricultural Research and Development Fund, and in part by Israel Science Foundation Grant No. 38606, De Ruiter Seeds, Enza Zaden, Keygene, Rijk Zwaan, Sakata Seed Corporation, Semillas Fito, Syngenta Seeds and Vilmorin Clause & Cie. AD was supported by a JAE-Doc contract from "Consejo Superior de Investigaciones Cientificas" (CSIC-Spain). MF was supported by a postdoctoral contract from CRAG. The research carried out at YX's laboratory was supported by Chinese funds (Grant No. 2008-Z42(3), 5100001, 2010AA101907).Díaz Bermúdez, A.; Fergany, M.; Formisano, G.; Ziarsolo, P.; Blanca Postigo, JM.; Fei, Z.; Staub, JE.... (2011). A consensus linkage map for molecular markers and Quantitative Trait Loci associated with economically important traits in melon. BMC Plant Biology. 11. https://doi.org/10.1186/1471-2229-11-111S1

Anchoring the consensus ICuGI genetic map to the melon (Cucumis melo L.) genome

Melon (Cucumis melo L.) genetic maps were compiled by the International Cucurbit Genomics Initiative (ICuGI) before the release of the melon genome. However, due to the use of different marker sets, the position of ICuGI markers in the genome remained unknown, complicating the integration of previous genetic mapping studies in the genome. We looked for the genome position of 870 simple sequence repeat and single nucleotide polymorphism (SNP) markers from the ICuGI map, locating 836 of them in the melon pseudochromosomes v3.5.1, and integrating them with previously available SNPs to reach a total of 1850 markers mapped in the genome sequence. The number of markers per scaffold ranged from 1 to 105, with an average of 13, thus improving on the previous studies in melon. Twenty-three of the markers mapped on virtual chromosome "0'', twelve of them being included in the ICuGI map, which could assist in the anchoring of some unanchored contigs and scaffolds. Genetic and physical distance comparison showed a good collinearity between them, confirming the quality of the ICuGI map. A higher recombination rate was also usually found at the ends of the chromosomes, whereas a drastic reduction was observed in the putative pericentromeric regions. Quantitative trait loci (QTL) previously located in the ICuGI map were also anchored in the genome. This work offers the opportunity to supplement the genetic maps available up to now with the genomic resources resulting from the melon genome sequencing to facilitate comparative mapping in melon between past and new studies, and to overcome some of the current limitations in QTL gene identification.This work was funded in part by the project AGL2012-40130-C02-02 from Spanish Ministry of Economy and Competitiveness (MINECO) to AJM.Díaz Bermúdez, A.; Forment Millet, JJ.; Argyris, JM.; Fukino, N.; Tzuri, G.; Harel-Beja, R.; Katzir, N.... (2015). 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Expression patterns of the studied anthocyanin biosynthesis structural and regulatory genes in different tissues of the "white" pomegranate.

<p>The expression patterns of the structural and regulatory genes that are predicted to be involved in anthocyanin biosynthesis in pomegranate, are represented. The analysis was done on skin and arils of the "white" pomegranate, during fruit development [from flowers (stage 1) to fully mature fruit (stage 12)] and on young leaves. W, young leaves of the "white" pomegranate; R, young leaves of the red cv. Wonderful. Semi-quantitative RT-PCR analysis was performed as described in Materials and methods. Samples were normalized to 18S and 28S ribosomal RNA (<i>rRNA)</i> as the reference gene for constitutive expression. PCR products were separated on a 1% agarose gel.</p

Schematic representation of the flavonoid-biosynthesis pathway leading to the production of anthocyanins and proanthocyanidins.

<p>Enzyme name abbreviations are as follows: PAL, phenylalanine ammonia-lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; F3'5'H, flavonoid 3',5'-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; LDOX, leucoanthocyanidin oxidase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; UFGT, UDP glucose:flavonoid 3-O-glucosyltransferase.</p

Co-dominance primer markers that distinguish between heterozygote and homozygote alleles for the insert in the red F2 progeny.

<p>White numbers indicate progeny with "white" phenotype and red numbers indicate progeny with colored phenotype. The red lettering C/C indicates homozygocity for the SNP marker (1,008 bp downstream to the ATG, shown also in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142777#pone.0142777.s008" target="_blank">S3 Table</a>). A scheme of the location of the F18 and R7 primers with relevance to the insert is in the bottom (F18 primer is based on the 18bp that were sequencing from the insert, using the AFLP method). PCR products were separated in a 1% agarose gel.</p

PCR amplification for the absence of the insertion within the <i>PgLDOX</i> gene in the F2 progeny.

<p>White numbers indicate progeny with "white" phenotype and red numbers indicate progeny with colored phenotype. A scheme of the location of the primers (F2 and R2) with relevance to the insert is in the bottom. PCR products were separated in a 1% agarose gel.</p

Single-Nucleotide Polymorphism Markers from De-Novo Assembly of the Pomegranate Transcriptome Reveal Germplasm Genetic Diversity

<div><p>Pomegranate is a valuable crop that is grown commercially in many parts of the world. Wild species have been reported from India, Turkmenistan and Socotra. Pomegranate fruit has a variety of health-beneficial qualities. However, despite this crop's importance, only moderate effort has been invested in studying its biochemical or physiological properties or in establishing genomic and genetic infrastructures. In this study, we reconstructed a transcriptome from two phenotypically different accessions using 454-GS-FLX Titanium technology. These data were used to explore the functional annotation of 45,187 fully annotated contigs. We further compiled a genetic-variation resource of 7,155 simple-sequence repeats (SSRs) and 6,500 single-nucleotide polymorphisms (SNPs). A subset of 480 SNPs was sampled to investigate the genetic structure of the broad pomegranate germplasm collection at the Agricultural Research Organization (ARO), which includes accessions from different geographical areas worldwide. This subset of SNPs was found to be polymorphic, with 10.7% loci with minor allele frequencies of (MAF<0.05). These SNPs were successfully used to classify the ARO pomegranate collection into two major groups of accessions: one from India, China and Iran, composed of mainly unknown country origin and which was more of an admixture than the other major group, composed of accessions mainly from the Mediterranean basin, Central Asia and California. This study establishes a high-throughput transcriptome and genetic-marker infrastructure. Moreover, it sheds new light on the genetic interrelations between pomegranate species worldwide and more accurately defines their genetic nature.</p></div

HPLC chromatograms at 520 nm of methanolic extracts of different tissues of cv. Wonderful (right) and the "white" pomegranate (left).

<p>Chromatogram peak identities were based on the UV/Vis spectrum and retention times: (1) delphinidin 3,5-diglucoside; (2) cyanidin 3,5-diglucoside; (3) pelargonidin 3,5-diglucoside; (4) delphinidin 3-glucoside; (5) cyanidin 3-glucoside; (6) pelargonidin 3-glucoside.</p