Molecular characterization of eliminated chromosomes in Hessian fly (Mayetiola destructor (Say))

Abstract

Like other cecidomyiid Diptera, Hessian fly has stable S chromosomes and dispensable E chromosomes that are retained only in the germ line. Amplified fragment length polymorphisms (AFLP), suppressive subtractive hybridization (SSH), fluorescent in-situ hybridization (FISH), and sequencing were used to investigate similarities and differences between S and E chromosomes. More than 99.9% of AFLP bands were identical between separated ovary and somatic tissue, but one band was unique to ovary and resembled Worf, a non-LTR retrotransposon. Arrayed clones, derived by SSH of somatic from ovarian DNA, showed no clones that were unique to ovary. FISH with BAC clones revealed a diagnostic banding pattern of BAC positions on both autosomes and both sex chromosomes, and each E chromosome shared a pattern with one of the S chromosomes. Sequencing analysis showed that E chromosomes are nearly identical to S chromosomes, since no sequence could be confirmed to belong only to E chromosomes. There were a few questionably E-specific sequences that are candidates for further investigation. Thus, the E chromosomes appear to be derived from S chromosomes by the acquisition or conversion of sequences that produce the negatively heteropycnotic region around the centromere.

Keywords: DNA sequencing; Hessian fly; chromosome elimination; germline; supernumerary chromosome.

Fig. 1

Multiplexed FISH of polytene chromosomes in salivary glands using the chromosome-specific probes listed in Supplemental Table 1. Yellow signal is the nucleolar organizer region (NOR) on chromosome A1. Green signal is on the long arm of chromosome A2. Red signal is on the short arm of chromosome X1. Blue signal is on the short arm of chromosome X2. Bar represents 10 μm

Fig. 2

Above, metaphase of testis with 4 maternally derived S chromosomes (labeled s), 33 additional chromosomes with probe signals, and 5 additional chromosomes without signals. The probes and signal colors are the same as in Fig. 1. Below, the 37 chromosomes from above arranged by type of hybridization signal. The 5 chromosomes without signal were not included. Bar represents 10 μm

Fig. 3

Left, metaphase of testis with 4 maternally derived S chromosomes (labeled s) and 23 additional chromosomes. The probes and signal colors are the same as in Fig. 1. Right, all 27 chromosomes from left sorted by color of hybridization signal. Bar represents 10 μm

Fig. 4

Amplified fragment length polymorphism (AFLP) of ovarian (without superscript) and somatic (with superscript) DNA after digestion with different combinations of restriction endonucleases, E (EcoRI) and M (MseI), and selective bases. Red arrow denotes an extra band present only in ovaries. Leftmost lane is size markers. (A) E-AA/M-CAC, (B) E-TG/M-CAC, (C) E-AG/M-CTA, (D) E-AG/M-CAG (E) E-TG/M-CAG, (F) E-TG/M-CAT, (G) E-AC/M-CTA

Fig. 5

Autoradiograms of duplicate nylon macroarrays hybridized with ³²P-labeled ovarian DNA (left panel) or somatic DNA (right panel). Red arrows denote extra spots visible with the somatic DNA

Fig. 6

Ethidium-stained agarose gel with amplified product of primer pair HFE315 in ovarian DNA of four of seven families (left lanes of pairs) and not in head DNA (right lanes of pairs)

Fig. 7

Ethidium-stained agarose gel with amplified product of primer pair HFE174 in ovarian DNA of five of seven families and no lanes of head DNA