基于KASP技术的水稻高温抗性基因分子标记的开发OA

[Objective]High-temperature stress has become one of the critical abiotic stress factors restricting the safety of rice production.The development of efficient and accurate molecular markers is crucial for molecular marker-assisted selection(MAS)breeding for rice heat tolerance.This study aims to develop functional KASP markers closely linked to major genes conferring high-temperature resistance in rice,to provide a valuable reference for the identification and breeding of heat-tolerant rice varieties.[Method]SNP-based KASP markers were developed for six key rice thermotolerance genes(TT1,SLG1,SUS3,QT12,TT2,and TT3)through a process involving the alignment of sequence variations across multiple germplasms,utilizing public genomic databases(RiceVarMap v2.0 and MSU),and based on previously identified functional key SNPs.Multiple sets of KASP primers were designed for each gene locus.These primers were validated for marker usability in 16 rice germplasms(TT1,SLG1,SUS3)or 22 rice germplasms(QT12,TT2,TT3),respectively,to screen for the optimal primer combinations and conduct genotyping assays.[Result]Multiple KASP primer sets were designed for the functional SNPs of the six high-temperature resistance genes,and following validation with germplasm resources,the optimal primer combination for each gene was identified,enabling precise,efficient,and reliable discrimination of the target allelic variants.(1)The optimal primer combinations were TT1-F/TT1-R,SLG1-F/SLG1-R,SUS3-F/SUS3-R,QT12-F/QT12-R1,TT2-F/TT2-R2,and TT3.1-F/TT3.1-R2,which all produced clear cluster separation for genotyping the two allelic variants of their respective genes.(2)Genotyping results indicated that different rice varieties exhibited abundant genetic diversity at the six high-temperature resistance gene loci.Among the 16 germplasms,only one cultivated rice accession,ACC9,carried the favorable allele(A)of the TT1 gene;11 accessions(including heterozygotes)harbored the favorable allele(G)of SLG1;and 4 accessions possessed the favorable allele(T)of SUS3.Within the 22 germplasms,3 accessions carried the favorable allele(A)of QT12;6 accessions(including heterozygotes)contained the favorable allele(A)of TT2;while the favorable allele(T)of TT3.1 exhibited the highest distribution frequency,being present in 18 accessions.[Conclusion]This study developed KASP molecular markers for the six high-temperature rice resistance genes,which can be applied to the identification of germplasm resources and marker-assisted selection breeding of heat-tolerant rice varieties.