Dataset Information

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

ABSTRACT: The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompetitive Allele Specific PCR (KASP), and six simple sequence repeat (SSR) markers, together with previously identified polymorphic markers, saturated the genetic linkage map for Pm61, especially in the proximal side of the target gene that was short of gene-linked markers. In the newly established genetic linkage map, Pm61 was located in a 0.71 cM genetic interval and can be detected in a high throughput scale by the KASP markers Xicsk8 and Xicsk13 or by the standard PCR-based markers Xicscx497 and Xicsx538. The newly saturated genetic linkage map will be useful in molecular marker assisted-selection of Pm61 in breeding for disease resistant cultivar and in its map-based cloning.

SUBMITTER: Hu J

PROVIDER: S-EPMC6387370 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

Hu Jinghuang J Li Jingting J Wu Peipei P Li Yahui Y Qiu Dan D Qu Yunfeng Y Xie Jingzhong J Zhang Hongjun H Yang Li L Fu Tiantian T Yu Yawei Y Li Mengjuan M Liu Hongwei H Zhu Tongquan T Zhou Yang Y Liu Zhiyong Z Li Hongjie H

International journal of molecular sciences 20190211 3

The gene Pm61 that confers powdery mildew resistance has been previously identified on chromosome arm 4AL in Chinese wheat landrace Xuxusanyuehuang (XXSYH). To facilitate the use of Pm61 in breeding practices, the bulked segregant analysis-RNA-Seq (BSR-Seq) analysis, in combination with the information on the Chinese Spring reference genome sequence, was performed in the F2:3 mapping population of XXSYH × Zhongzuo 9504. Two single nucleotide polymorphism (SNP), two Kompe ...[more]

PMID: 30754626

Similar Datasets

Project description:BackgroundWheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important disease affecting wheat production. Planting resistant cultivars is an effective, safe, and economical method to control the disease. Map construction using next-generation sequencing facilitates gene cloning based on genetic maps and high-throughput gene expression studies. In this study, specific-locus amplified fragment sequencing (SLAF) was used to analyze Huixianhong (female parent), Hongyoumai (male parent) and two bulks (50 homozygous resistant and 50 susceptible F2:3 segregating population derived from Huixianhong × Hongyoumai to determine a candidate gene region for resistance to powdery mildew on the long arm of chromosome 7B in wheat landrace Hongyoumai. Gene expressions of candidate regions were obtained using bulked segregant RNA-seq in 10 homozygous resistant and 10 susceptible progeny inoculated by Bgt.. Candidate genes were obtained using homology-based cloning in two parents.ResultsA 12.95 Mb long candidate region in chromosome 7BL was identified, and five blocks in SLAF matched the scaffold of the existing co-segregation marker Xmp1207. In the candidate region, 39 differentially expressed genes were identified using RNA-seq, including RGA4 (Wheat_Chr_Trans_newGene_16173)-a disease resistance protein whose expression was upregulated in the resistant pool at 16 h post inoculation with Bgt. Quantitative reverse transcription (qRT)-PCR was used to further verify the expression patterns in Wheat_Chr_Trans_newGene_16173 that were significantly different in the two parents Hongyoumai and Huixianhong. Two RGA4 genes were cloned based on the sequence of Wheat_Chr_Trans_newGene_16173, respectively from two parent and there was one amino acid mutation: S to G in Huixianhong on 510 loci.ConclusionThe combination of SLAF and BSR-seq methods identified a candidate region of pmHYM in the chromosome 7BL of wheat landrace cultivar Hongyoumai. Comparative analysis between the scaffold of co-segregating marker Xmp1207 and SLAF-seq showed five matching blocks. qRT-PCR showed that only the resistant gene Wheat_Chr_Trans_newGene_16173 was significantly upregulated in the resistant parent Hongyoumai after inoculation with Bgt, and gene cloning revealed a difference in one amino acid between the two parent genes, indicating it was involved in the resistance response and may be the candidate resistance gene pmHYM.

Project description:Powdery mildew resistance gene Pm4b, originating from Triticum persicum, is effective against the prevalent Blumeria graminis f. sp. tritici (Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification of Pm4b during the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-Seq technique and the bulked segregant analysis (BSR-Seq) was applied in an F2:3 mapping population (237 families) derived from a pair of isogenic lines VPM1/7∗Bainong 3217 F4 (carrying Pm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-Seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3 families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8 and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, 46 variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. Four Pm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking the Pm4b gene. Three SSR markers, Xics13, Xics43, and Xics76, were incorporated in the new genetic linkage map, which located Pm4b in a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a collinear relationship with Brachypodium distachyon chromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included C2 domain protein, peroxidase activity protein, protein kinases of PKc_like super family, Mlo family protein, and catalytic domain of the serine/threonine kinases (STKc_IRAK like super family). The markers developed in the present study facilitate identification of Pm4b during its MAS practice.

Project description:BackgroundMaize 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.ResultsA 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.ConclusionsThese 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.

Project description:Powdery mildew resistance gene Pm4b, originating from Triticum perscicum, is effective against the prevalent Blumeria graminis f. sp. tritici (Bgt) isolates from certain regions of wheat production in China. The lack of tightly linked molecular markers with the target gene prevents the precise identification of Pm4b during the application of molecular marker-assisted selection (MAS). The strategy that combines the RNA-seq technique and the bulked segregant analysis (BSR-seq) was applied in an F2:3 mapping population (237 families) derived from a pair of isogenic lines VPM1/7*Bainong 3217 F4 (carrying Pm4b) and Bainong 3217 to develop more closely linked molecular markers. RNA-seq analysis of the two phenotypically contrasting RNA bulks prepared from the representative F2:3 families generated 20,745,939 and 25,867,480 high-quality read pairs, and 82.8% and 80.2% of them were uniquely mapped to the wheat whole genome draft assembly for the resistant and susceptible RNA bulks, respectively. Variant calling identified 283,866 raw single nucleotide polymorphisms (SNPs) and InDels between the two bulks. The SNPs that were closely associated with the powdery mildew resistance were concentrated on chromosome 2AL. Among the 84 variants that were potentially associated with the disease resistance trait, forty-six variants were enriched in an about 25 Mb region at the distal end of chromosome arm 2AL. Four Pm4b-linked SNP markers were developed from these variants. Based on the sequences of Chinese Spring where these polymorphic SNPs were located, 98 SSR primer pairs were designed to develop distal markers flanking the Pm4b gene. Three markers, Xics13, Xics43 and Xics76, were incorporated in the new genetic linkage map, which located Pm4b in a 3.0 cM genetic interval spanning a 6.7 Mb physical genomic region. This region had a co-linear relationship with Brachypodium distachyum chromosome 5, rice chromosome 4, and sorghum chromosome 6. Seven genes associated with disease resistance were predicted in this collinear genomic region, which included protein kinases of PKc_like super family, C2 domain protein, peroxidase activity protein, Mlo family protein, and catalytic domain of the serine/threonine kinases (PKc_like super family). The markers developed in the present study facilitate identification of Pm4b during its MAS practice.

Dataset Information

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene Pm61.

Publications

Development of SNP, KASP, and SSR Markers by BSR-Seq Technology for Saturation of Genetic Linkage Map and Efficient Detection of Wheat Powdery Mildew Resistance Gene <i>Pm61</i>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets