Whole-genome Sequencing And Genetic Diversity Of 'Candidatus Liberibacter Asiaticus'

Citrus Huanglongbing(HLB)is one of the most destructive diseases in citrus industry worldwide.Currently,HLB has been prevalent in more than fifty countries/areas distributed in Asia,Africa,South America,North America and Oceanica.Although the pathogens of HLB can not be cultured yet,three liberibacters,?Candidatus Liberibacter asiaticus?(Las),?Ca.L.africanus?(Laf)and ?Ca.L.americanus?(Lam),were found closely associated with HLB and were considered as HLB pathogens.Among the three liberibactes,Las was distributed most widely and was also the agent of HLB occurring in China.Although HLB was first reported in southern China one century ago,the complete genome of Las was still unknown.In this study,the whole genome of Las strain gxpsy from Guangxi,China was sequenced.Based on the complete genome,molecular markers with polymorphism were developed and applied for analysis of genetic diversity of Las population worldwide.Additionally,based on the unique sequence feature of a long tandem repeat in the genome of Las,a molecular methodology with high sensitivity was developed for the detection of Las.The main results were as follows:1.The first whole genome of Las from China(strain gxpsy)was successfully sequenced.The genome size of strain gxpsy was 1,268,237 bp.This genome comprised a G+C content of 36.5%,3 ribosomal RNA operons,44 tRNAs and 1,141 predicted coding sequences,368 of which were hypothetical genes.The number,component,distri-bution of genes in the genome of Las strain gxpsy were nearly identical to those in the genome of Las strain psy62 from Florida.However,in contrast to the psy62 genome,the genome of gxpsy was found to contain two tandem prophage regions.The prophage region located in nucleotide position from 1,223,783 to 1,263,670 in the gxpsy genome was highly similar to the only prophage region in psy62 genome with sequence identity of 94.4%.2.A phylogenetic tree was constructed using a cleaned and concatenated alignment of 50 orthologous proteins and covered 59 alphaproteobacterias whose whole genomes had been sequenced.The tree showed that in liberibacters evolution of the four uncultured liberibacters,including Las,Laf,Lam and ?Ca.L.solanacearum?(Lso)prevalent in some solanaceae crops,was relatively quick,while the evolution of liberibacter L.crescens(Lcr),the only culturable liberibacter known yet,was relatively slow.Additionally,Las showed the closest genetic relationship to Laf,then to Lso,Lam and Lcr,respectively.No obvious genetic differentiation was found between Las strains gxpsy and psy62 based on the phylogenetic analysis.3.Eight loci of variable-number tandem repeat(VNTR)were identified in Las genome,and were used for multilocus VNTR analysis(MLVA)of 436 Las isolates collected worldwide.The dendrogram from UPGMA clustering of the VNTR allele patterns obtained from MLVA showed the tested isolates could be divided into five genetic groups named as group A,B,C,D and E,respectively.Group A was composed of the majority of Florida isolates,some California isolates and all isolates from Texas,Mexico and Belize.Group B was composed of the majority of isolates from Southeast-Asian countries,some California isolates and one Indian isolate.All India isolates except one separately composed Group C.Two isolates from China composed Group D.Group E was composed of all Brazilian isolates and a few isolates from Florida and Southeast-Asian countries.These results indicated that two genetic groups of Las,A and E,were prevalent in Florida.According to the distribution of the individual of this two groups in Florida,it was inferred that Las of group A was mainly spreading northward along the east coast of Florida,while Las of group E was mainly spreading towards northwest inland of Florida.There were also two Las genetic groups,A and B,found in California.Additionally,results from MLVA showed that 12.8% HLB samples were infected by mixed strains of Las.Further analysis indicated that incidence of mixed-strain Las infection in an area was proportional to its HLB history.4.Two loci of insertion and deletion with large DNA fragment(LF-InDel),LF-InDel-3 and LF-InDel-4,were identified in Las genome.A total of 504 Las isolates collected worldwide were analyzed for this two loci.Results showed that in locus LF-InDel-3,DNA fragment deletion(DFD)-type Las was only found in North America and was predominant in its population in this area,While all Las from Asia and South America was DNA fragment insertion(DFI)-type.In locus LF-InDel-4,DFI-type Las group was predominant in its population in high altitude areas with relatively low annual average temperature,while DFD-type Las group was predominant in its population in areas with relatively high annual average temperature.Additionally,DFD in locus LF-InDel-3 would led to DFD event in locus LF-InDel-4,and DFI in locus LF-InDel-4 would led to DFI event in locus LF-InDel-3.5.Based on the study of tandem repeat(TR)in Las genome,a TR-based polymerase chain displacement reaction(TR-PCDR)method was developed for the detection of Las.A unique primer set was designed for TR-PCDR.The former primer was designed on tandem repeat region in Las genome,while the reverse primer was designed on conserved sequence region close to the selected tandem repeat region in Las genome.Primed with a thermostable Taq DNA polymerase mutant with strand displacement activity but lacking 5? to 3? exonuclease activity,TR-PCDR could produce more than two amplicons after each amplification cycle.Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment.The sensitive level was proven to be 100 times higher than conventional PCR and similar to real-time PCR.Data from the detection of Las with field samples using the above three methods also showed similar results.As TR-PCDR can be performed on any regular thermal cycler,application prospect for this methodology will be bright.