abstract
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Gene cloning is the basement of function analysis. However, isolation of new genes is still in deep water, especially for the complex genome, such as wheat. In the Yr27 and TaQsd1 clone study, the authors cloned the candidate gene based on the genome de novo assembly, which significantly accelerated the pace of gene cloning. For the Yr27 gene, though it is an allele of the leaf rust resistance gene Lr13, the result provides the chance of manipulation the sequence to create new R gene. We also cloned the Yr27 gene based on three populations. In our previous study, three QTLs (YrAc and YrNP63 in CIMMYT cultivar Yaco “S” and Napo63, YrSK99 in Chinese wheat line Shanken99) for stripe rust resistance were localized on chromosome 2BS. After fine mapping, we found YrAc, YrNP63 and YrSK99 shared 190 Kb physical interval. The interval contains three high confidence genes based on IWGSC RefSeq v1.0, encoding Cellulose synthase-like protein (TraesCS2B01G182700), NBS-LRR disease resistance protein (Lr13/Ne2) (TraesCS2B01G182800) and Ribonuclease (TraesCS2B01G182900), respectively. The VIGS confirmed that TraesCS2B01G182800 was the candidate Yr27. After comparing the Sanger sequence result, YrAc, YrNP63, YrSK99 and Yr27 were the same gene and exactly the same as reported Yr27 in Kariega. After inoculation at a different stage, the resistance of YrAc was related to the growth period of wheat. The resistance of YrSK99 and YrAc were influenced by genetic background. Sixteen haplotypes were identified for TraesCS2B01G182800 based on SNPs contained in wheat 660K SNP array. The Hap-5 (Yr27) was the excellent haplotype. The specific KASP marker of Yr27 was also developed and can be used in molecular assisted breeding. We also transferred Yr27 to ‘Fielder’ and edited this gene by CRISPR-Cas9. However, the results of stable transformation still need few months. In addition, the Lr13 transgene lines were treated by stripe rust races and they showed susceptible. To recover whether Yr27 confer the resistance to leaf rust, the virulence and avirulence race to Lr13 was challenged on Yr27 and the Yr27 line displayed susceptible. Different Yr27 alleles confer resistance to different pathogens. In the future, elucidating multiple recognition specificities of different alleles will provide insight into resistance mechanism.