Research

Development of Microsatellite DNA Markers and Their Use for Determination of Genetic Variablity
in Two Geographic Races of Anopheles maculatus Population in Thailand

ABSTRACT

The microsatellite DNA marker has been developed for distinguishing between the two races ( E and B) of An..maculatus Theobal 1901 s.s.mosquito that transmits malaria in Thailand. Dinucleotide and trinucleotide microsatellites were characterized from An. maculatus prior to their use as genetic markers. A partial genomic library of An. maculatus, consisting of 39,600 kb, was screened with either
(GT)n, (CT)n or (CCT)n synthetic oligonucleotide probes. Approximately 1.6% of recombinants contained sequences that hybridized to either (GT)12 or (CT)12 dinucleotide and (CCT)7 trinucleotide probes, suggesting that microsatellites are abundant in the genome of An. maculatus. Estimation of abundance of dinucleotide and trinucleotide repeats revealed that (GT)n/(CA)n microsatellites occurs on average every 68 kb, (CT)n/(GA)n repeats every 495 kb. and (CCT)n/(GGA)n repeats every 792 kb.

Of 38 putative clones were sequenced, 23 loci of dinucleotide repeats were found (20 loci were (GT)n/(CA)n repeats, 3 loci were (CT)n/(GA)n repeats) and microsatellite primers were constructed related to sequences that flanking dinucleotide repeat regions.

Four microsatellite markers were selected to perform PCR scoring for determination of genetic variability in two geographic races of An. maaculatus population; one from Uthai Thani province, and other from Narathiwat which were distinguished as races B and E, respectively, on the basis of ovarian polytene chromosome rearrangement. A high level of polymorphism was observed in all 4 microsatellite loci analyzed in both populations. The number of alleles detected at each locus ranged from 12 to 15 and the mean heterozygosity per locus was 0.591 (S.E.= 0.019) in the Uthai Thani population, and 0.451 (S.E = 0.060) in The Narathiwat population. A total of 39 alelles were found among four loci in the Uthai Thani population and 41 alleles in Narathiwat population. The mean number of alleles per locus in the Uthai Thani and Narathiwat were 9.75 nad 10.25, respectively. The large number of alleles and polymorphic nature resolved from microsatellite loci, including the ability to detect heterozygosity made these markers valuable for the study of population genetics

Microsatellite polymorphism in Anopheles maculatus, a malaria vector in Thailand.

Rongnoparut P, Yaicharoen R, Sirichotpakorn N, Rattanarithikul R, Lanzaro GC, Linthicum KJ.

Department of Entomology, United States Army Medical Component-Armed Forces Research Institute of Medical Sciences, (USAMC-AFRIMS), Bangkok, Thailand.Am J Trop Med Hyg 1996 Dec;55(6):589-94

ABSTRACT

Dinucleotide microsatellites were characterized from Anopheles maculatus, a species of mosquito that transmits malaria. A partial genomic library of An. maculatus, consisting of 3,960 kilobases (kb), was screened with either (GT)12 or (CT)12 probes. Approximately 1.5% of the recombinants contained sequences that hybridized to either (GT)12 or (CT)12 dinucleotide probes, suggesting that
microsatellites are abundant in the genome of An. maculatus. Estimation of abundance of the two dinucleotide repeats revealed that (GT)n or (CA)n microsatellites occur on average every 68 kb and (CT)n or (GA)n repeats every 495 kb. Among 23 microsatellite loci sequenced, four loci were selected to synthesize primers to perform polymerase chain reaction scoring for genetic polymorphism in a population of An. maculatus. A high level of polymorphism was observed with all four microsatellite loci analyzed. The number of alleles detected at each locus ranged from eight to 12 and the heterozygosities ranged from 0.25 to 0.54. A total of 42 alleles were found among four microsatellite loci. The large number of alleles and polymorphic nature resolved from microsatellite loci make these markers valuable for the study of population genetic structure and gene flow. Knowledge of gene flow is required to develop vector control strategies using genetic manipulations of malaria vector populations.

Estimates of gene flow among Anopheles maculatus populations in Thailand
using microsatellite analysis.

Rongnoparut P, Sirichotpakorn N, Rattanarithikul R, Yaicharoen R, Linthicum KJ
Am J Trop Med Hyg 1999 Mar 60:508-15

ABSTRACT

We report an analysis of seven microsatellite loci in eight populations of Anopheles maculatus mosquitoes dispersed over a distance of approximately 1,100 km in Thailand. A wide spectrum of genetic variability, with mean heterozygosities ranging from 0.738 to 0.847 were found. Based on microsatellite analysis, geographic populations of An. maculatus can be grouped into two clusters; one includes upper and lower northern populations that extend from approximately 11 degrees to 16 degrees north latitude, and the other (southern populations) extends south from about 7 degrees to 6 degrees north latitude. Wright's F(ST) and Slatkins's R(ST) for all seven microsatellite loci indicated low estimates of differentiation among all populations (mean values of F(ST) and R(ST) = 0.0406 and 0.051, respectively, corresponding to the Nm values of 5.91 and 4.65, respectively), and suggested that gene flow occurs among populations. However, there is some restriction of gene flow between the northern and southern populations. Geographic barriers could be limiting factors for greater gene flow between populations.