It is imperative to identify highly polymorphic and tightly linked markers of a known trait for molecular marker-assisted selection. Potyvirus resistance 4 (Pvr4) locus in pepper confers resistance to three pathotypes of potato virus Y and to pepper mottle virus. We describe the use of next-generation sequencing technology to generate molecular markers tightly linked to Pvr4. Initially, comparative genomics was carried out, and a syntenic region of tomato on chromosome ten was used to generate PCR-based markers and map Pvr4. Subsequently, the genomic sequence of pepper was used, and more than 5000 single-nucleotide variants (SNVs) were identified within the interval. In addition, we identified nucleotide binding site–leucine-rich repeat-type disease resistance genes within the interval. Several of these SNVs were converted to molecular markers desirable for large-scale molecular breeding programmes. Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0294-5) contains supplementary material, which is available to authorized users.
Modern plant breeding heavily relies on the use of molecular markers. In recent years, next generation sequencing (NGS) emerged as a powerful technology to discover DNA sequence polymorphisms and generate molecular markers very rapidly and cost effectively, accelerating the plant breeding programmes. A single dominant locus, Frl, in tomato provides resistance to the fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL), causative agent of Fusarium crown and root rot. In this study, we describe the generation of molecular markers associated with the Frl locus. An F2 mapping population between an FORL resistant and a susceptible cultivar was generated. NGS technology was then used to sequence the genomes of a susceptible and a resistant parent as well the genomes of bulked resistant and susceptible F2 lines. We zoomed into the Frl locus and mapped the locus to a 900 kb interval on chromosome 9. Polymorphic single-nucleotide polymorphisms (SNPs) within the interval were identified and markers co-segregating with the resistant phenotype were generated. Some of these markers were tested successfully with commercial tomato varieties indicating that they can be used for marker-assisted selection in large-scale breeding programmes.Electronic supplementary materialThe online version of this article (10.1007/s00122-018-3136-0) contains supplementary material, which is available to authorized users.
During a study of Sclerotinia sclerotiorum populations on Ranunculus acris (meadow buttercup) in the UK, dead and dying flowers collected from plants in a Herefordshire meadow were incubated at 20°C in damp conditions. Fungal growth typical of S. sclerotiorum (white fluffy mycelium ⁄ sclerotia) emerging from individual flowers was transferred to potato dextrose agar. DNA was extracted from cultures and the ITS regions of the rDNA amplified and sequenced. BLAST analysis showed that of 32 isolates selected at random, only 17 were S. sclerotiorum while 15 were identified as S. subarctica nom. prov. (= Sclerotinia species 1; HolstJensen et al., 1998;Winton et al., 2006Winton et al., , 2007. All 15 S. subarctica ITS sequences were identical (GenBank Accession No. GU018183). The identity of the S. subarctica isolates was further confirmed by the lack of an intron in the LSU rDNA compared with S. sclerotiorum (Holst-Jensen et al., 1998).A formal description and host range for S. subarctica has yet to be published but it is reported to be morphologically indistinguishable from S. sclerotiorum (Holst-Jensen et al., 1998). However, the cultures of S. subarctica in this study produced fewer but larger sclerotia in vitro (ca. 5AE8 ± 1AE0 mm diameter) than S. sclerotiorum (ca. 4AE1 ± 0AE9 mm). Sclerotinia subarctica has only been reported on wild hosts in Norway (Holst-Jensen et al., 1998) and causing white mould disease on vegetable crops in Alaska (as does S. sclerotiorum; Winton et al., 2006). As oilseed rape is the major crop host of S. sclerotiorum in the UK, its susceptibility to S. subarctica was tested by inoculation of plants and detached leaves. Stem and leaf lesions were identical to those caused by S. sclerotiorum although stem lesions of S. subarctica were slower to develop. This is the first time that infection of oilseed rape by S. subarctica has been demonstrated. The presence of S. subarctica in the UK indicates that this pathogen is not confined to the 'High North' as suggested previously (Winton et al., 2007). Significantly, as S. subarctica causes symptoms very similar to S. sclerotiorum, it may currently be undetected in white mould affected crops in the UK and hence further work to establish its distribution on crops and wild hosts is merited. The presence of S. subarctica (and S. sclerotiorum) on R. acris also suggests that this wild host is a reservoir of inoculum for crop plants.
Cucumber is a widely grown vegetable crop plant and a host to many different plant pathogens. Cucumber vein yellowing virus (CVYV) causes economic losses on cucumber crops in Mediterranean countries and in some part of India such as West Bengal and in African countries such as Sudan. CVYV is an RNA potyvirus transmitted mechanically and by whitefly (Bemisia tabaci) in a semipersistent manner. Control of this virus is heavily dependent on the management of the insect vector and breeding virus-resistant lines. DNA markers have been used widely in conventional plant breeding programs via marker-assisted selection (MAS). However, very few resistance sources against CVYV in cucumber exist, and also the lack of tightly linked molecular markers to these sources restricts the rapid generation of resistant lines. In this work, we used genomics coupled with the bulked segregant analysis method and generated the MAS-friendly Kompetitive allele specific PCR (KASP) markers suitable for CsCvy-1 selection in cucumber breeding using a segregating F2 mapping population and commercial plant lines. Variant analysis was performed to generate single-nucleotide polymorphism (SNP)-based markers for mapping the population and genotyping the commercial lines. We fine-mapped the region by generating new markers down to 101 kb with eight genes. We provided SNP data for this interval, which could be useful for breeding programs and cloning the candidate genes.
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