A 1,681-locus consensus genetic map of cultivated cucumber including 67 NB-LRR resistance gene homolog and ten gene loci

Luming Yang 0 Dawei Li 0 3 Yuhong Li 0 3 Xingfang Gu 2 Sanwen Huang 2 Jordi Garcia-Mas 4 Yiqun Weng 0 1 0 Horticulture Department, University of Wisconsin , Madison WI 53706 , USA 1 USDA-ARS Vegetable Crops Research Unit, Horticulture Department, University of Wisconsin , Madison, WI 53706 , USA 2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences , Beijing 100018 , China 3 Horticulture College, Northwest A&F University , Yangling 712100 , China 4 IRTA, Center for Research in Agricultural Genomics CSIC-IRTA-UAB-UB , 08193, Bellaterra, Barcelona , Spain Background: Cucumber is an important vegetable crop that is susceptible to many pathogens, but no disease resistance (R) genes have been cloned. The availability of whole genome sequences provides an excellent opportunity for systematic identification and characterization of the nucleotide binding and leucine-rich repeat (NB-LRR) type R gene homolog (RGH) sequences in the genome. Cucumber has a very narrow genetic base making it difficult to construct high-density genetic maps. Development of a consensus map by synthesizing information from multiple segregating populations is a method of choice to increase marker density. As such, the objectives of the present study were to identify and characterize NB-LRR type RGHs, and to develop a high-density, integrated cucumber genetic-physical map anchored with RGH loci. Results: From the Gy14 draft genome, 70 NB-containing RGHs were identified and characterized. Most RGHs were in clusters with uneven distribution across seven chromosomes. In silico analysis indicated that all 70 RGHs had EST support for gene expression. Phylogenetic analysis classified 58 RGHs into two clades: CNL and TNL. Comparative analysis revealed high-degree sequence homology and synteny in chromosomal locations of these RGH members between the cucumber and melon genomes. Fifty-four molecular markers were developed to delimit 67 of the 70 RGHs, which were integrated into a genetic map through linkage analysis. A 1,681-locus cucumber consensus map including 10 gene loci and spanning 730.0 cM in seven linkage groups was developed by integrating three component maps with a bin-mapping strategy. Physically, 308 scaffolds with 193.2 Mbp total DNA sequences were anchored onto this consensus map that covered 52.6% of the 367 Mbp cucumber genome. Conclusions: Cucumber contains relatively few NB-LRR RGHs that are clustered and unevenly distributed in the genome. All RGHs seem to be transcribed and shared significant sequence homology and synteny with the melon genome suggesting conservation of these RGHs in the Cucumis lineage. The 1,681-locus consensus genetic-physical map developed and the RGHs identified and characterized herein are valuable genomics resources that may have many applications such as quantitative trait loci identification, map-based gene cloning, association mapping, marker-assisted selection, as well as assembly of a more complete cucumber genome. - Background Over the last decade, many plant pathogen resistance (R) genes or quantitative trait loci (QTL) have been cloned. The largest class of known R genes encodes proteins with a central nucleotide binding (NB) domain and a C-terminal leucine-rich repeat (LRR) domain [1]. Based on the amino-terminal domain feature, the NB-LRR proteins can be divided into two classes: TNL (TIR-NB-LRR) and CNL (CC-NB-LRR) in which the R proteins possess, respectively, either the Toll/Interleukin-1 Receptor (TIR) domain or a coiled-coil (CC) domain [2]. The NB domain seems to have NTP-hydrolyzing activity for regulating signal transduction through conformational changes [2]. The LRR domain contains tandemly arrayed repeats that is involved in the specific recognition of pathogen effectors [3]. Both TIR and CC domains are assumed to be involved in protein-protein interactions and signal transduction [4,5]. Due to the availability of whole genome sequences, NB-encoding resistance gene homolog (RGH) sequences have been annotated and mapped in a number of plant species such as Arabidopsis thaliana [6], poplar (Populus trichocarpa) [7], potato (Solanum tuberosum) [8,9], rice (Oryza sativa) [10], sorghum (Sorghum bicolor) [11], grapevine (Vitis vinifera) [12], coffee tree (Coffea arabica) [13], Medicago truncatula [14], and papaya (Carica papaya) [15]. While NB-LRR genes are widely distributed among plant genomes, their numbers vary greatly in different species. For example, the papaya and grapevine genome contains 55 and 535 NB-LRR RGHs representing 0.2% and 1.8% of their total genes, respectively [12,15]. A lack of recent genome duplication was believed to be the reason of the overall low NB-LRR gene numbers in papaya [16]. NB-encoding genes are unevenly distributed in the plant genome and are mainly organized in multi-gene clusters. The clustered distribution of R-genes is assumed to provide a reservoir of genetic variations from which new pathogen specificity can evolve via gene duplication, unequal crossing-over, ectopic recombination or diversifying selection [17,18]. In addition, nucleotide polymorphism analyses demonstrated extremely high level of inter- and intra-specific variations of NB-LRR genes, which presumably evolve rapidly in response to changes in pathogen populations [12,19]. Nevertheless, conservation of synteny for NBLRR disease resistance genes among phylogenetically related species was also observed [20,21]. However, the extent of genome-wide conservation and synteny of NB-LRR RGHs between different species is not well documented. Cucumber, Cucumis sativus L. (2n = 2x =14) is an economically important vegetable crop and a system of choice for studying several important biological processes [22]. In recent years, application of next generation sequencing technologies enabled release of draft genomes of three cucumber lines (9930, Gy14 and B10) [23-25] providing powerful tools for understanding the structure and organization of R genes in the cucumber genome. In the 9930 draft genome, 61 NB-containing RGHs were identified [23], but no details were given for these RGHs, and the RGH numbers seem to be underestimated as compared with an improved annotation of the 9930 genome (Version 2.0) [26]. Thus, one objective of the present study was to conduct genome wide identification and characterization of NB-LRR type RGHs in the Gy14 draft genome assembly (Version 1.0) [27]. Since the ratio of genetic to physical distances varies along the chromosomes (for example, [28]), the information of genetic map locations of RGHs, especially on a high-density reference genetic map, is very useful for map-based cloning of R genes or association mapping through the candidate gene approach. The association of RGHs with candidate disease resistance genes has been well established in a number of crops such as melon (Cucumis melo) [29,30], wheat (Triticum aestivum) [31], cucumber [32], sunflower (Helianthus annuus) [33], and potato [34]. T (...truncated)