Development Of Microsatellite Markers And Construction Of Genetic Linkage Map In Brown Planthopper

Brown planthopper (Nilaparvata lugens Stal)(Hemiptera:Delphacidae) is economically important pest on rice that feed directly or serve as vectors of pathogenic microorganisms and viruses to crops, resulting in significant damage and yield losses for farmers. In recent decades, brown planthopper has become notorious due to the emergence of new, virulent populations that can overcome host resistance. The variations in virulent population of brown planthopper are revealed by studying in molecular genetics. Molecular markers are useful tools for studying the distribution of genetic diversity and understanding population genetics. In this study, a larger number of microsatellite molecular markers were developed by using two different ways, and then they were used for assessing genetic diversity in populations of the brown planthopper and constructing genetic linkage map of N. lugens. Besides, we mapped the brown planthopper resistance gene Bph9of the rice variety Pokkali.To assess genetic diversity in populations of the brown planthopper, we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). Out of12,303sequences from UNKA (BPH) EST database,1,969SSRs were identified, accounting for an average density of one SSR per3.91kb EST sequences. The results indicate that the EST sequences of brown planthopper are rich in SSRs. In the SSRs identified, trinucloetide repeats were the most abundant among all five repeat types, accounting for67.39%. AAT and AG were the predominant trinucleotide and dinucleotide motifs, respectively, and GC-rich SSR motifs were less frequent. Three hundred and fifty one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri based on the cross-amplification success (92.3%). And then a subset of61polymorphic SSRs was selected for evaluating genetic diversity and population structure among individuals of four brown planthopper populations. It was clear that genetic diversity of biotype1feeding on susceptible rice TN1was lower than that of other three virulent populations feeding on resistant rice varieties. The results of unweighted pair group method with arithmetic means (UPGMA) dendrogram and the principal coordinates analysis (PCoA) showed that individuals from the same population were grouped into one cluster, and the genetic relationship among four populations was consistent with the differences of resistance of host rice varieties. The analysis of molecular variance (AMOVA) suggested that there was host resistance-based genetic differentiation among different brown planthopper populations.The enriched genomic library containing the microsatellite sequences was constructed by using the FIASCO (Fast Isolation by AFLP of Sequence COntaining Repeats) protocol, and then we developed the genomic microsatellites. In this study, the three of (AG)13,(AC)13and (AAG)8-enriched partial genomic libraries were constructed, and there were151sequences with microsatellites after sequencing and analyzing390clones, finally236primer pairs were developed. Of the96primers from (AG)13-enriched partial genomic library,80successfully yielded amplification products. As a result,17new developed markers showed polymorphism among the samples of natural population of N. lugens collected from Wuyishan and were used to assess genetic diversity. These markers revealed3to28alleles, with an average of17.3alleles per locus. The expected and observed heterozygosities ranged from0.157to0.959and from0.167to1.000, respectively. At the same time, all these markers can yielde amplification products in the closely related planthopper N. muiri, showing high cross-transferability level.The two F1families for mapping populations were established by crossing of both parernts, as which individuals of biotype1and individuals of hiotype2were served. The one population is consisting of154individuals, and the other is consisting of51individuals. Then, the336polymorphism SSR markers between both parernts of one or both F1populations were used for genotyping in the two mapping populations. The segregation data was analysed by using JoinMap4software and basing on the two way pseudo-testcross strategy. MapChart2.2software was used for graphical visualization of the linkage groups. In order to increase the resolution of genetic linkage map, linkage mapping was performed using tow F1outbred families, respectively, and a composite linkage map of N. lugens was generated by incorporating map information from the two families. On the TM6linkage map spanning813.4cM, there were265SSR markers and17linkage groups. The coverage of TM6map was86.4%. On the MT5linkage map spanning794.0cM, there were236SSR markers and16linkage groups. The coverage of MT5map was86.2%. A total of314markers were placed on the composite map, and14linkage groups were formed, which were consistent with the haploid autosome number of N. lugens. The composite map spaned797.8cM with an average spacing of2.5cM, the number of per group varied from2to60, and the average spacing of per group ranged from1.0cM to4.5cM.In the previous studies, the Bph9has been mapped on the long arm of chromosome12, whereas the reports about high resolution mapping or cloning of this gene were not found. In this study, we constructed an F2segregating population of crossing between resistence rice variety Pokkali and susceptible rice variety9311. The bulked seedling test method was used to evaluate the resistance of136F2.3families in Wuhan and Hainan, respectively and the scores of them were obtained. The resistant, moderate-resistant and susceptible families segregated in a1:2:1ratio (Wuhan:31:63:42,χc2=2.9<χ20.05,2=5.99; Hainan:42:60:34,χc2=3.1<χ20.05,2=5.99) in the F2population, indicating that a single dominant gene conferred the BPH-resistance in Pokkali. And then the linkage markers with the resistance gene were found in the rice genome by using bulked segregation analysis method. The partial linkage map of rice chromosome12was constracted and the QTL screening was carried out based on the genotype of each F2plant and the phenotype of corresponding F23families. Finally, the resistance gene Bph9was located on the region between SSR markers RM403and RM4557, and this region was0.3cM in genetic length and250kb in physical length (according to the sequence information of Nipponbare). The Bph9gene is explained77.8%of the phenotypic variance of BPH resistance in F2populations.