Detection of Blackleg Resistance Gene Rlm1 in Double-Low Rapeseed Accessions from Sichuan Province, by Kompetitive Allele-Specific PCR.
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ABSTRACT:
SUBMITTER: Chai L
PROVIDER: S-EPMC8053842 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Genetic resistance to blackleg (Leptosphaeria maculans, Lm) of canola (Brassica napus, Bn) has been extensively studied, but the mechanisms underlying the host-pathogen interaction are still not well understood. Here, a comparative transcriptome analysis was performed on a resistant doubled haploid Bn line carrying the resistance gene Rlm1 following inoculation with a virulent (avrLm1) or avirulent (AvrLm1) Lm isolate on cotyledons. A total of 6999 and 3015 differentially expressed genes (DEGs) were identified, respectively, in inoculated local tissues with compatible (susceptible) and incompatible (resistant) interactions. Functional enrichment analysis found several biological processes, including protein targeting to membrane, ribosome and negative regulation of programmed cell death, were over-represented exclusively among up-regulated DEGs in the resistant reaction, whereas significant enrichment of salicylic acid (SA) and jasmonic acid (JA) pathways observed for down-regulated DEGs occurred only in the susceptible reaction. A heat-map analysis showed that both biosynthesis and signaling of SA and JA were induced more significantly in the resistant reaction, implying that a threshold level of SA and JA signaling is required for the activation of Rlm1-mediated resistance. Co-expression network analysis revealed close correlation of a gene module with the resistance, involving DEGs regulating pathogen-associated molecular pattern recognition, JA signaling and transcriptional reprogramming. Substantially fewer DEGs were identified in mock-inoculated (control) cotyledons, relative to those in inoculated local tissues, including those involved in SA pathways potentially contributing to systemic acquired resistance (SAR). Pre-inoculation of cotyledon with either an avirulent or virulent Lm isolate, however, failed to induce SAR on remote tissues of same plant despite elevated SA and PR1 protein. This study provides insights into the molecular mechanism of Rlm1-mediated resistance to blackleg.
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Project description:BackgroundOwing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs.ResultsThe present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2*/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2*/Dx5) and weak-gluten (Null/Dx2) types also increased.ConclusionA cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.
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Project description:Temperate japonica rice varieties exhibit wide variation in the phenotypes of several important agronomic traits, including disease resistance, pre-harvest sprouting resistance, plant architecture, and grain quality, indicating the presence of genes contributing to favorable agronomic traits. However, gene mapping and molecular breeding has been hampered as a result of the low genetic diversity among cultivars and scarcity of polymorphic DNA markers. Single nucleotide polymorphism (SNP)-based kompetitive allele-specific PCR (KASP) markers allow high-throughput genotyping for marker-assisted selection and quantitative trait loci (QTL) mapping within closely related populations. Previously, we identified 740,566 SNPs and developed 771 KASP markers for Korean temperate japonica rice varieties. However, additional markers were needed to provide sufficient genome coverage to support breeding programs. In this study, the 740,566 SNPs were categorized according to their predicted impacts on gene function. The high-impact, moderate-impact, modifier, and low-impact groups contained 703 (0.1%), 20,179 (2.7%), 699,866 (94.5%), and 19,818 (2.7%) SNPs, respectively. A subset of 357 SNPs from the high-impact group was selected for initial KASP marker development, resulting in 283 polymorphic KASP markers. After incorporation of the 283 markers with the 771 existing markers in a physical map, additional markers were developed to fill genomic regions with large gaps between markers, and 171 polymorphic KASP markers were successfully developed from 284 SNPs. Overall, a set of 1225 KASP markers was produced. The markers were evenly distributed across the rice genome, with average marker density of 3.3 KASP markers per Mbp. The 1225 KASP markers will facilitate QTL/gene mapping and marker-assisted selection in temperate japonica rice breeding programs.
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Project description: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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