Yuhong Yang

The genome of the cucumber, Cucumis sativus L.

Cucumber is an economically important crop as well as a model system for sex determination studies and plant vascular biology. Here we report the draft genome sequence of Cucumis sativus var. sativus L., assembled using a novel combination of traditional Sanger and next-generation Illumina GA sequencing technologies to obtain 72.2-fold genome coverage. The absence of recent whole-genome duplication, along with the presence of few tandem duplications, explains the small number of genes in the cucumber. Our study establishes that five of the cucumbers seven chromosomes arose from fusions of ten ancestral chromosomes after divergence from Cucumis melo. The sequenced cucumber genome affords insight into traits such as its sex expression, disease resistance, biosynthesis of cucurbitacin and fresh green odor. We also identify 686 gene clusters related to phloem function. The cucumber genome provides a valuable resource for developing elite cultivars and for studying the evolution and function of the plant vascular system.

Cloning and Characterization of R3b; Members of the R3 Superfamily of Late Blight Resistance Genes Show Sequence and Functional Divergence

Massive resistance (R) gene stacking is considered to be one of the most promising approaches to provide durable resistance to potato late blight for both conventional and genetically modified breeding strategies. The R3 complex locus on chromosome XI in potato is an example of natural R gene stacking, because it contains two closely linked R genes (R3a and R3b) with distinct resistance specificities to Phytophthora infestans. Here, we report about the positional cloning of R3b. Both transient and stable transformations of susceptible tobacco and potato plants showed that R3b conferred full resistance to incompatible P. infestans isolates. R3b encodes a coiled-coil nucleotide-binding site leucine-rich repeat protein and exhibits 82% nucleotide identity with R3a located in the same R3 cluster. The R3b gene specifically recognizes Avr3b, a newly identified avirulence factor from P. infestans. R3b does not recognize Avr3a, the corresponding avirulence gene for R3a, showing that, despite their high sequence similarity, R3b and R3a have clearly distinct recognition specificities. In addition to the Rpi-mcd1/Rpi-blb3 locus on chromosome IV, the R3 locus on chromosome XI is the second example of an R-gene cluster with multiple genes recognizing different races of P. infestans.

Chromosomal mapping and QTL analysis of resistance to downy mildew in Cucumis sativus

Downy mildew of cucumber (Cucumis sativus), caused by Pseudoperonospora cubensis, is a major foliar disease worldwide. The cucumber inbred lines K8 (resistant to downy mildew) and K18 (susceptible) were used to study the inheritance of resistance to downy mildew. Chromosomal mapping of the resistance genes was completed to provide a theoretical basis for the resistance mechanisms and for marker assisted selection (MAS). Inoculation was used to test the level of resistance to P. cubensis in the F2 and F2:3 families derived from the cross K8 × K18. Simple sequence repeat (SSR) analysis, combined with bulked segregation analysis (BSA), was done with the DNA of F2 plants using 2,360 pairs of SSR primers. JoinMap Version 3.0 and MapInspect were used to construct SSR linkages and to verify the relationships between these SSR linkages and cucumber chromosomes. Quantitative trait locus (QTL) analysis of downy mildew resistance was done using MapQTL Version 4.0. Inheritance of resistance to downy mildew in K8 was quantitative. Five QTLs for resistance to downy mildew were detected: dm1.1, dm5.1, dm5.2, dm5.3, and dm6.1. The loci of dm1.1 and dm6.1 were on chromosomes 1 and 6, respectively. The loci of dm5.1, dm5.2, and dm5.3 were on chromosome 5, and were linked. Six linked SSR markers for these five QTLs were identified: SSR31116, SSR20705, SSR00772, SSR11012, SSR16882, and SSR16110. Six and four nucleotide binding site (NBS)-type resistance gene analogs (RGAs) were predicted in the region of dm5.2 and dm5.3, respectively. These results will be of benefit for fine-mapping the major QTLs for downy mildew resistance, and for MAS in cucumber.

Fine genetic mapping localizes cucumber scab resistance gene Ccu into an R gene cluster

Scab, caused by Cladosporium cucumerinum, is an important disease of cucumber, Cucumis sativus. In this study, we conducted fine genetic mapping of the single dominant scab resistance gene, Ccu, with 148 F9 recombinant inbred lines (RILs) and 1,944 F2 plants derived from the resistant cucumber inbred line 9110Gt and the susceptible line 9930, whose draft genome sequence is now available. A framework linkage map was first constructed with simple sequence repeat markers placing Ccu into the terminal 670xa0kb region of cucumber Chromosome 2. The 9110Gt genome was sequenced at 5× genome coverage with the Solexa next-generation sequencing technology. Sequence analysis of the assembled 9110Gt contigs and the Ccu region of the 9930 genome identified three insertion/deletion (Indel) markers, Indel01, Indel02, and Indel03 that were closely linked with the Ccu locus. On the high-resolution map developed with the F2 population, the two closest flanking markers, Indel01 and Indel02, were 0.14 and 0.15xa0cM away from the target gene Ccu, respectively, and the physical distance between the two markers was approximately 140xa0kb. Detailed annotation of the 180xa0kb region harboring the Ccu locus identified a cluster of six resistance gene analogs (RGAs) that belong to the nucleotide binding site (NBS) type R genes. Four RGAs were in the region delimited by markers Indel01 and Indel02, and thus were possible candidates of Ccu. Comparative DNA analysis of this cucumber Ccu gene region with a melon (C. melo) bacterial artificial chromosome (BAC) clone revealed a high degree of micro-synteny and conservation of the RGA tandem repeats in this region.

Development of InDel markers linked to Fusarium wilt resistance in cabbage

Cabbage Fusarium wilt (CFW) is a destructive disease causing great losses to cabbage (Brassica oleracea L. var. capitata L.) production worldwide. At present, there are few reports concerning molecular marker research on cabbage resistance to CFW. In this study, 160 double haploid (DH) lines were obtained from the F1 population of a 99–77 (highly resistant to CFW)xa0×xa099–91 (highly susceptible to CFW) cross. Insertion–deletion (InDel) markers were designed according to the reference genome sequence of cabbage and the whole-genome re-sequencing data of the two parents. A genetic map of chromosome C06 including seven InDel markers was constructed based on the DH population. Thus, FOC (resistance gene to Fusarium oxysporum f. sp. conglutinans) was located on chromosome C06 and two InDel markers out of the seven, M10 and A1, flanked the gene at 1.2 and 0.6xa0cM, respectively. Marker A1 revealed a significant consistency with the phenotype assay in the F2 population as well as in 40 inbred lines (96 and 82xa0%, respectively). This study lays the foundation for fine mapping and cloning of the FOC gene and for marker-assisted selection in cabbage resistance breeding.

Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato

Late blight, caused by the oomycete pathogen Phytophthora infestans (Mont.) de Bary, is a devastating disease for tomato and potato crops. In the past decades, many late blight resistance (R) genes have been characterized in potato. In contrast, less work has been conducted on tomato. The Ph-3 gene from Solanum pimpinellifolium was introgressed into cultivated tomatoes and conferred broad-spectrum resistance to P. infestans. It was previously assigned to the long arm of chromosome 9. In this study, a high-resolution genetic map covering the Ph-3 locus was constructed using an F2 population of a cross between Solanum lycopersicum CLN2037B (containing Ph-3) and S. lycopersicum LA4084. Ph-3 was mapped in a 0.5xa0cM interval between two markers, Indel_3 and P55. Eight putative genes were found in the corresponding 74xa0kb region of the tomato Heinz1706 reference genome. Four of these genes are resistance gene analogs (RGAs) with a typical nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 domain. Each RGA showed high homology to the late blight R gene Rpi-vnt1.1 from Solanum venturii. Transient gene silencing indicated that a member of this RGA family is required for Ph-3-mediated resistance to late blight in tomato. Furthermore, this RGA family was also found in the potato genome, but the number of the RGAs was higher than in tomato.

Identification and mapping of quantitative resistance to late blight (Phytophthora infestans) in Solanum habrochaites LA1777

Late blight (Phytophthora infestans) can have devastating effects on tomato production over the whole world. Most of the commercial cultivars of tomato, Solanum lycopersicum, are susceptible. Qualitative and quantitative resistance has been described in wild relatives of tomato. In general qualitative resistance can more easily be overcome by newly evolved isolates. Screening of three S. habrochaites accessions (LA1033, LA2099 and LA1777) through a whole plant assay showed that accession LA1777 had a good level of resistance to several isolates of P. infestans. To explore the potential in this wild species, an introgression line (IL) population of S. habrochaites LA1777 was used to screen individual chromosome regions of the wild species by a detached leaf assay. Two major isolates (T1,2 and T1,2,4) were used and two parameters were measured: lesion size (LS), and disease incidence (DI). Substantial variation was observed between the individual lines. QTLs were identified for LS but not for DI. The presence of five QTLs derived from LA1777 (Rlbq4a, Rlbq4b, Rlbq7, Rlbq8 and Rlbq12) results in unambiguous higher levels of resistance. All QTLs co-localized with previously described QTLs from S. habrochaites LA2099 except QTL Rlbq4b, which is therefore a novel QTL.

Mapping and analysis of a novel candidate Fusarium wilt resistance gene FOC1 in Brassica oleracea

BackgroundCabbage Fusarium wilt is a major disease worldwide that can cause severe yield loss in cabbage (Brassica olerecea). Although markers linked to the resistance gene FOC1 have been identified, no candidate gene for it has been determined so far. In this study, we report the fine mapping and analysis of a candidate gene for FOC1 using a double haploid (DH) population with 160 lines and a F2 population of 4000 individuals derived from the same parental lines.ResultsWe confirmed that the resistance to Fusarium wilt was controlled by a single dominant gene based on the resistance segregation ratio of the two populations. Using InDel primers designed from whole-genome re-sequencing data for the two parental lines (the resistant inbred-line 99–77 and the highly susceptible line 99–91) and the DH population, we mapped the resistance gene to a 382-kb genomic region on chromosome C06. Using the F2 population, we narrowed the region to an 84-kb interval that harbored ten genes, including four probable resistance genes (R genes): Bol037156, Bol037157, Bol037158 and Bol037161 according to the gene annotations from BRAD, the genomic database for B. oleracea. After correcting the model of the these genes, we re-predicted two R genes in the target region: re-Bol037156 and re-Bol0371578. The latter was excluded after we compared the two genes’ sequences between ten resistant materials and ten susceptible materials. For re-Bol037156, we found high identity among the sequences of the resistant lines, while among the susceptible lines, there were two types of InDels (a 1-bp insertion and a 10-bp deletion), each of which caused a frameshift and terminating mutation in the cDNA sequences. Further sequence analysis of the two InDel loci from 80 lines (40 resistant and 40 susceptible) also showed that all 40 R lines had no InDel mutation while 39 out of 40xa0S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.ConclusionsThis work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.

A major quantitative trait locus conferring resistance to fusarium wilt was detected in cucumber by using recombinant inbred lines

Fusarium wilt is an important root disease of cucumber throughout the world. There are no accurate simple sequence repeat (SSR) markers for use in molecular breeding of fusarium wilt resistance and no studies on chromosomal mapping of the resistance in cucumber. In this paper, a set of 148 F9 recombinant inbred lines derived from the cross 9110Gtxa0×xa09930 and a total of 2,416 pairs of SSR primers were used to study the inheritance of fusarium wilt resistance and to detect quantitative trait loci (QTLs) conferring the resistance in cucumber. Genetic analysis indicated that the resistance to fusarium wilt in 9110Gt was quantitative. One major QTL, Foc2.1, was screened in the years 2007, 2009 and 2012. It accounted for phenotypic variances of 64.2, 32.2 and 38.8xa0% with logarithm of odds scores of 32.78, 12.51 and 15.15 in the three years, respectively. The major QTL was placed in the region of SSR03084–SSR17631 within a genetic distance of 2.4xa0cM on chromosome 2. The physical length of the genomic region harboring Foc2.1 was 751.6xa0kb and there were seven predicted nucleotide binding site resistance genes. The validation of SSR17631 linked to Foc2.1 was tested using 46 diverse germplasms. SSR17631 had an accuracy rate of 87.88xa0% for selecting resistant materials and it could be used to screen cucumber resources with fusarium wilt resistance in molecular marker-assisted selection breeding. The major QTL identified in this paper will help to understand the genetic basis of fusarium wilt resistance. The present study has provided a firm step for fine mapping and gene cloning of fusarium wilt resistance in cucumber in the future.

Genetic analysis and gene mapping of papaya ring spot virus resistance in cucumber

The papaya ring spot virus (PRSV) causes significant fruit yield loss in cucurbit crops. Understanding of the inheritance and molecular mapping of PRSV resistance will facilitate development of resistant varieties to control this disease. In the present study, an F2 population was developed from the cross between susceptible ‘65G’ and resistant ‘02245’ cucumber inbred lines. Genetic analysis of PRSV resistance in 144 F2:3-derived F3 families showed that resistance is controlled by a single recessive gene which was designated as prsv02245. Simple sequence repeat (SSR) markers were employed in polymorphism screening between PRSV-susceptible and resistant DNA pools. The PRSV resistance gene, prsv02245, was mapped on chromosome 6 that was flanked by two SSR markers, SSR11-177 and SSR11-1, which was 1.1 and 2.9xa0cM away from the prsv02245 locus, respectively. The physical distance between the two markers was approximately 600xa0kb. The accuracy rate of marker-assisted selection of PRSV resistance among 35 cucumber lines using the marker, SSR11-177 was more than 80xa0%. Results from this study provide a valuable tool for fine mapping, gene cloning, and marker-assisted breeding for PRSV resistance in cucumber.