Fine mapping of Brassica napus blackleg resistance gene Rlm1 through bulked segregant RNA sequencing

Abstract The fungal pathogen Leptosphaeria maculans causes blackleg disease on canola and rapeseed (Brassica napus) in many parts of the world. A B. napus cultivar, ‘Quinta’, has been widely used for the classification of L. maculans into pathogenicity groups. In this study, we confirmed the presence of Rlm1 in a DH line (DH24288) derived from B. napus cultivar ‘Quinta’. Rlm1 was located on chromosome A07, between 13.07 to 22.11 Mb, using a BC1 population made from crosses of F1 plants of DH16516 (a susceptible line) x DH24288 with bulked segregant RNA Sequencing (BSR-Seq). Rlm1 was further fine mapped in a 100 kb region from 19.92 to 20.03 Mb in the BC1 population consisting of 1247 plants and a F2 population consisting of 3000 plants using SNP markers identified from BSR-Seq through Kompetitive Allele-Specific PCR (KASP). A potential resistance gene, BnA07G27460D, was identified in this Rlm1 region. BnA07G27460D encodes a serine/threonine dual specificity protein kinase, catalytic domain and is homologous to STN7 in predicted genes of B. rapa and B. oleracea, and A. thaliana. Robust SNP markers associated with Rlm1 were developed, which can assist in introgression of Rlm1 and confirm the presence of Rlm1 gene in canola breeding programs.

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Development of genic KASP SNP markers from RNA-Seq data for map-based cloning and marker-assisted selection in maize

BMC Plant Biology ◽

10.1186/s12870-021-02932-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Zhengjie Chen ◽

Dengguo Tang ◽

Jixing Ni ◽

Peng Li ◽

Le Wang ◽

...

Keyword(s):

Marker Assisted Selection ◽

Inbred Lines ◽

Average Density ◽

Snp Markers ◽

Data Sets ◽

Rna Seq ◽

Specific Pcr ◽

Maize Inbred Lines ◽

Allele Specific ◽

Allele Specific Pcr

Abstract Background Maize is one of the most important field crops in the world. Most of the key agronomic traits, including yield traits and plant architecture traits, are quantitative. Fine mapping of genes/ quantitative trait loci (QTL) influencing a key trait is essential for marker-assisted selection (MAS) in maize breeding. However, the SNP markers with high density and high polymorphism are lacking, especially kompetitive allele specific PCR (KASP) SNP markers that can be used for automatic genotyping. To date, a large volume of sequencing data has been produced by the next generation sequencing technology, which provides a good pool of SNP loci for development of SNP markers. In this study, we carried out a multi-step screening method to identify kompetitive allele specific PCR (KASP) SNP markers based on the RNA-Seq data sets of 368 maize inbred lines. Results A total of 2,948,985 SNPs were identified in the high-throughput RNA-Seq data sets with the average density of 1.4 SNP/kb. Of these, 71,311 KASP SNP markers (the average density of 34 KASP SNP/Mb) were developed based on the strict criteria: unique genomic region, bi-allelic, polymorphism information content (PIC) value ≥0.4, and conserved primer sequences, and were mapped on 16,161 genes. These 16,161 genes were annotated to 52 gene ontology (GO) terms, including most of primary and secondary metabolic pathways. Subsequently, the 50 KASP SNP markers with the PIC values ranging from 0.14 to 0.5 in 368 RNA-Seq data sets and with polymorphism between the maize inbred lines 1212 and B73 in in silico analysis were selected to experimentally validate the accuracy and polymorphism of SNPs, resulted in 46 SNPs (92.00%) showed polymorphism between the maize inbred lines 1212 and B73. Moreover, these 46 polymorphic SNPs were utilized to genotype the other 20 maize inbred lines, with all 46 SNPs showing polymorphism in the 20 maize inbred lines, and the PIC value of each SNP was 0.11 to 0.50 with an average of 0.35. The results suggested that the KASP SNP markers developed in this study were accurate and polymorphic. Conclusions These high-density polymorphic KASP SNP markers will be a valuable resource for map-based cloning of QTL/genes and marker-assisted selection in maize. Furthermore, the method used to develop SNP markers in maize can also be applied in other species.

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Detection of Blackleg Resistance Gene Rlm1 in Double-Low Rapeseed Accessions from Sichuan Province, by Kompetitive Allele-Specific PCR

The Plant Pathology Journal ◽

10.5423/ppj.oa.10.2020.0204 ◽

2021 ◽

Vol 37 (2) ◽

pp. 194-199

Author(s):

Liang Chai ◽

Jinfang Zhang ◽

Wannakuwattewaduge Gerard Dilantha Fernando ◽

Haojie Li ◽

Xiaoqin Huang ◽

...

Keyword(s):

Resistance Gene ◽

Sichuan Province ◽

Blackleg Resistance ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr

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Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. sylvestris

Genome ◽

10.1139/g07-103 ◽

2008 ◽

Vol 51 (1) ◽

pp. 64-72 ◽

Cited By ~ 56

Author(s):

Fengqun Yu ◽

Derek J. Lydiate ◽

S. Roger Rimmer

Keyword(s):

Brassica Napus ◽

Linkage Group ◽

Resistance Gene ◽

Leptosphaeria Maculans ◽

Resistance Locus ◽

Dominant Allele ◽

Breeding Lines ◽

Disease Reaction ◽

Blackleg Resistance ◽

Map Location

The spectrum of resistance to isolates of Leptosphaeria maculans and the map location of a new blackleg resistance gene found in the canola cultivar Brassica napus ‘Surpass 400’ are described. Two blackleg resistance genes, LepR1 and LepR2, from B. rapa subsp. sylvestris and introgressed in B. napus were identified previously. ‘Surpass 400’ also has blackleg resistance introgressed from B. rapa subsp. sylvestris. Using 31 diverse isolates of L. maculans, the disease reaction of ‘Surpass 400’ was compared with those of the resistant breeding lines AD9 (which contains LepR1), AD49 (which contains LepR2), and MC1-8 (which contains both LepR1 and LepR2). The disease reaction on ‘Surpass 400’ was different from those observed on AD9 and MC1-8, indicating that ‘Surpass 400’ carries neither LepR1 nor both LepR1 and LepR2 in combination. Disease reactions of ‘Surpass 400’ to most of the isolates tested were indistinguishable from those of AD49, which suggested ‘Surpass 400’ might contain LepR2 or a similar resistance gene. Classical genetic analysis of F1 and BC1 plants showed that a dominant allele conferred resistance to isolates of L. maculans in ‘Surpass 400’. The resistance gene, which mapped to B. napus linkage group N10 in an interval of 2.9 cM flanked by microsatellite markers sR12281a and sN2428Rb and 11.7 cM below LepR2, was designated LepR3. A 9 cM region of the B. napus genome containing LepR3 was found to be syntenic with a segment of Arabidopsis chromosome 5.

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Breakdown of a Brassica rapa subsp. sylvestris Single Dominant Blackleg Resistance Gene in B. napus Rapeseed by Leptosphaeria maculans Field Isolates in Australia

Plant Disease ◽

10.1094/pdis.2003.87.6.752a ◽

2003 ◽

Vol 87 (6) ◽

pp. 752-752 ◽

Cited By ~ 85

Author(s):

H. Li ◽

K. Sivasithamparam ◽

M. J. Barbetti

Keyword(s):

Brassica Napus ◽

Resistance Gene ◽

Disease Severity ◽

Brassica Rapa ◽

Oilseed Rape ◽

Leptosphaeria Maculans ◽

Blackleg Disease ◽

Field Isolates ◽

Western Australian ◽

Lower Stem

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus) grown in Canada, Europe, and Australia. Cv. Surpass 400 was released in Australia in 2000 as the most resistant cultivar to L. maculans. It carries a single dominant resistance gene from B. rapa subsp. sylvestris. This cultivar usually shows a hypersensitive response to L. maculans characterized by small, dark brown lesions that are necrotic, localized, and without pycnidia on cotyledons, leaves, and stems. However, in 2001 on a Western Australian experimental farm, a small proportion of the lesions on the lower stem and crown region of cv. Surpass 400 were typical of those observed in susceptible cultivars, which were brown, necrotic lesions with a darker margin, but they contained fewer pycnidia. Forty seedlings of cv. Surpass 400 and susceptible cv. Westar were inoculated with pycnidiospore suspensions (106/ml) of each of 18 isolates taken from lesions on cv. Surpass 400. All 18 isolates caused collapse of cotyledons of susceptible cv. Westar. Four of these isolates caused large cotyledon lesions with some pycnidia on cv. Surpass 400. Three of these four isolates were subsequently inoculated onto 60 seedlings per isolate, at each of the four cotyledon lobes of each seedling of the two cultivars. Inoculated plants were assessed for disease severity on cotyledons and transplanted to the field 14 days after inoculation. The cotyledons of inoculated cv. Surpass 400 showed characteristic large, necrotic lesions with pycnidia, while the cotyledons of cv. Westar had collapsed and contained a mass of pycnidia. Blackleg disease severity in the crown region of the stem was assessed at 2 weeks before harvest. Fifty-four percent of the cv. Surpass 400 transplanted inoculated plants subsequently developed susceptible symptoms of crown cankers on stems. These symptoms were deep, girdling, brown lesions on the plant crowns with some pycnidia. One hundred percent of cv. Westar plants were infected and dead at this stage. This confirmed the ability of these field isolates to overcome the single dominant resistance gene present in cv. Surpass 400. To our knowledge, this is the first report of breakdown of a single dominant B. rapa subsp. sylvestris gene based resistance to blackleg in oilseed rape in the field.

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Transcriptome sequencing assisted discovery and computational analysis of novel SNPs associated with flowering in Raphanus sativus in-bred lines for marker-assisted backcross breeding

Horticulture Research ◽

10.1038/s41438-019-0200-0 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Jinhee Kim ◽

Abinaya Manivannan ◽

Do-Sun Kim ◽

Eun-Su Lee ◽

Hye-Eun Lee

Keyword(s):

Genetic Selection ◽

Inbred Lines ◽

Snp Markers ◽

Functional Annotations ◽

Specific Pcr ◽

Polymorphic Snps ◽

Allele Specific ◽

Seed Purity Test ◽

Backcross Breeding ◽

Marker Assisted Backcross Breeding

Abstract The sequencing of radish genome aids in the better understanding and tailoring of traits associated with economic importance. In order to accelerate the genomics assisted breeding and genetic selection, transcriptomes of 33 radish inbred lines with diverse traits were sequenced for the development of single nucleotide polymorphic (SNP) markers. The sequence reads ranged from 2,560,543,741 bp to 20,039,688,139 bp with the GC (%) of 47.80–49.34 and phred quality score (Q30) of 96.47–97.54%. A total of 4951 polymorphic SNPs were identified among the accessions after stringent filtering and 298 SNPs with efficient marker assisted backcross breeding (MAB) markers were generated from the polymorphic SNPs. Further, functional annotations of SNPs revealed the effects and importance of the SNPs identified in the flowering process. The SNPs were predominantly associated with the four major flowering related transcription factors such as MYB, MADS box (AG), AP2/EREB, and bHLH. In addition, SNPs in the vital flowering integrator gene (FT) and floral repressors (EMBRYONIC FLOWER 1, 2, and FRIGIDA) were identified among the radish inbred lines. Further, 50 SNPs were randomly selected from 298 SNPs and validated using Kompetitive Allele Specific PCR genotyping system (KASP) in 102 radish inbred lines. The homozygosity of the inbred lines varied from 56 to 96% and the phylogenetic analysis resulted in the clustering of inbred lines into three subgroups. Taken together, the SNP markers identified in the present study can be utilized for the discrimination, seed purity test, and adjusting parental combinations for breeding in radish.

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A linked SNP marker to genotype Fr-B2 in wheat

Crop and Pasture Science ◽

10.1071/cp18248 ◽

2018 ◽

Vol 69 (9) ◽

pp. 859

Author(s):

H. A. Eagles ◽

J. Hyles ◽

Jayne Wilson ◽

Karen Cane ◽

K. L. Forrest ◽

...

Keyword(s):

Triticum Aestivum L ◽

Frost Tolerance ◽

Large Deletion ◽

Wheat Breeding ◽

Snp Markers ◽

Breeding Lines ◽

Specific Pcr ◽

Chromosome 5B ◽

Allele Specific ◽

Specific Pcr Assay

Fr-B2 is a complex locus on chromosome 5B that affects frost tolerance, days to heading, grain yield and probably other traits of commercial importance in wheat (Triticum aestivum L.). It interacts epistatically with other major genes, especially VRN1. There are two known alleles of Fr-B2: an intact, wild-type allele, and an allele with a large deletion. Published methods for identifying these alleles are slow and expensive, making the development of a high-throughput, co-dominant SNP (single-nucleotide polymorphism) marker highly desirable, especially for commercial wheat breeding. A diverse panel of cultivars and breeding lines was characterised for SNPs and alleles of Fr-B2. Four SNP markers co-segregated as a haplotype block with Fr-B2 across unrelated cultivars and related backcrosses differing for alleles of Fr-B2. A robust KASP (Kompetitive allele-specific PCR) assay was developed for one of the SNPs, KASP_IWB26333, which should facilitate the inclusion of Fr-B2 on genotyping platforms for breeding and research.

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Validation of SNP markers associated with ascochyta blight resistance in pea

Canadian Journal of Plant Science ◽

10.1139/cjps-2018-0211 ◽

2019 ◽

Vol 99 (2) ◽

pp. 243-249

Author(s):

Ambuj B. Jha ◽

Krishna K. Gali ◽

Sabine Banniza ◽

Thomas D. Warkentin

Keyword(s):

Association Studies ◽

Ascochyta Blight ◽

Snp Markers ◽

Blight Resistance ◽

Maturity Stage ◽

Single Nucleotide ◽

Specific Pcr ◽

Allele Specific ◽

Allele Specific Pcr ◽

Important Disease

Ascochyta blight of pea is an important disease that can cause severe yield loss. Our previous studies identified several closely linked single nucleotide polymorphism (SNP) markers associated with ascochyta blight resistance. The objective of this study was to validate SNP markers in 36 cultivars from the Saskatchewan pea regional variety trial. Ascochyta blight scores ranged from 1.0 to 9.0 at the physiological maturity stage under field conditions across the 25 site–years in Saskatchewan from 2013 to 2017. Based on Kompetitive Allele-Specific PCR assays, six SNP markers were used for an association study. SNP markers RGA-G3Ap103, PsC8780p118, and PsC22609p103 were significantly (P < 0.05) associated with ascochyta blight scores in 2013 and 2016 at Saskatoon. PsC8780p118 was significantly associated with ascochyta blight scores at Milden in 2014 and Rosthern in 2017. Furthermore, RGA-G3Ap103 showed significant association at Milden in 2014. Based on association studies, RGA-G3Ap103 and PsC8780p118 may have some potential as markers for pea breeding.

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Genetic mapping of the Leptosphaeria maculans avirulence gene corresponding to the LepR1 resistance gene of Brassica napus

Theoretical and Applied Genetics ◽

10.1007/s00122-011-1724-3 ◽

2011 ◽

Vol 124 (3) ◽

pp. 505-513 ◽

Cited By ~ 15

Author(s):

Kaveh Ghanbarnia ◽

Derek J. Lydiate ◽

S. Roger Rimmer ◽

Genyi Li ◽

H. Randy Kutcher ◽

...

Keyword(s):

Brassica Napus ◽

Genetic Mapping ◽

Resistance Gene ◽

Leptosphaeria Maculans ◽

Avirulence Gene

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Genetic Control and Host Range of Avirulence Toward Brassica napus Cultivars Quinta and Jet Neuf in Leptosphaeria maculans

Phytopathology ◽

10.1094/phyto.2001.91.1.70 ◽

2001 ◽

Vol 91 (1) ◽

pp. 70-76 ◽

Cited By ~ 99

Author(s):

M. H. Balesdent ◽

A. Attard ◽

D. Ansan-Melayah ◽

R. Delourme ◽

M. Renard ◽

...

Keyword(s):

Brassica Napus ◽

Resistance Gene ◽

Single Gene ◽

Leptosphaeria Maculans ◽

Avirulence Gene ◽

Gene Interactions ◽

Deleterious Gene ◽

Or Genes ◽

Avr Gene ◽

Avr Genes

Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 × avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance gene, termed Rlm4, present in the Quinta line analyzed and linked to Rlm1.

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