Quantitative Trait Locus Mapping of Resistance to Turnip Yellows Virus in Brassica rapa and Brassica oleracea and Introgression of These Resistances by Resynthesis Into Allotetraploid Plants for Deployment in Brassica napus

Turnip yellows virus (TuYV) is aphid-transmitted and causes considerable yield losses in oilseed rape (OSR, Brassica napus, genome: AACC) and vegetable brassicas. Insecticide control of the aphid vector is limited due to insecticide resistance and the banning of the most effective active ingredients in the EU. There is only one source of TuYV resistance in current commercial OSR varieties, which has been mapped to a single dominant quantitative trait locus (QTL) on chromosome A04. We report the identification, characterisation, and mapping of TuYV resistance in the diploid progenitor species of OSR, Brassica rapa (genome: AA), and Brassica oleracea (genome: CC). Phenotyping of F1 populations, produced from within-species crosses between resistant and susceptible individuals, revealed the resistances were quantitative and partially dominant. QTL mapping of segregating backcross populations showed that the B. rapa resistance was controlled by at least two additive QTLs, one on chromosome A02 and the other on chromosome A06. Together, they explained 40.3% of the phenotypic variation. In B. oleracea, a single QTL on chromosome C05 explained 22.1% of the phenotypic variation. The TuYV resistance QTLs detected in this study are different from those in the extant commercial resistant varieties. To exploit these resistances, an allotetraploid (genome: AACC) plant line was resynthesised from the interspecific cross between the TuYV-resistant B. rapa and B. oleracea lines. Flow cytometry confirmed that plantlets regenerated from the interspecific cross had both A and C genomes and were mixoploid. To stabilise ploidy, a fertile plantlet was self-pollinated to produce seed that had the desired resynthesised, allotetraploid genome AACC. Phenotyping of the resynthesised plants confirmed their resistance to TuYV. Genotyping with resistance-linked markers identified during the mapping in the progenitors confirmed the presence of all TuYV resistance QTLs from B. rapa and B. oleracea. This is the first report of TuYV resistance mapped in the Brassica C genome and of an allotetraploid AACC line possessing dual resistance to TuYV originating from both of its progenitors. The introgression into OSR can now be accelerated, utilising marker-assisted selection, and this may reduce selection pressure for TuYV isolates that are able to overcome existing sources of resistance to TuYV.

Genetic Dissection of the Breakdown of Durable Resistance in Indica Rice Variety PTB33 to Brown Planthoppers Nilaparvata Lugens (Stål)

BackgroundTo avoid and delay the resistance breakdown of varieties against pathogens and insect pests, broad-spectrum and durable resistance by multiple genes pyramiding are expected to be one of the practical approaches. The indica rice variety PTB33 (Oryza sativa L.) shows high and durable resistance to the brown planthopper (BPH, Nilaparvata lugens Stål). However, this variety gradually lost its resistance against the recent virulence development of BPH. However, breakdown processes are not fully elucidated by individual genetic loci. ResultsEffective QTLs were explored across the whole genomic region against four BPH populations collected in Japan in 1988, 1989, 1999, and 2013 using high-density single-nucleotide polymorphism (SNP) markers obtained by genotyping-by-sequencing. Among seven genomic regions of PTB33 likely conferring BPH resistance, four QTLs, qFSA4a, qFSA6, qFSA11, and qFSA12 on chromosomes 4, 6, 11, and 12, respectively, were validated as BPH resistance QTLs. The PTB33 alleles at the four QTLs positively contributed to BPH resistance. Infestation of monogenic segregating lines showed that the PTB33 alleles at qFSA11, qFSA12, and qFSA4a lost resistance effects at least in 1989, 1999, and 2013, respectively. ConclusionThis study showed breakdown of durable resistance in PTB33 was explained by step-by-step losses of genetic effects at each resistance locus and probably multiple acquisitions of virulence genes in BPH in a gene-by-gene specific manner.

Mapping pre-harvest sprouting resistance loci in AAC Innova × AAC Tenacious spring wheat population

Background Pre-harvest sprouting (PHS) is a major problem for wheat production due to its direct detrimental effects on wheat yield, end-use quality and seed viability. Annually, PHS is estimated to cause > 1.0 billion USD in losses worldwide. Therefore, identifying PHS resistance quantitative trait loci (QTLs) is crucial to aid molecular breeding efforts to minimize losses. Thus, a doubled haploid mapping population derived from a cross between white-grained PHS susceptible cv AAC Innova and red-grained resistant cv AAC Tenacious was screened for PHS resistance in four environments and utilized for QTL mapping. Results Twenty-one PHS resistance QTLs, including seven major loci (on chromosomes 1A, 2B, 3A, 3B, 3D, and 7D), each explaining ≥10% phenotypic variation for PHS resistance, were identified. In every environment, at least one major QTL was identified. PHS resistance at most of these loci was contributed by AAC Tenacious except at two loci on chromosomes 3D and 7D where it was contributed by AAC Innova. Thirteen of the total twenty-one identified loci were located to chromosome positions where at least one QTL have been previously identified in other wheat genotype(s). The remaining eight QTLs are new which have been identified for the first time in this study. Pedigree analysis traced several known donors of PHS resistance in AAC Tenacious genealogy. Comparative analyses of the genetic intervals of identified QTLs with that of already identified and cloned PHS resistance gene intervals using IWGSC RefSeq v2.0 identified MFT-A1b (in QTL interval QPhs.lrdc-3A.1) and AGO802A (in QTL interval QPhs.lrdc-3A.2) on chromosome 3A, MFT-3B-1 (in QTL interval QPhs.lrdc-3B.1) on chromosome 3B, and AGO802D, HUB1, TaVp1-D1 (in QTL interval QPhs.lrdc-3D.1) and TaMyb10-D1 (in QTL interval QPhs.lrdc-3D.2) on chromosome 3D. These candidate genes are involved in embryo- and seed coat-imposed dormancy as well as in epigenetic control of dormancy. Conclusions Our results revealed the complex PHS resistance genetics of AAC Tenacious and AAC Innova. AAC Tenacious possesses a great reservoir of important PHS resistance QTLs/genes supposed to be derived from different resources. The tracing of pedigrees of AAC Tenacious and other sources complements the validation of QTL analysis results. Finally, comparing our results with previous PHS studies in wheat, we have confirmed the position of several major PHS resistance QTLs and candidate genes.

Genomic regions associated with chocolate spot (Botrytis fabae Sard.) resistance in faba bean (Vicia faba L.)

Chocolate spot (CS), caused by Botrytis fabae Sard., is an important threat to global faba bean production. Growing resistant faba bean cultivars is, therefore, paramount to preventing yield loss. To date, there have been no reported quantitative trait loci (QTLs) associated with CS resistance in faba bean. The objective of this study was to identify genomic regions associated with CS resistance using a recombinant inbred line (RIL) population derived from resistant accession ILB 938. A total of 165 RILs from the cross between Melodie/2 and ILB 938/2 were genotyped and evaluated for CS reactions under replicated, controlled climate conditions. QTL analysis identified five loci contributing to CS resistance on faba bean chromosomes 1 and 6, accounting for 5.0-23.4% of the total phenotypic variance. The sequences of SNP markers linked to resistance QTLs on chromosome 1 that have the largest effects encode multiple classes of putative disease and/or defense-related genes. The results of this study not only provide insight into disease-resistance QTLs, but can also be used as potential targets for marker-assisted breeding in faba bean genetic improvement for CS resistance. Keywords: Botrytis fabae; chocolate spot; faba bean; disease resistance; QTL mapping; candidate gene

Mapping Blast Resistance Genes in Rice Varieties ‘Minghui 63’ and ‘M-202’

Rice blast disease caused by the fungus Magnaporthe oryzae (syn. M. grisea) is one of the most lethal diseases for sustainable rice production worldwide. Blast resistance mediated by major resistance genes are often broken-down after a short period of deployment, while minor blast resistance genes, each providing a small effect on disease reactions, are more durable. In the present study, we first evaluated disease reactions of two rice breeding parents ‘Minghui 63’ and ‘M-202’ with 11 US blast races, IA45, IB1, IB45, IB49, IB54, IC1, IC17, ID1, IE1, IG1, and IH1 commonly found under greenhouse conditions using a category disease rating resembling infection types under field conditions. ‘Minghui 63’ exhibited differential resistance responses in comparison with that of ‘M-202’ to the tested blast races. A recombinant inbred line (RIL) population of 275 lines from a cross between ‘Minghui 63’ and ‘M-202’ was also evaluated with the above mentioned blast races. The population was genotyped with 156 simple sequence repeat (SSR) and insertion and deletion (Indel) markers. A linkage map with a genetic distance of 1022.84 cM was constructed using inclusive composite interval mapping (ICIM) software. A total of 10 resistance QTLs, eight from ‘Minghui 63’ and two from ‘M-202’, were identified. One major QTL, qBLAST2 on chr 2, was identified by seven races/isolates. The remaining nine minor resistance QTLs were mapped on chromosome 1, 3, 6, 9, 10, 11 and 12. These findings provide useful genetic markers and resources to tag minor blast resistance genes for marker assisted selection in rice breeding program and for further studies of underlying genes.

RNAseq-based gene expression profiling of the Anopheles funestus pyrethroid resistant strain FUMOZ highlights the predominant role of the duplicated CYP6P9a/b cytochrome P450s

Insecticide-based interventions, notably long-lasting insecticidal nets (LLINs), against mosquito vectors of malaria are currently threatened by pyrethroid resistance. Here, we contrasted RNAseq-based gene expression profiling of laboratory resistant (FUMOZ) and susceptible (FANG) strains of the major malaria vector Anopheles funestus. Cytochrome P450 genes were the predominant over-expressed detoxification genes in FUMOZ, with high expression of the duplicated CYP6P9a (fold-change of 82.23 versus FANG) and CYP6P9b (FC 11.15). Other over-expressed P450s belonged to the same cluster of P450s corresponding to the resistance to pyrethroid 1 (rp1) QTL on chromosome 2R. Several Epsilon class glutathione s-transferases were also over-expressed in FUMOZ, as was the ATP-binding cassette transporter AFUN019220 (ABCA) which also exhibited between-strain alternative splicing events at exon 7. Significant differences in SNP frequencies between strains occurred in resistance QTLs rp1 (CYP6P9a/b, CYP6AA1), rp2 on chromosome 2 L (CYP6Z1, CYP6M7, CYP6Z3) and rp3 on chromosome 3R (CYP9J5, CYP9J4 and CYP9J3). Differences were also detected in CYP4G17 and CYP4G16 genes on the X chromosome, both of which are associated with cuticular resistance in An. gambiae. A close analysis of nonsynonymous diversity at the CYP6P9a/b loci revealed a drastic loss of diversity in FUMOZ with only a single polymorphism and 2 haplotypes vs 18 substitutions and 8 haplotypes in FANG. By contrast, a lowly expressed cytochrome P450 (CYP4C36) did not exhibit diversity differences between strains. We also detected the known pyrethroid resistance conferring amino acid change N384S in CYP6P9b. This study further elucidates the molecular bases of resistance in An. funestus, informing strategies to better manage widespread resistance across Africa.

Role of methylation-related genes and microRNA revealed from RNA-Seq study of FHB susceptible hexaploid wheat NIL in response to pathogen stress

Wheat (Triticum aestivum L.), a most important crop among the cereals, accounts for over 20% of the total calories and protein available in human food. However, Fusarium graminearum, a devastating Fusarium head blight (FHB) pathogen of wheat, poses a greater threat to quality wheat grain production as it produces obnoxious mycotoxins. Presently, FHB is a minor disease in India but can cause significant yield and quality losses if rain occurs during mid-anthesis. In plants, epigenetics including DNA methylation and sRNA accumulation regulates gene expression and plays a definitive role in the adaptation to adverse environmental conditions. In a previous study response of wheat to F. graminearum was investigated through transcriptome analysis of lines differing for 2DL FHB resistance QTLs. However, a comprehensive analysis of methylation-related genes in response to pathogen was not performed in that study or any other studies. We re-analyzed the RNA-Seq data of said study to reveal the roles of methylation-related genes in response to F. graminearum. Sixteen methylation-related genes were down-regulated in a susceptible line, 2-2890 (pedigree of line). GO associated these genes with L-methionine salvage from methylthioadenosine and S-adenosylmethionine and steroid biosynthesis (p-value 0.001). Co-expression analysis of this NIL with methionine S-methyl-transferase gene (MSM; TraesCS1A02G013800) resulted in 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR; TraesCS5A02G269300). HMGCR was negatively correlated (-1.00) with genes encoding pathogenesis-related and detoxification proteins and xylanase inhibitors. GO associated these genes with methionine S-methyl transferase (p-value 0.001). Expression of HMGCR was higher (Log2 levels from 3.25 to 4.00) in pathogen inoculated compared to MSM (Log2 levels from 1.25 to 3.25) in mock-inoculated samples. 43 genes were down-regulated by miR9678. These genes are associated with responses to biotic stimulus and glucan endo-1,4-beta-glucanase in GO.

Identification of Root Rot Resistance QTLs in Pea Using Fusarium solani f. sp. pisi-Responsive Differentially Expressed Genes

Pisum sativum (pea) yields in the United States have declined significantly over the last decades, predominantly due to susceptibility to root rot diseases. One of the main causal agents of root rot is the fungus Fusarium solani f. sp. pisi (Fsp), leading to yield losses ranging from 15 to 60%. Determining and subsequently incorporating the genetic basis for resistance in new cultivars offers one of the best solutions to control this pathogen; however, no green-seeded pea cultivars with complete resistance to Fsp have been identified. To date, only partial levels of resistance to Fsp has been identified among pea genotypes. SNPs mined from Fsp-responsive differentially expressed genes (DEGs) identified in a preceding study were utilized to identify QTLs associated with Fsp resistance using composite interval mapping in two recombinant inbred line (RIL) populations segregating for partial root rot resistance. A total of 769 DEGs with single nucleotide polymorphisms (SNPs) were identified, and the putative SNPs were evaluated for being polymorphic across four partially resistant and four susceptible P. sativum genotypes. The SNPs with validated polymorphisms were used to screen two RIL populations using two phenotypic criteria: root disease severity and plant height. One QTL, WB.Fsp-Ps 5.1 that mapped to chromosome 5 explained 14.8% of the variance with a confidence interval of 10.4 cM. The other four QTLs located on chromosomes 2, 3, and 5, explained 5.3–8.1% of the variance. The use of SNPs derived from Fsp-responsive DEGs for QTL mapping proved to be an efficient way to identify molecular markers associated with Fsp resistance in pea. These QTLs are potential candidates for marker-assisted selection and gene pyramiding to obtain high levels of partial resistance in pea cultivars to combat root rot caused by Fsp.

Molecular Breeding Strategy and Challenges Towards Improvement of Downy Mildew Resistance in Cauliflower (Brassica oleracea var. botrytis L.)

Cauliflower (Brassica oleracea var. botrytis L.) is one of the important, nutritious and healthy vegetable crops grown and consumed worldwide. But its production is constrained by several destructive fungal diseases and most importantly, downy mildew leading to severe yield and quality losses. For sustainable cauliflower production, developing resistant varieties/hybrids with durable resistance against broad-spectrum of pathogens is the best strategy for a long term and reliable solution. Identification of novel resistant resources, knowledge of the genetics of resistance, mapping and cloning of resistance QTLs and identification of candidate genes would facilitate molecular breeding for disease resistance in cauliflower. Advent of next-generation sequencing technologies (NGS) and publishing of draft genome sequence of cauliflower has opened the flood gate for new possibilities to develop enormous amount of genomic resources leading to mapping and cloning of resistance QTLs. In cauliflower, several molecular breeding approaches such as QTL mapping, marker-assisted backcrossing, gene pyramiding have been carried out to develop new resistant cultivars. Marker-assisted selection (MAS) would be beneficial in improving the precision in the selection of improved cultivars against multiple pathogens. This comprehensive review emphasizes the fascinating recent advances made in the application of molecular breeding approach for resistance against an important pathogen; Downy Mildew (Hyaloperonospora parasitica) affecting cauliflower and Brassica oleracea crops and highlights the QTLs identified imparting resistance against this pathogen. We have also emphasized the critical research areas as future perspectives to bridge the gap between availability of genomic resources and its utility in identifying resistance genes/QTLs to breed downy mildew resistant cultivars. Additionally, we have also discussed the challenges and the way forward to realize the full potential of molecular breeding for downy mildew resistance by integrating marker technology with conventional breeding in the post-genomics era. All this information will undoubtedly provide new insights to the researchers in formulating future breeding strategies in cauliflower to develop durable resistant cultivars against the major pathogens in general and downy mildew in particular.