The nuclear genome of the phytoseiid Metaseiulus occidentalis (Acari: Phytoseiidae) is among the smallest known in arthropods.
The genome size of the phytoseiid Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt) needs to be estimated before the whole nuclear genome can be sequenced. Two different procedures were used to estimate the genome size of M. occidentalis; (1) flow cytometry (Marescalchi et al. in Genome 33:789-793, 1990) and (2) quantitative real-time PCR (qRT-PCR) (Wilhelm et al. in Nucleic Acids Res 31:e56, 2003). Fluorescence intensity of propidium iodide-stained nuclei of M. occidentalis was measured by flow cytometry using females, males, and eggs. Only the eggs yielded peaks, which ranged in size from 35 to 160 Mb, with a tall peak of 140 Mb in 1-day-old eggs and 65 Mb in 2-day-old eggs, respectively. However, the peaks are broad and do not provide an accurate estimate. The qRT-PCR procedure required single-copy nuclear gene sequences from this phytoseiid. This was accomplished by designing degenerate primers, amplifying the Actin and EF1alpha sequences from M. occidentalis, and then designing M. occidentalis-specific primers that amplified a unique sequence. The standard qRT-PCR protocol was inefficient and amplification failed frequently, so we developed a high-fidelity qRT-PCR protocol, which utilizes a mix of two DNA polymerases (Taq and a proof-reading Tgo or ACCUZYME) to consistently amplify sequences. This allowed us to estimate the nuclear genome size of M. occidentalis as 88-90 +/- 5 Mb. When compared to other arthropod genomes, this appears to be very small.
Jeyaprakash
Ayyamperumal
A
Department of Entomology and Nematology, University of Florida, Gainesville, FL, 32611, USA. ajey@ifas.ufl.edu
Hoy
Marjorie A
MA
D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D003001:Cloning, Molecular; D005260:Female; D005434:Flow Cytometry; D016678:Genome; D023281:Genomics / Q000379:methods; D008297:Male; D008925:Mites / Q000235:genetics*; D010063:Ovum / Q000166:cytology; D016133:Polymerase Chain Reaction