Cloning And Molecular Evolution Analysis Of NBS Class Resistance Gene Analogs From Four Species Of Bamboo Of Bambusoideae

China is the richest countries of bamboo resources, which are widely distributed.Sooty blotch、leaf spot blight and other diseases seriously affect its ornamentalcharacteristics and survival rate, resulting in economic losses. According to the conservedmotifs in the NBS type disease resistance genes, two pairs of primers are designed andused to clone resistance gene analogs from Phyllostachys nigra、Dendrocalamussemiscandens、Bambusa multiplex cv. Fernleef and Thyrsostachys siamensis. Thephylogenetic trees are built using the nucleotide sequences based on the NJ method. All thenucleotide sequences are divided into groups based on the differences of the geneticdistance. The domain value of genetic distance is greater than0.400. Detecting the positiveselection sites of the groups which contains more than3sequences.Six models which areM0、M1a、M2a、M3、M7、M8of the site models in software PAML are used to to explorethe selection pressure of RGAs in four types of bamboo. Gene conversion is detected byGENECONV. Preliminary study if the molecular clock hypothesis is existed by testing4kinds of bamboo RGA nucleotide sequences with ORF and pseudogenes.72NBS class RGAs with ORF are cloned From4kinds of bamboo in Bambusoideae,among them, Phyllostachys nigra contains33sequences, Dendrocalamus semiscandenscontains19sequences, Bambusa multiplex cv. Fernleef contains12sequences,Thyrsostachys siamensis contains8sequences.Sequence analysis showed that RGAscomprised the conserved domains P-loop, RNBS-A, Kinase-2a, Kinase-3a, HD, andRNBS-D, all of which are non-TIR-NBS type RGA.33RGAs in Phyllostachys nigra aredivided into10groups;19RGAs in Dendrocalamus semiscandens were divided into9groups;12RGAs in Bambusa multiplex cv. Fernleef were divided into8groups;8RGAsin Thyrsostachys siamensis were divided into5groups.The numbers of RGAs in differentgroups have significant difference. The result of selection pressure test shows that, NBSclass RGAs in Phyllostachys nigra and Dendrocalamus semiscandens are under positiveselection pressure.There is no statistically significant positive selection sites in Bambusamultiplex cv. Fernleef. There are10sequeces and12sites in Phyllostachys nigra,4sequeces and7sites in Dendrocalamus semiscanden,which detected statistically significantpositive selection effect, based on the BEB and LRT detecting.The results of positiveselection sites locating show that positive selection sites rarely appeared in the NBS conserved motifs. The results of purifying selected sites testing shows that, there are84purifying selected sites detected in Phyllostachys nigra group4and group5, among them16sites located in the NBS conserved motifs. There are no purifying selected sites detectedin other group. The results of gene conversion detection show that, gene conversion eventsare detected in group8、group10of Phyllostachys nigra and group4、group6ofDendrocalamus semiscandens. No clear correlation is found between positive selection andgene conversion.The sequences which undergo positive selection pressure, as well as geneconversion in Phyllostachys nigra and Dendrocalamus semiscandens, might be functionalresistance genes. The molecular clock hypothesis were rejected in RGA nucleotidesequences with ORF in4kinds of bamboos, but accepted in RGA pseudogene sequenceswhich under small selection pressure.