L. asiaticus’, it should be noted that broader population analyses using a larger array of molecular markers will help resolved the questions on the origin and dissemination of HLB-associated ‘Ca. L. asiaticus. Methods Sample collection/DNA extraction DNA from HLB-affected samples from Asia (India, China,
Cambodia, Vietnam, Thailand, Taiwan, and Japan), North America (Florida, USA) find more and South America (State of São Paulo, Brazil) were extracted from the respective sources and sent as microbially-sterile and non-infectious samples. HLB-associated Liberibacter-free DNA samples were used as negative controls. Basically, leaf samples were collected from citrus trees with blotchy mottle and blotchy mottle-like symptoms. Leaves were washed under running tap water and blotted dry with paper towels. The midribs were then excised from the leaf blade. Total genomic DNA was extracted from 4-5 midribs per sample. Pexidartinib order Samples were ground in liquid nitrogen and DNA was extracted using the CTAB method. Precipitated DNA was dissolved
in 100 μl of TE buffer. The quality of DNA samples was checked by electrophoresis in 1.2% agarose gels. DNA samples were diluted 30 times with water for PCR. Microsatellite marker development To identify putative microsatellite regions in the ‘Ca. L. asiaticus’ genome, we used the program ‘Tandem Repeats Finder’ . Following the identification of these regions, primers were designed (Eurofins-Operon) that flanked the prospective repeat sequence to generate a FK228 concentration product of 150-400 base pairs. Over 100 primer sets were tested using multiple DNA samples obtained from HLB-affected plants from India, China, Brazil and Florida. We postulated that polymorphisms,
if present, should be observed within this pilot sample due to their geographic separation. Following amplification of regions containing putative microsatellite using the test primers, the products of each reaction were then run on 5% of polyacrylamide gels. Silver staining was then used to visualize polymorphic alleles. This screening procedure identified seven loci with amplified sequence length variability. To facilitate high-throughput genotyping analysis, each of seven forward primers was labelled with a fluorescent Idoxuridine dye (Table 1). Amplified products were analyzed by an ABI 3130 xl Genetic Analyser (Applied Biosystems, Foster City, CA). PCR based genotyping PCR was performed in 20 μl containing 2 μl of 10× reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA), 2.5 pmole of each of SSR primer pairs and 2 μl of diluted DNA sample. PCR was conducted in the following conditions: 94°C for 4 minutes; 40 cycles consisting of 94°C for 45 seconds, annealing temperature (Table 1) for 45 seconds, and 72°C for 45 seconds; then a final extension at 72°C for 7 minutes. The successes of amplifications were checked running 5 μl of amplified products in agarose gel electrophoresis using 2.5% agarose-TBE gels.