Identification Of The Loci And Development Of CAPS Marker For Freezing Tolerance In Common Wheat

The freezing damage seriously affects high yield and stable yield of wheat.Identification of the loci for freezing tolerance can contribute to clone candidate genes and provide useful information for understanding the molecular regulatory mechanism of freezing tolerance,also help to accelerate freezing tolerance breeding in wheat.In this study,147 wheat varieties with different level of freezing tolerance were used to detect the loci for freezing tolerance using association analysis,combining with the freezing tolerance phenotypes in the field investigated in 12/2015,1/2016 and 2/2016,and then CAPS markers were developed for freezing tolerance.The main results are as follows:(1)A total of 147 wheat varieties with different levels of freezing tolerance was genotyped using 460 pairs of SSR markers uniformly distributed on wheat 21 chromosomes,combining with freezing tolerance traits investigated in the field,11 SSR markers were significantly associated with freezing tolerance(P < 0.01)by both GLM and MLM methods,explaining 4.1-18.9% of phentopic variations.Particularly,3 loci were detected at three stages,and located on chromosomes 2B(wmc474),4A(wmc262)and 6B(wmc702).(2)Based on genetic backgrounds of 403 breeding materials collected in our group,125 varieties with different levels of freezing tolerance were to identify SNP markers associated with freezing tolerance by association analysis.In total,25015 SNP markers from Illumina wheat 90 K chip were used to genotype the panel.The results showed that 98 SNP markers were significantly associated with freezing tolerance(P < 0.01).In particular,SNP markers significantly associated with freezing tolerance at three stages were detected on chromosomes 1B(11),2A(19)and 7A(22),explaining 8.8-18.0% of the phenotypic variations,indicating that the three chromosomes maybe harbor the major loci for freezing tolerance.(3)According to SNP markers information identified here,the SNP wsnp?BG605368A?Ta?2?4 on chromosome 2A was further transformed to the CAPS marker,designated CAPS-Ta-2-4.Using 2.0% agarose gel electrophoresis,our results indicated that varieties carrying the A allele was amplified the fragment length of 185 bp and the G allele for the 70 bp product through the CAPS-Ta-2-4 primer.In the natural population consisted of 403 breeding materials,there was a significant difference in freezing tolerance between the two alleles of CAPS-Ta-2-4(P<0.01),the allele A was related to strong freezing tolerance,while the G was for weak freezing tolerance.(4)The SNP mutation significantly related to freezing tolerance occurred in the gene encoding polygalacturonase.In order to further verify the relationship between the gene expression and freezing tolerance based on the transcription level,three varieties carrying the allele A with strong freezing tolerance and three varieties carrying the allele G with weak freezing tolerance were used to investigate the expression levels of the target gene in different treatments.After growing at 20 ?to the third leaf stage,they were exercised at 4 for 7d,then transferred to ?-5 for 10 hours to 20 hours,and then transferred to ?-9 ?for 10 hours to 20 hours.The results showed that the relative expression levels of the polygalacturonase gene in varieties with strong freezing tolerance was significantly lower than that in varieties with weak freezing tolerance,indicating that the gene negatively regulated the freezing tolerance of wheat.