Saiful Islam, Jiana Li, Jie Zheng, Yingtao Zuo, Ying Zou, Tao Yu, Mohammad Ataur Rahman, Xianzhou Nie, Zhiguo Fang, Tao Luo, Botao Song, Bihua Nie
{"title":"利用优化的PCR和KASP标记提高四倍体马铃薯抗病基因Rx1和Rx2的检测效率","authors":"Saiful Islam, Jiana Li, Jie Zheng, Yingtao Zuo, Ying Zou, Tao Yu, Mohammad Ataur Rahman, Xianzhou Nie, Zhiguo Fang, Tao Luo, Botao Song, Bihua Nie","doi":"10.1016/j.hpj.2025.01.014","DOIUrl":null,"url":null,"abstract":"Potato virus X (PVX) seriously threatens global potato cultivation, necessitating a deeper understanding of the genetic mechanisms responsible for resistance. The <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes play a pivotal role in breeding potatoes with broad-spectrum PVX resistance. However, current DNA markers for these genes present uncertainties, as they are found in both resistant and susceptible materials, complicating the identification of the responsible resistance gene. This study aimed to develop robust high-throughput molecular markers, specifically Kompetitive Allele-Specific PCR (KASP) markers, to reliably detect the <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes. Genetic polymorphism analysis of seven resistant and four susceptible potato genotypes revealed seven single nucleotide polymorphisms (SNPs) and one insertion-deletion (Indel), along with corresponding amino acid changes in the Rx1 and Rx2 protein sequences. Based on these variations, high-throughput molecular markers were developed. Notably, the primer pairs Rx1-1R-F + Rx1-5R-R and Rx2-1R2-F + Rx2-2R2-R showed high accuracy, detecting <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes without false positives or false negatives. Optimized multiplex PCR protocols also demonstrated strong potential in simultaneously detecting both genes. KASP markers targeting SNPs at positions 2 515 (G to C) in the <ce:italic>Rx1</ce:italic> CDS and 2 445 (C to G) in the <ce:italic>Rx2</ce:italic> CDS efficiently differentiated between heterozygous resistant and homozygous susceptible genotypes. Furthermore, these markers effectively identified <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genotypes in 243 and 133 progenies across multiple crosses. Progenies that tested positive for <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> through PCR or KASP assays exhibited extreme resistance (ER) to PVX, while negative progeny was susceptible. These findings highlight the potential of the developed markers to accelerate the breeding cycle and improve the production of PVX-resistant potato varieties.","PeriodicalId":13178,"journal":{"name":"Horticultural Plant Journal","volume":"26 1","pages":""},"PeriodicalIF":6.2000,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Improving detection of potato virus X resistance genes Rx1 and Rx2 in tetraploid potato using optimized PCR and KASP markers\",\"authors\":\"Saiful Islam, Jiana Li, Jie Zheng, Yingtao Zuo, Ying Zou, Tao Yu, Mohammad Ataur Rahman, Xianzhou Nie, Zhiguo Fang, Tao Luo, Botao Song, Bihua Nie\",\"doi\":\"10.1016/j.hpj.2025.01.014\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Potato virus X (PVX) seriously threatens global potato cultivation, necessitating a deeper understanding of the genetic mechanisms responsible for resistance. The <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes play a pivotal role in breeding potatoes with broad-spectrum PVX resistance. However, current DNA markers for these genes present uncertainties, as they are found in both resistant and susceptible materials, complicating the identification of the responsible resistance gene. This study aimed to develop robust high-throughput molecular markers, specifically Kompetitive Allele-Specific PCR (KASP) markers, to reliably detect the <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes. Genetic polymorphism analysis of seven resistant and four susceptible potato genotypes revealed seven single nucleotide polymorphisms (SNPs) and one insertion-deletion (Indel), along with corresponding amino acid changes in the Rx1 and Rx2 protein sequences. Based on these variations, high-throughput molecular markers were developed. Notably, the primer pairs Rx1-1R-F + Rx1-5R-R and Rx2-1R2-F + Rx2-2R2-R showed high accuracy, detecting <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genes without false positives or false negatives. Optimized multiplex PCR protocols also demonstrated strong potential in simultaneously detecting both genes. KASP markers targeting SNPs at positions 2 515 (G to C) in the <ce:italic>Rx1</ce:italic> CDS and 2 445 (C to G) in the <ce:italic>Rx2</ce:italic> CDS efficiently differentiated between heterozygous resistant and homozygous susceptible genotypes. Furthermore, these markers effectively identified <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> genotypes in 243 and 133 progenies across multiple crosses. Progenies that tested positive for <ce:italic>Rx1</ce:italic> and <ce:italic>Rx2</ce:italic> through PCR or KASP assays exhibited extreme resistance (ER) to PVX, while negative progeny was susceptible. 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Improving detection of potato virus X resistance genes Rx1 and Rx2 in tetraploid potato using optimized PCR and KASP markers
Potato virus X (PVX) seriously threatens global potato cultivation, necessitating a deeper understanding of the genetic mechanisms responsible for resistance. The Rx1 and Rx2 genes play a pivotal role in breeding potatoes with broad-spectrum PVX resistance. However, current DNA markers for these genes present uncertainties, as they are found in both resistant and susceptible materials, complicating the identification of the responsible resistance gene. This study aimed to develop robust high-throughput molecular markers, specifically Kompetitive Allele-Specific PCR (KASP) markers, to reliably detect the Rx1 and Rx2 genes. Genetic polymorphism analysis of seven resistant and four susceptible potato genotypes revealed seven single nucleotide polymorphisms (SNPs) and one insertion-deletion (Indel), along with corresponding amino acid changes in the Rx1 and Rx2 protein sequences. Based on these variations, high-throughput molecular markers were developed. Notably, the primer pairs Rx1-1R-F + Rx1-5R-R and Rx2-1R2-F + Rx2-2R2-R showed high accuracy, detecting Rx1 and Rx2 genes without false positives or false negatives. Optimized multiplex PCR protocols also demonstrated strong potential in simultaneously detecting both genes. KASP markers targeting SNPs at positions 2 515 (G to C) in the Rx1 CDS and 2 445 (C to G) in the Rx2 CDS efficiently differentiated between heterozygous resistant and homozygous susceptible genotypes. Furthermore, these markers effectively identified Rx1 and Rx2 genotypes in 243 and 133 progenies across multiple crosses. Progenies that tested positive for Rx1 and Rx2 through PCR or KASP assays exhibited extreme resistance (ER) to PVX, while negative progeny was susceptible. These findings highlight the potential of the developed markers to accelerate the breeding cycle and improve the production of PVX-resistant potato varieties.
期刊介绍:
Horticultural Plant Journal (HPJ) is an OPEN ACCESS international journal. HPJ publishes research related to all horticultural plants, including fruits, vegetables, ornamental plants, tea plants, and medicinal plants, etc. The journal covers all aspects of horticultural crop sciences, including germplasm resources, genetics and breeding, tillage and cultivation, physiology and biochemistry, ecology, genomics, biotechnology, plant protection, postharvest processing, etc. Article types include Original research papers, Reviews, and Short communications.