Biodiversity of Asian rice gall midge (Orseolia oryzae Wood Mason) from five countries examined by AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis was used to assess t he biodiversity of one of the most important dipteran pests of cereals, the Asian rice gall midge (Orseolia oryzae Wood Mason). Larvae and pupae were collected at 15 locations in five Asian countries and preserved in 95% etha nol for storage, shipment, and DNA extraction using cetyltrimethylammonium bromide (CTAB). Although only similar to 1 mu g of DNA was extracted from a single pupa or larva, the use of several AFLP primers in various combinati ons meant that this amount of DNA was sufficient to allow many DNA fingerpr ints to be made per individual. Fingerprints were sufficiently reproducible , especially during selective amplification, to allow the genetic diversity within a field population to be characterized. Extraction of DNA from a po ol of 20 insects yielded AFLP fingerprints in which variation among individ uals was sacrificed in favor of detecting differences among populations. Fo r each location, pooled DNA was amplified with three primer pairs. A total of 261 distinct AFLP bands were identified for the 45 fingerprints. Cluster analysis, performed by the unweighted pair-group method (UPGMA), separated the populations into two distinct groups. Group I included two populations from Guangdong province of southern China and one each from Laos and Impha l in northeastern India, while group II was comprised of eleven populations from elsewhere in India (Assam, Orissa, Madhya Pradesh, Andhra Pradesh, an d Kerala) and from Nepal and Sri Lanka. AFLP analysis provided insight into the origins of gall midge biotypes. In 1992, the prevailing biotype in Imp hal changed from Indian biotype 3 to a new biotype 3M. Our data show that b iotype 3M belongs to group I and did not arise by a recent mutation from bi otype 3, which belongs to group II. By contrast, Indian biotypes 2 and 4 ar e likely to have diverged through recent mutation and selection, as are Chi nese biotypes 1 and 4. The almost simultaneous emergence of new biotypes in Kerala and Sri Lanka during 1985-1988 was most probably coincidental, beca use these biotypes are not closely related. AFLP fingerprints were also abl e to detect sexual dimorphism in the DNA of adult gall midges and to distin guish gall midge from its major parasite Platygaster oryzae.