| The nucleotide-binding site (NBS) disease-resistance genes are the largest category inthe plant disease resistance gene (R gene).Through the analysis of same domain class ofthe R genes, the disease resistance gene analogs (RGA) could be found and verified, whichlay a foundation and safeguards for future studing the mechanisms of plant diseaseresistance. The complete sets of disease-resistance candidate genes, which encode the NBSsequence, were filtered in the genome of two varieties of foxtail millet (Yugu No.1andZhanggu). According to the statistical analysis by geNorm, NormFinder, and BestKeepersoftwares, selected of the optimal reference genes, which were used gene expression inQ-PCR, from6reference genes,25S rRNA,18S rRNA,17S rRNA, GAPDH, ACTB andUBQ, in foxtail millet. The main results were summarized as followings:1.269NBS-type resistance gene analogs (RGAs) in Yugu No.1and281NBS-typeresistance gene analogs in Zhanggu were obtained by the method of homologous sequencealignment. To blast the obtained RGAs, this study identified72identical genes and164genes with more than90%similarity between in Yugu No.1and Zhanggu.2. RGAs were divided into six categories: CC-NBS, CC-NBS-LRR, NBS, NBS-LRR,TIR-NBS and Others. The number of the CC-NBS-LRR type RGAs was most commontype and the number of the TIR-NBS class RGAs was mini-mum in both foxtail milletvarieties used. The numbers were87and81, accounted for35%and29%of the totalnumber RGAs, respectively. The Yugu No.1RGAs had only two TIR-NBS type RGAs, butthe Zhanggu RGAs contained all the categories except the TIR-NBS type.3. The chromosome distributions of the identified NBS type RGAs in Yugu No.1RGAs showed that chromosome8contained the largest number of the NBS genes, It wasalso interesting that chromosome9was the longest chromosome in both the Zhanggu andYugu No.1RGAs, but it contained the lowest number of NBS-coding sequences. Whilecomparing these chromosomes in the two RGAs, it can be seen that most genes were in thesame position but few of genes were end to end. In Yugu No.1RGAs, it was found that34gene clusters contained a total number of161genes. In Zhanggu RGAs, it was found that28gene clusters contained a total number of151genes. The largest gene cluster contained12and16genes and both located on chromosome8in Yugu No.1and Zhanggu RGAs,respectively. 4. The goal of this study was to determine the optimal reference gene for RT-qPCRanalysis of gene expression in different varieties and different tissues of foxtail millet. Weanalyzed6candidate reference genes and found that25S rRNA was the best choice fornormalization between leaf samples from7different varieties, if only one reference genewas required; however, for greater accuracy, a combination of18S rRNA and25S rRNAshould be considered. Our results also suggested that18S rRNA would be an appropriatereference gene to normalize expression in Yugu No.1leaves infected by rust, as well as indifferent tissues of Yugu No.1, although18S rRNA was not necessarily the most stablereference gene in these experiments.5. Analysis on NBS-1(NBS type RGAs) and NBS-2(NBS-LRR type RGAs)expression by Q-PCR，NBS-1expression pattern was very similar to that of NBS-2in bothof resistant variety (Shilixiang) and susceptible variety (Yugu No.1). They were firstly bothdecreased significantly after connect rust. Subsequently, they were decreased significantly.And expression levels were significantly higher in the resistant varieties than that insusceptible varieties. So NBS-1and NBS-2genes were foxtail millet Rust resistance relatedgene. |
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