Genetic Diversity Of Zizania Latifolia Griseb(Turcz) And Functional Analysis Of ZlBBR1

Zizania latifolia is not only a natural genetic resource for rice breeding,but also plays a significant ecological role and has potential economic value.At present,the basic information,such as genetic diversity,growth condition,evolution and genetic structure of Z.latifolia is still not very clear.The evolutionary relationships of resistance genes between rice and Z.latifolia are barely understand.In addition,the progress of resistance gene cloning and utilization from Z.latifolia has been very slow.In this research,seven SSR markers derived from Z.latifolia were used for genetic diversity and genetic structure analysis of 26 wild populations from the Middle and East of China.ZlBBR1 was cloned from Z.latifolia according to reverse genetics method and the functional characterization of this gene was carried out.The homologous genes of ZlBBR1 were cloned from national samples of Z.latifolia and a set of rice germplasm resources based on homology cloning method.Bioinformatic analysis of these ZlBBR1 homologous genes were carried out and the main results are as follows:1.There was rich genetic diversity in the wild populations of Z.latifolia from the Middle-East China(PPB = 92.19%,Ao = 1.922,Ae = 1.454,He = 0.274,I = 0.422,Gst = 0.673)and the genetic similarity coefficient and genetic distance were between 0.5569~0.9981 and 0.0019~0.5854,respectively.The evolution analysis showed that there was no significant association between the phylogenetic relationship and the geographic locations in the Z.latifolia populations.26 populations were likely originated from a same ancestor and evolved into two different branches.2.The ZlBBR1 gene was cloned from Z.latifolia through reverse genetics method and the alleles of ZlBBR1 was cloned by homologous cloning method from nationwide samples.ZlBBR1 is higly conserved,there are only two alleles of ZlBBR1 at the amino acid level,i.e ZlBBR1-1 and ZlBBR1-2 which encode 1078 AA and 1080 AA,respectively.3.The ZlBBR1 gene which encodes an CC-NBS-LRR type R-protein was mainly expressed in the aerial part of Z.latifolia plant and its transient expression showed that ZlBBR1 protein was located in cell membranes.4.Driven by its own promoter,the transgenic ZlBBR1 plants showed significantly increased resistance to bacterial blight PXO71 in T0 and the pure line generations.However,ZlBBR1 overexpression plants did not show any enhanced resistance to PXO71.The SA content of three transgenic pure lines were significantly increased after 48 h inoculation compared to control materials,in contrast,the JA contents were significantly decreased.Upregulation of the pathogenesis-related genes and plant hormone signal transduction related gene were observed in L1 line 72 h after inoculation when compared to 0h;horizontally,after 72 h inoculation L1 line showed super oxide metabolism related genes significantly enriched compared to the same treatment in the control materials.Twelve pathogenesis-related genes and nine plant hormone signal transduction related gene were involved,of which seven genes belong to the TIFY family.5.There are mutations/SNPs in all domains of Os BBR1,of which most variations are presented within the NB-ARC domain and the least variations are identified in the coiled-coil domain.Os BBR1 gene does not follow the neutral evolution,and is likely due to limited ancestors of japonica subspecies domestication.These findings,on the one hand deepens our understanding about the genetic evolution of Z.latifolia in the Middle-East China and at the same time provides certain theoretical basis for protection of the wild rice germplasm resource,on the other hand provide new valuable materials for theoretical study of rice breeding,then deepen our knowledge of resistant gene evolution.