High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping

Background: Cucurbita pepo is amember of the Cucurbitaceae family, the second-most important horticultural family in terms of economic importance after Solanaceae. The ¿summer squash¿ types, including Zucchini and Scallop, rank among the highest-valued vegetables worldwide. There are few genomic tools available for this species. The first Cucurbita transcriptome, along with a large collection of Single Nucleotide Polymorphisms (SNP), was recently generated using massive sequencing. A set of 384 SNP was selected to generate an Illumina GoldenGate assay in order to construct the first SNP-based genetic map of Cucurbita and map quantitative trait loci (QTL). Results: We herein present the construction of the first SNP-based genetic map of Cucurbita pepo using a population derived from the cross of two varieties with contrasting phenotypes, representing the main cultivar groups of the species¿ two subspecies: Zucchini (subsp. pepo) ¿ Scallop (subsp. ovifera). The mapping population was genotyped with 384 SNP, a set of selected EST-SNP identified in silico after massive sequencing of the transcriptomes of both parents, using the Illumina GoldenGate platform. The global success rate of the assay was higher than 85%. In total, 304 SNP were mapped, along with 11 SSR from a previous map, giving a map density of 5.56 cM/marker. This map was used to infer syntenic relationships between C. pepo and cucumber and to successfully map QTL that control plant, flowering and fruit traits that are of benefit to squash breeding. The QTL effects were validated in backcross populations. Conclusion: Our results show that massive sequencing in different genotypes is an excellent tool for SNP discovery, and that the Illumina GoldenGate platform can be successfully applied to constructing genetic maps an performing QTL analysis in Cucurbita. This is the first SNP-based genetic map in the Cucurbita genus and is an invaluable new tool for biological research, especially considering that most of these markers are located in the coding regions of genes involved in different physiological processes. The platform will also be useful for future mapping and diversity studies, and will be essential in order to accelerate the process of breeding new and better adapted squash varieties.This research was funded by the INIA projects RTA2008-00035-C02-01/02 and RTA2011-00044-C02-1/2 of the Spanish Instituto Nacional de Investigacion y Tecnologia Agraria and FEDER funds (EU). The NVD grant was supported by the Programa de Formacion del Personal Tecnico e Investigador from IFAPA, co-financed with European Social Funds. The authors wish to thank P. Salas and E. Martinez Perez for their technical assistance in the fruit characterization. We are thankful for the kindly suggestions of Dr. Harry Paris for the F2 C. pepo mapping population.Esteras Gómez, C.; Gómez, P.; Monforte Gilabert, AJ.; Blanca Postigo, JM.; Vicente-Dolera, N.; Roig Montaner, MC.; Nuez Viñals, F.... (2012). High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genomics. 13(80):1-21. https://doi.org/10.1186/1471-2164-13-80S121138

First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding.Financial support was provided by the Spanish Project INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Almentaria) RTA2011-00044C02-01, the ANR MELODY (ANR-11-BSV7-0024), the European Research Council (ERCSEXYPARTH), FEDER, and FSE funds. NVD has been awarded a grant by the Andalusian Institute of Agronomy Research IFAPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Vicente-Dolera, N.; Troadec, C.; Moya, M.; Río-Celestino, MD.; Pomares-Viciana, T.; Bendahmane, A.; Picó Sirvent, MB.... (2014). First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement. PLoS ONE. 9(11):112743-112743. https://doi.org/10.1371/journal.pone.0112743S112743112743911Paris, H. S., Yonash, N., Portnoy, V., Mozes-Daube, N., Tzuri, G., & Katzir, N. (2002). Assessment of genetic relationships in Cucurbita pepo (Cucurbitaceae) using DNA markers. Theoretical and Applied Genetics, 106(6), 971-978. doi:10.1007/s00122-002-1157-0Parry, M. A. J., Madgwick, P. J., Bayon, C., Tearall, K., Hernandez-Lopez, A., Baudo, M., … Phillips, A. L. (2009). Mutation discovery for crop improvement. Journal of Experimental Botany, 60(10), 2817-2825. doi:10.1093/jxb/erp189Gilchrist, E., & Haughn, G. (2010). 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K., Tsai, H., Berardi, S., … Dubcovsky, J. (2009). A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biology, 9(1), 115. doi:10.1186/1471-2229-9-115Kumar, A. P., Boualem, A., Bhattacharya, A., Parikh, S., Desai, N., Zambelli, A., … Bendahmane, A. (2013). SMART -- Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biology, 13(1), 38. doi:10.1186/1471-2229-13-38Kurowska, M., Daszkowska-Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., & Maluszynski, M. (2011). TILLING - a shortcut in functional genomics. Journal of Applied Genetics, 52(4), 371-390. doi:10.1007/s13353-011-0061-1Rigola, D., van Oeveren, J., Janssen, A., Bonné, A., Schneiders, H., van der Poel, H. J. A., … van Eijk, M. J. T. (2009). High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint™ Technology. PLoS ONE, 4(3), e4761. doi:10.1371/journal.pone.0004761González, M., Xu, M., Esteras, C., Roig, C., Monforte, A. J., Troadec, C., … Picó, B. (2011). Towards a TILLING platform for functional genomics in Piel de Sapo melons. BMC Research Notes, 4(1). doi:10.1186/1756-0500-4-289Elias, R., Till, B. J., Mba, C., & Al-Safadi, B. (2009). Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Research Notes, 2(1), 141. doi:10.1186/1756-0500-2-141Dahmani-Mardas, F., Troadec, C., Boualem, A., Lévêque, S., Alsadon, A. A., Aldoss, A. A., … Bendahmane, A. (2010). Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach. PLoS ONE, 5(12), e15776. doi:10.1371/journal.pone.0015776Boualem, A., Fleurier, S., Troadec, C., Audigier, P., Kumar, A. P. K., Chatterjee, M., … Bendahmane, A. (2014). Development of a Cucumis sativus TILLinG Platform for Forward and Reverse Genetics. 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Summary of the <i>Cucurbita pepo</i> mutant collection development.

<p>Summary of the <i>Cucurbita pepo</i> mutant collection development.</p

Sequence and structural analysis of APRX.

<p>(A) Amino acid alignment of <i>Cucurbita pepo</i> APRX and homologous proteins from <i>Arabidopsis thaliana</i> (AAM66044.1), <i>Capsicum annun</i> (AFU51540.1), <i>Cucumis sativus</i> (AAA33128.1), <i>Phaseolus vulgaris</i> (AAD37427.1) and <i>Pytum sativus</i> (BAD97438.1). Numbers above the alignment indicate the amino acid positions along the APRX protein. The EMS-induced stop mutation in W132 is shown above the alignment in red. (B) Superposition of the predicted 3D structure model of APRX WT protein indicated in grey and the mutant protein indicated in blue. The proximal heme pocket is represented in red and the two calcium ions in green. The position of the induced stop codon mutation indentified in Cp-161 family (W132*) is indicated in red. The APRX model was determined using the Geno3D server (<a href="http://geno3d-pbil.ibcp.fr" target="_blank">http://geno3d-pbil.ibcp.fr</a>). (C) Wild type and albino phenotypes observed in line Cp-161.</p

Gene structure of the target genes screened in the <i>C. pepo</i> population (PSY, LCYb, ETR1, ERS1, and APRX).

<p>This drawing was made using the PARSESNP program, which maps the mutation on a gene model to illustrate the distribution of mutations. Orange boxes represent exons and orange lines introns, the size (pb) is indicated in blue. Dashed lines in red and black indicate amplicons analyzed by TILLING. Purple triangles represent silent mutations and black and red triangles represent missense and truncation mutations, respectively. Mutations before ATG and after STOP codon are not shown.</p

Tilled genes and mutation frequency in the <i>C. pepo</i> mutant collection with 768 M2 families screened.

a<p>nucleic acid transition is a non-synonymous mutation and induce amino acid change in the translated protein.</p>b<p>nucleic acid transition produces a stop codon and may induce a truncated protein.</p>c<p>nucleic acid transition is located in splicing motif.</p>d<p>nucleic acid transition induces a synonymous mutation and then no change in the translated protein.</p>e<p>nucleid acid transition is located in an intronic or promotor region.</p><p>Tilled genes and mutation frequency in the <i>C. pepo</i> mutant collection with 768 M2 families screened.</p

Examples of morphological mutants observed within the <i>C. pepo</i> TILLING population.

<p>(a) Plant affected in cotyledon colour, albino. (b) Plant affected in cotyledon colour, chlorotic. (c) Plant affected in cotyledon number. (d–e) Female flowers with abnormal stigma. (f–h) Different coloured fruits. (i–k) Semi-dwarf plants with bushy and hyper compact architecture. (l) Albino dwarf plant. (m) Different size and colour leaves.</p

Effect of EMS dose on mutation frequency in <i>C. pepo</i> mutant collection.

<p><b>*</b>Number of identified mutations in all the families for this EMS dose after screening five genes.</p><p>Effect of EMS dose on mutation frequency in <i>C. pepo</i> mutant collection.</p