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

Background

Cabbage 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.

Results

We 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 40 S lines matched the two types of loci. Thus re-Bol037156 was identified as a likely candidate gene for FOC1 in cabbage.

Conclusions

This work may lay the foundation for marker-assisted selection as well as for further function analysis of the FOC1 gene.     

An Improved Brassica rapa Genetic Linkage Map and Locus-specific Variations in a Doubled Haploid Population

In this study, we describe the construction of an improved Chinese cabbage genetic linkage map by integrating simple sequence repeats (SSRs) and insertion/deletion polymorphisms (InDels) into a previously published map of a doubled haploid (DH) population. The population was derived from a cross between the Chinese cabbage line BY (Brassica rapa ssp. pekinensis) and a European turnip line MM (Brassica rapa L. ssp. rapifera). A total of 629 markers were aligned to ten linkage groups, with a total map length of 1,173.8 cM, and an average distance between markers of 1.87 cm. Of the 126 SSRs and 133 InDels mapped, 46 and 34 were novel, respectively. A comparison of the linkage map with the B. rapa genome showed that more than 93 % of the markers, including 112 SSRs and 129 InDels, could be anchored unambiguously to a specific location on one of the ten chromosomes. In most cases, the order of markers on the linkage map and physical map was similar; however, the majority of linkage groups contained a number of markers whose positions were either transposed or had moved slightly forwards or backwards. During microspore culture, it was observed that 11 SSRs and one InDel showed either variation in size, or the appearance of new marker bands in the DH lines. As a first step to addressing this SSR/InDel marker instability, six SSR and one InDel loci were sequenced, which revealed that the size variation was due mainly to changes in repeat-motif number or to the insertion/deletion of new fragments of DNA.     

Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa

The use of clubroot resistance (CR) genes is an effective and economical approach for controlling Plasmodiophora brassicae, the causal agent of clubroot disease in Chinese cabbage (Brassica rapa) and other Brassica crops. In a previous study, we identified and mapped the CRb locus on chromosome A03 of B. rapa in the doubled-haploid (DH) line ‘CR Shinki DH line’ of Chinese cabbage. In this study, CRb, a dominant gene conferring resistance to pathotype 4 of P. brassicae, was finely mapped in combination with bulked segregant analysis and bioinformatics analysis (BIA). Using 1,486 highly susceptible individuals and 2,896 individuals from two separate F₂ populations of ‘702-5’ (B. rapa ssp. chinensis) ×  ‘CR Shinki DH line,’ the CRb locus was narrowed to a region of approximately 0.14 cM between two flanking markers, TCR79 and TCR108. The sequences of seven newly developed markers linked to CRb were landed on bacterial artificial chromosome (BAC) of the reference B. rapa ‘Chiifu-401-42’ by BIA, and a physical map consisting of three BAC clones was constructed. The CRb locus was defined as an interval of approximately 83.5 kb on a BAC clone (KBrB085J21). The target interval contained one Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) gene, one NBS–LRR gene, and several putative regulatory genes in the B. rapa genome. The CRb gene was tightly linked to two other CR genes, CRa and CRb ^Kato . These results provide useful information for isolation of the CRb gene and tightly linked molecular markers for breeding CR in B. rapa.     

QTL Analysis of Head Splitting Resistance in Cabbage (Brassica oleracea L. var. capitata) Using SSR and InDel Makers Based on Whole-Genome Re-Sequencing

Head splitting resistance (HSR) in cabbage is an important trait closely related to both quality and yield of head. However, the genetic control of this trait remains unclear. In this study, a doubled haploid (DH) population derived from an intra-cross between head splitting-susceptible inbred cabbage line 79–156 and resistant line 96–100 was obtained and used to analyze inheritance and detect quantitative trait loci (QTLs) for HSR using a mixed major gene/polygene inheritance analysis and QTL mapping. HSR can be attributed to additive-epistatic effects of three major gene pairs combined with those of polygenes. Negative and significant correlations were also detected between head Hsr and head vertical diameter (Hvd), head transverse diameter (Htd) and head weight (Hw). Using the DH population, a genetic map was constructed with simple sequence repeat (SSR) and insertion–deletion (InDel) markers, with a total length of 1065.9 cM and average interval length of 4.4 cM between adjacent markers. Nine QTLs for HSR were located on chromosomes C3, C4, C7, and C9 based on 2 years of phenotypic data using both multiple-QTL mapping and inclusive composite interval mapping. The identified QTLs collectively explained 39.4 to 59.1% of phenotypic variation. Three major QTLs (Hsr 3.2, 4.2, 9.2) showing a relatively larger effect were robustly detected in different years or with different mapping methods. The HSR trait was shown to have complex genetic mechanisms. Results from QTL mapping and classical genetic analysis were consistent. The QTLs obtained in this study should be useful for molecular marker-assisted selection in cabbage breeding and provide a foundation for further research on HSR genetic regulation.     

Linkage map construction using InDel and SSR markers and QTL analysis of heading traits in Brassica oleracea var. capitata L.

Cabbage heading traits are important quantitative traits that greatly affect both quality and yield of cabbage. However, the genetic control of these traits remains unclear. To detect quantitative trait loci (QTLs) associated with heading traits, a double haploid (DH) population with 196 lines was created from a cabbage hybrid 01–20 × 96–100. A genetic map with insertion–deletion and simple sequence repeat markers was constructed based on the DH population, with a total length of 934.06 cM and average interval length of 2.3 cM between adjacent markers. Field experiments in three seasons were carried out to evaluate the heading traits, including head mature period (Hm), head weight (Hw), core length (Cl), head vertical diameter (Hvd), and the ratio of Cl to Hvd (Cl/Hvd). Using the map and the trait data, 13 reliable QTLs in total were identified and 5 were found in more than one season based on the adjusted means of three seasons. Major QTLs were identified for Hm (R ² = 40.4, LOD = 14.84), Hw (R ² = 28.6, LOD = 9.83), Cl (R ² = 38.8, LOD = 15.73), Hvd (R ² = 19.2, LOD = 9.26), and Cl/Hvd (R ² = 38.8, LOD = 12.75). The most significant QTLs were Hm3.1, Cl3.1, and Cl/Hvd3.1, which were detected in three seasons with the maximum contribution rate of almost 40 %. Six active regions that harbored more than one QTL were identified on five chromosomes, and one of them contained major QTLs associated with five traits. The QTLs obtained in this study should be useful for marker-assisted selection in cabbage breeding and for understanding the genetic control of these traits.     

Genetic and molecular analyses of resistance to a variant of Puccinia striiformis in barley

Seedlings of 62 Australian barley cultivars and two exotic barley genotypes were assessed for resistance to a variant of Puccinia striiformis, referred to as “Barley Grass Stripe Rust” (BGYR), first detected in Australia in 1998, which is capable of infecting wild Hordeum species and some genotypes of cultivated barley. Fifty-three out of 62 cultivated barley cultivars tested were resistant to the pathogen. Genetic analyses of seedling resistance to BGYR in six Australian barley cultivars and one Algerian barley landrace indicated that they carried either one or two major resistance genes to the pathogen. A single recessive seedling resistance gene, rpsSa3771, identified in Sahara 3771, was located on the long arm of chromosome 1 (7 H), flanked by the restriction fragment length polymorphism (RFLP) markers Xwg420 and Xcdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using the doubled haploid (DH) population Clipper × Sahara 3771 identified two major quantitative trait loci (QTL), one on the long arm of chromosome 3 (3 H) and the second on the long arm of chromosome 1 (7 H), accounting for 26 % and 18 % of the total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to seedling resistance gene rpsSa3771 and the second QTL was concluded to correspond to a single APR gene, designated rpsCl, contributed by cultivar Clipper.     

Construction of a sequence-based bin map and mapping of QTLs for downy mildew resistance at four developmental stages in Chinese cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. ssp. <Emphasis Type="Italic">pekinensis</Emphasis>)

Specific-locus amplified fragment sequencing is a high-resolution method for genetic mapping, genotyping, and single nucleotide polymorphism (SNP) marker discovery. Previously, a major QTL for downy mildew resistance, BraDM, was mapped to linkage group A08 in a doubled-haploid population derived from Chinese cabbage lines 91–112 and T12–19. The aim of the present study was to improve the linkage map and identify the genetic factors involved in downy mildew resistance. We detected 53,692 high quality SLAFs, of which 7230 were polymorphic, and 3482 of the polymorphic markers were used in genetic map construction. The final map included 1064 bins on ten linkage groups and was 858.98 cM in length, with an average inter-locus distance of 0.81 cM. We identified six QTLs that are involved in downy mildew resistance. The four major QTLs, sBrDM8, yBrDM8, rBrDM8, and hBrDM8, for resistance at the seedling, young plant, rosette, and heading stages were mapped to A08, and are identical to BraDM. The two minor resistance QTLs, rBrDM6 (A06) and hBrDM4 (A04), were active at the rosette and heading stages. The major QTL sBrDM8 defined a physical interval of ~228 Kb on A08, and a serine/threonine kinase family gene, Bra016457, was identified as the possible candidate gene. We report here the first high-density bin map for Chinese cabbage, which will facilitate mapping QTLs for economically important traits and SNP marker development. Our results also expand knowledge of downy mildew resistance in Chinese cabbage and provide three SNP markers (A08-709, A08-028, and A08-018) that we showed to be effective when used in MAS to breed for downy mildew resistance in B. rapa.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

Accumulation of quantitative trait loci conferring broad-spectrum clubroot resistance in Brassica oleracea

Throughout the world, clubroot disease is one of the most damaging diseases affecting Brassica oleracea. To develop marker-assisted selection (MAS) that could assist the incorporation of durable clubroot resistance (CR) into cultivars, previous genetic analyses have identified several CR quantitative trait loci (CR–QTL). However, the independent and cumulative effects of each CR locus against various isolates have rarely been tested. Previously, we identified one major CR–QTL and four minor CR–QTL in the F2 plants from broccoli doubled haploid (DH) line × cabbage DH line of B. oleracea. In the present study, to clarify their effectiveness for controlling disease involving various isolates, inoculation testing was conducted in genotypes with various combinations of the CR genes, which were selected using the DNA markers closely associated with each CR–QTL. In exploring the overall disease incidence, it was apparent that a single involvement of the major CR gene located in the PbBo(Anju)1 locus, or accumulation of CR genes in the minor CR–QTL, is not enough to confer sufficient resistance. One major CR gene in the QTL PbBo(Anju)1 locus plus two to three minor CR genes conferred moderate resistance. The genotype in which all of the CR genes locating in the five QTL including PbBo(Anju)1 were accumulated showed the highest resistance, and it was broadly resistant against six isolates. Accumulation of several CR genes by MAS is necessary to conduct CR breeding in B. oleracea. Our developed DNA markers can be used efficiently to make selections of required loci for the acquisition of resistance, and use of these markers will be a powerful tool for CR breeding in B. oleracea.     

Effect of a rye dwarfing gene on plant height,heading stage,and Fusarium head blight in triticale (×Triticosecale Wittmack)

Key message

The rye-derived dwarfing gene Ddw1 on chromosome 5R acts in triticale in considerably reducing plant height, increasing FHB severity and delaying heading stage.

Abstract

Triticale, an amphiploid hybrid between durum wheat and rye, is an European cereal mainly grown in Germany, France, Poland, and Belarus for feeding purposes. Dwarfing genes might further improve the genetic potential of triticale concerning lodging resistance and yield. However, they might have pleiotropic effects on other, agronomically important traits including Fusarium head blight. Therefore, we analyzed a population of 199 doubled haploid (DH) lines of the cross HeTi117-06 × Pigmej for plant height, heading stage, and FHB severity across 2 locations and 2 years. The most prominent QTL was detected on chromosome 5R explaining 48, 77, and 71 % of genotypic variation for FHB severity, plant height, and heading stage, respectively. The frequency of recovery in cross validation was ≥90 % for all three traits. Because the markers that detect dwarfing gene Ddw1 in rye are also in our population the most closely linked markers, we assume that this major QTL resembles Ddw1. For FHB severity two, for plant height three, and for heading stage five additional QTL were detected. Caused by the considerable genetic variation for heading stage and FHB severity within the progeny with the dwarfing allele, short-strawed, early heading and FHB-resistant lines can be developed when population size is large enough.     

Mapping and candidate-gene screening of the novel Turnip mosaic virus resistance gene retr02 in Chinese cabbage (Brassica rapa L.)

The extreme resistance to Turnip mosaic virus observed in the Chinese cabbage (Brassica rapa) line, BP8407, is monogenic and recessive. Bulked segregant analysis was carried out to identify simple sequence repeat and Indel markers linked to this recessive resistance gene, termed recessive Turnip mosaic virus resistance 02 (retr02). Mapping of PCR-specific Indel markers on 239 individuals of a BP8407 × Ji Zao Chun F₂ population, located this resistance gene to a 0.9-cM interval between two Indel markers (BrID10694 and BrID101309) and in scaffold000060 or scaffold000104 on chromosome A04 of the B. rapa genome. Eleven eukaryotic initiation factor 4E (eIF4E) and 14 eukaryotic initiation factor 4G (eIF4G) genes are predicted in the B. rapa genome. A candidate gene, Bra035393 on scaffold000104, was predicted within the mapped resistance locus. The gene encodes the eIF(iso)4E protein. Bra035393 was sequenced in BP8407 and Ji Zao Chun. A polymorphism (A/G) was found in exon 3 between BP8407 and Ji Zao Chun. This gene was analysed in four resistant and three susceptible lines. A correlation was observed between the amino acid substitution (Gly/Asp) in the eIF(iso)4E protein and resistance/susceptibility. eIF(iso)4E has been shown previously to interact with the TuMV genome-linked protein, VPg.     

Comparative mapping of Raphanus sativus genome using Brassica markers and quantitative trait loci analysis for the Fusarium wilt resistance trait

Fusarium wilt (FW), caused by the soil-borne fungal pathogen Fusarium oxysporum is a serious disease in cruciferous plants, including the radish (Raphanus sativus). To identify quantitative trait loci (QTL) or gene(s) conferring resistance to FW, we constructed a genetic map of R. sativus using an F₂ mapping population derived by crossing the inbred lines ‘835’ (susceptible) and ‘B2’ (resistant). A total of 220 markers distributed in 9 linkage groups (LGs) were mapped in the Raphanus genome, covering a distance of 1,041.5 cM with an average distance between adjacent markers of 4.7 cM. Comparative analysis of the R. sativus genome with that of Arabidopsis thaliana and Brassica rapa revealed 21 and 22 conserved syntenic regions, respectively. QTL mapping detected a total of 8 loci conferring FW resistance that were distributed on 4 LGs, namely, 2, 3, 6, and 7 of the Raphanus genome. Of the detected QTL, 3 QTLs (2 on LG 3 and 1 on LG 7) were constitutively detected throughout the 2-year experiment. QTL analysis of LG 3, flanked by ACMP0609 and cnu_mBRPGM0085, showed a comparatively higher logarithm of the odds (LOD) value and percentage of phenotypic variation. Synteny analysis using the linked markers to this QTL showed homology to A. thaliana chromosome 3, which contains disease-resistance gene clusters, suggesting conservation of resistance genes between them.     

Fine mapping and identification of candidate Br-or gene controlling orange head of Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Orange head Chinese cabbage accumulates significant amounts of carotenoids with enhanced nutritional quality. To develop molecular markers for breeding of Chinese cabbage lines with high carotenoid content and to isolate the candidate gene underlying carotenoid synthesis, we performed fine mapping of the orange locus in a F₂S₄ mapping population. Genetic analysis revealed that the phenotype of the orange head trait was controlled by a single recessive gene, Br-or. The F₂S₄ mapping population consisting of 1,724 individuals was developed from the cross between parental lines 11J16 and 11S39-2 by continuous selfing of a single heterozygous individual. Twenty-one tightly linked simple sequence repeat (SSR) and insertion/deletion polymorphism (InDel) markers were obtained. High-resolution genetic mapping of these markers in the F₂S₄ mapping population placed Br-InDel2 and Br-InDel1 at genetic distances of 0.1 and 0.2 cM, respectively, on either side of the Br-or locus. Based on comparison of these two marker sequences with the fully sequenced Brassica rapa genome, the Br-or locus was delimited to a 16.7 kb genomic region. Three open reading frames (ORFs) were predicted in the target region. ORF1 encoded carotenoid isomerase, which is involved in the isomerization of carotenoids. ORF1 was found to be co-segregated with the Br-or locus and was thus the most likely candidate gene for Br-or. The information obtained here will facilitate the breeding of nutrient-enriched Chinese cabbage through marker-assisted selection and provide a platform for gaining a better understanding of the regulation of carotenoid biosynthesis in these plants.     

Inheritance and QTL mapping of Fusarium wilt race 4 resistance in cotton

Diseases such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] represent expanding threats to cotton production. Integrating disease resistance into high-yielding, high-fiber quality cotton (Gossypium spp.) cultivars is one of the most important objectives in cotton breeding programs worldwide. In this study, we conducted a comprehensive analysis of gene action in cotton governing FOV race 4 resistance by combining conventional inheritance and quantitative trait loci (QTL) mapping with molecular markers. A set of diverse cotton populations was generated from crosses encompassing multiple genetic backgrounds. FOV race 4 resistance was investigated using seven parents and their derived populations: three intraspecific (G. hirsutum × G. hirsutum L. and G. barbadense × G. barbadense L.) F₁ and F₂; five interspecific (G. hirsutum × G. barbadense) F₁ and F₂; and one RIL. Parents and populations were evaluated for disease severity index (DSI) of leaves, and vascular stem and root staining (VRS) in four greenhouse and two field experiments. Initially, a single resistance gene (Fov4) model was observed in F₂ populations based on inheritance of phenotypes. This single Fov4 gene had a major dominant gene action and conferred resistance to FOV race 4 in Pima-S6. The Fov4 gene appears to be located near a genome region on chromosome 14 marked with a QTL Fov4-C14 ₁ , which made the biggest contribution to the FOV race 4 resistance of the generated F₂ progeny. Additional genetic and QTL analyses also identified a set of 11 SSR markers that indicated the involvement of more than one gene and gene interactions across six linkage groups/chromosomes (3, 6, 8, 14, 17, and 25) in the inheritance of FOV race 4 resistance. QTLs detected with minor effects in these populations explained 5–19 % of the DSI or VRS variation. Identified SSR markers for the resistance QTLs with major and minor effects will facilitate for the first time marker-assisted selection for the introgression of FOV race 4 resistance into elite cultivars during the breeding process.     

Genetic and comparative genomics mapping reveals that a powdery mildew resistance gene Ml3D232 originating from wild emmer co-segregates with an NBS-LRR analog in common wheat (Triticum aestivum L.)

Powdery mildew caused by Blumeria graminis f. sp. tritici is one of the most important wheat diseases worldwide and breeding for resistance using diversified disease resistance genes is the most promising approach to prevent outbreaks of powdery mildew. A powdery mildew resistance gene, originating from wild emmer wheat (Triticum turgidum var. dicoccoides) accessions collected from Israel, has been transferred into the hexaploid wheat line 3D232 through crossing and backcrossing. Inoculation results with 21 B. graminis f. sp. tritici races indicated that 3D232 is resistant to all of the powdery mildew isolates tested. Genetic analyses of 3D232 using an F₂ segregating population and F₃ families indicated that a single dominant gene, Ml3D232, confers resistance in the host seedling stage. By applying molecular markers and bulked segregant analysis (BSA), we have identified polymorphic simple sequence repeats (SSR), expressed sequence tags (EST) and derived sequence tagged site (STS) markers to determine that the Ml3D232 is located on chromosome 5BL bin 0.59–0.76. Comparative genetic analyses using mapped EST markers and genome sequences of rice and Brachypodium established co-linearity of the Ml3D232 genomic region with a 1.4 Mb genomic region on Brachypodium distachyon chromosome 4, and a 1.2 Mb contig located on the Oryza sativa chromosome 9. Our comparative approach enabled us to develop new EST–STS markers and to delimit the genomic region carrying Ml3D232 to a 0.8 cM segment that is collinear with a 558 kb region on B. distachyon. Eight EST markers, including an NBS-LRR analog, co-segregated with Ml3D232 to provide a target site for fine genetic mapping, chromosome landing and map-based cloning of the powdery mildew resistance gene. This newly developed common wheat germplasm provides broad-spectrum resistance to powdery mildew and a valuable resource for wheat breeding programs.     

Construction of a genetic map based on high-throughput SNP genotyping and genetic mapping of a TuMV resistance locus in Brassica rapa

Brassica rapa is a member of the Brassicaceae family and includes vegetables and oil crops that are cultivated worldwide. The introduction of durable resistance against turnip mosaic virus (TuMV) into agronomically important cultivars has been a significant challenge for genetic and horticultural breeding studies of B. rapa. Based on our previous genome-wide analysis of DNA polymorphisms between the TuMV-resistant doubled haploid (DH) line VC40 and the TuMV-susceptible DH line SR5, we constructed a core genetic map of the VCS-13M DH population, which is composed of 83 individuals derived from microspore cultures of a F₁ cross between VC40 and SR5, by analyzing the segregation of 314 sequence-characterized genetic markers. The genetic markers correspond to 221 SNPs and 31 InDels of genes as well as 62 SSRs, covering 1,115.9 cM with an average distance of 3.6 cM between the adjacent marker loci. The alignment and orientation of the constructed map showed good agreement with the draft genome sequence of Chiifu, thus providing an efficient strategy to map genic sequences. Using the genetic map, a novel dominant TuMV resistance locus (TuMV-R) in the VCS-13M DH population was identified as a 0.34 Mb region in the short arm of chromosome A6 in which four CC–NBS–LRR resistance genes and two pathogenesis-related-1 genes reside. The genetic map developed in this study can play an important role in the genetic study of TuMV resistance and the molecular breeding of B. rapa.     

Development of InDel markers for Brassica rapa based on whole-genome re-sequencing

Genome-wide detection of short insertion/deletion length polymorphisms (InDels, <5 bp) in Brassica rapa (named the A genome) was performed by comparing whole-genome re-sequencing data from two B. rapa accessions, L144 and Z16, to the reference genome sequence of Chiifu-401-42. In total, we identified 108,558 InDel polymorphisms between Chiifu-401-42 and L144, 26,795 InDels between Z16 and Chiifu-401-42, and 26,693 InDels between L144 and Z16. From these, 639 InDel polymorphisms of 3–5 bp in length between L144 and Z16 were selected for experimental validation; 491 (77 %) yielded single PCR fragments and showed polymorphisms, 7 (1 %) did not amplify a product, and 141 (22 %) showed no polymorphism. For further validation of these intra-specific InDel polymorphisms, 503 candidates, randomly selected from the 639 InDels, were screened across seven accessions representing different B. rapa cultivar groups. Of these assayed markers, 387 (77 %) were polymorphic, 111 (22 %) were not polymorphic and 5 (1 %) did not amplify a PCR product. Furthermore, we randomly selected 518 InDel markers to validate their polymorphism in B. napus (the AC genome) and B. juncea (the AB genome), of which more than 90 % amplified a PCR product; 132 (25 %) showed polymorphism between the two B. napus accessions and 41 (8 %) between the two B. juncea accessions. This set of novel PCR-based InDel markers will be a valuable resource for genetic studies and breeding programs in B. rapa.     

Linkage mapping and identification of QTL affecting deoxynivalenol (DON) content (Fusarium resistance) in oats (Avena sativa L.)

Mycotoxins caused by Fusarium spp. is a major concern on food and feed safety in oats, although Fusarium head blight (FHB) is often less apparent than in other small grain cereals. Breeding resistant cultivars is an economic and environment-friendly way to reduce toxin content, either by the identification of resistance QTL or phenotypic evaluation. Both are little explored in oats. A recombinant-inbred line population, Hurdal × Z595-7 (HZ595, with 184 lines), was used for QTL mapping and was phenotyped for 3 years. Spawn inoculation was applied and deoxynivalenol (DON) content, FHB severity, days to heading and maturity (DH and DM), and plant height (PH) were measured. The population was genotyped with DArTs, AFLPs, SSRs and selected SNPs, and a linkage map of 1,132 cM was constructed, covering all 21 oat chromosomes. A QTL for DON on chromosome 17A/7C, tentatively designated as Qdon.umb-17A/7C, was detected in all experiments using composite interval mapping, with phenotypic effects of 12.2–26.6 %. In addition, QTL for DON were also found on chromosomes 5C, 9D, 13A, 14D and unknown_3, while a QTL for FHB was found on 11A. Several of the DON/FHB QTL coincided with those for DH, DM and/or PH. A half-sib population of HZ595, Hurdal × Z615-4 (HZ615, with 91 lines), was phenotyped in 2011 for validation of QTL found in HZ595, and Qdon.umb-17A/7C was again localized with a phenotypic effect of 12.4 %. Three SNPs closely linked to Qdon.umb-17A/7C were identified in both populations, and one each for QTL on 5C, 11A and 13A were identified in HZ595. These SNPs, together with those yet to be identified, could be useful in marker-assisted selection to pyramiding resistance QTL.     

High-resolution mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.)

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.     

Molecular mapping of leaf rust resistance gene Lr15 in hexaploid wheat

Leaf rust is a widespread and commonly occurring rust disease of wheat. Genetic resistance is the most economical method of reducing losses due to leaf rust. Lr15 has been shown to be present on wheat chromosome 2D and is reported to be a seedling resistance gene. However, tightly linked markers associated with Lr15 have not been reported to date. To identify molecular markers linked to Lr15, an F₂ mapping population of Thatcher × Thatcher-Lr15 was generated. Available wheat simple sequence repeat markers were utilized in parental screening and polymorphic markers were used to analyze the entire population of 221 plants. Phenotypic evaluations of the F₂-derived F₃ progenies with Puccinia triticina Eriks. pathotype 162A (93R15) confirmed the monogenic inheritance of Lr15. The linkage group representing chromosome 2DS was constructed at LOD 4.0 which revealed the closest flanking markers Xgwm4562 and Xgwm102 at a distance of 3.1 and 9.3 cM, respectively. Furthermore, utilization of these flanking markers in combination has successfully identified wheat lines with or without Lr15. These markers could potentially be useful in gene pyramiding with other genes to enhance rust resistance in wheat.