Progress 08/01/03 to 07/31/05
Outputs
Efforts supported by this grant produced a very high quality karyotype of the honey bee that is based on measurements of all morphologically distinct features of the chromosomes in 74 well-spread DAPI-stained haploid chromosomes prepared from testis in drone pupae of the sequenced (DH4 honey bee strain. Included are measurements of centromere positions, overall chromosome length, position of the ribosomal organizer regions and position and extent of AT rich, DAPI positive bands. The total length of the 16 chromosome haploid complement at meiotic pre-metaphase is 30.1 microns. Of this, 36 percent is relatively AT rich, DAPI-positive sequence that surrounds each centromere. The chromosomes are ordered by length, ranging in size from 3.48 to 1.16 microns, with chromosomes #2 - #13 similar, ranging from 1.5 and 2.5 microns in length. The morphology and order is different from that in earlier papers based on smaller data sets. Chromosomes 2, 5 and 7 agree with Beye &
Moritz. (1995. Heredity 86, 145-50); the designation of chromosomes 3, 9, 14, and 16 differ, while chromosome 8 fits characteristics of their chromosome 9. Beye & Moritz chromosomes #4 and #11 that hybridize with their rDNA probes match arm ratio, and DAPI banding of chromosomes 6 and 12, with the ribosomal signal on telomeric positions in both chromosomes. The BAC-FISH effort hybridized fluorescent labeled, large DNA fragments isolated from bacterial artificial chromosomes (BACs) to well spread mitotic and meiotic chromosome preparations as above. The chromosomal location of hybridized fluorescent BAC DNA sequence was used to anchor the BACs to a chromosome in the honey bee karyotype. Each BAC contained a known simple tandem repeat sequence marker (STS) whose position on the honey genetic map of M. Solignac was known (Solignac et al. 2004. Genetics 167, 253-62). Honey bee sequence contigs that contained one or more of these same STS markers were anchored to the genetic map as part of
the honey bee sequencing effort, integrating thereby the cytological, genetic and physical maps. All chromosomes (plus the NOR regions on chromosomes 6 and 12) were identified by BACFISH using one or more of the BACs. The exceptions are chromosomes 14 and 16 that both hybridize to the same BAC. The karyotype and BACFISH effort indicates that the relatively uniform distribution of centromeric AT rich bands across all chromosomes produces overall chromosome lengths that agree very well with the relative genetic map length of each chromosome. The only exceptions are linkage group 3, which FISH places on chromosome 2, and linkage group 2 that FISH places on chromosome 4. Thus linkage groups 1-16 to correspond to chromosomes 1, 4, 3, 2, 5-16 in the karyotype. A number of BACs hybridize to more than one chromosome, but, except for the assignment of chromosomes 2 and 4 to linkage groups 4 and 2, there have been no BAC combinations that fail to hybridize to the predicted chromosome in the
relative order predicted by the genetic map. Thus, there appears to be excellent chromosomal level synteny between the strains used in genetic mapping and the strain used to produce the assembled map sequences.
Impacts
This grant effort anchored the cytological map to the genetic maps and physical map of the completed honey bee genome project. This is an essential part of every sequencing project. The honey bee physical map and two available genetic maps were based on several different bees subspecies. Since the genetic maps were used to assemble sequence contigs into the final chromosomal physical maps of the completed honey bee genome, it was very important that this grant effort showed synteny of chromosome length and genetic map length and verified the use of the genetic map in the assembly process. Chromosomes 2 and 4 were assigned to linkage groups 4 and 2, while all other chromosomes were assigned to the corresponding genetic and physical chromosomal maps. The high resolution honey bee karyotype produced in this project showed centromere position, DAPI bright (AT rich) regions and the position of rDNA on chromosome 6 and 12, and updated the karyotypes from earlier studies.
This will facilitate future reverse genetics (sequence related back to gene function) in the honey bee.
Publications
- Robinson, G. et. al. 2006. The genome of a highly social species, the honey bee Apis mellifera. Nature issue devoted to the complete honey bee genomic sequence.
- Joseph J. Gillespie, J. J., J. S. Johnston, J. J. Cannone, R. R. Gutell. 2006. Characteristics of the nuclear (18S, 5.8S, 28S, and 5S) and mitochondrial (16S and 12S) rDNA genes of Apis mellifera (Insecta: Hymenoptera): Structure, conservation, organization, and retrotransposition. J. Insect Molecular Biology. Companion papers.
- Johnston, J. S. and G. Aquino-Perez. 2006. Karyotype and BACFISH integration of the cytological, genetic and physical honey bee maps. J. Insect Molecular Biology. Companion papers.
|
Progress 01/01/04 to 12/31/04
Outputs
The development of an integrated cytomolecular map was initiated by hiring research scientist, Mr. Gildarda Aqino-Perez. Mr. Aquino is an accomplished insect cytogenticist and was ideal for the project. In June, Mr. Aquino changed his status from Research Associate to Ph. D. candidate, which resulted in salary reductions and permitted the extension of the useful grant period. In the first year of the project, we greatly modified existing honey bee cytogenetic protocols, which resulted in greatly increased quality and quantity of honey bee drone (male) meiotic chromosome spreads. Our goal, to produce 5-8 well-separated complete spreads in each sweep across the slide at 1000X, was met. Following that, we began the process of matching at least one honey bee BAC to each chromosome. This was accomplished for nearly half the chromosomes, but was frustrated by the lack of morphological markers for different chromosomes, by hybridization to multiple chromosomes and by weak
fluorescence. To overcome these problems we initiated a series of tests to solve the various problems. Additional rounds of antibody amplification were found necessary to produce clear bright fluorescence. Extreme purity of the BAC DNA was found to reduce the spurious hybridization. Finally, familiarity with the differential condensation of meiotic chromosomes and of the DAPI bright bands in these chromesomes was found critical to correctly identify homologous chromosomes in different spreads. We tried to produce mitotic chromosomes, in the hopes they would have less condensation differences, but found (as have others) that the mitotic index is low in available tissues. With the resolution available from the improved FISH methods, we have begun to address differences in the genetic maps produced by the two primary recombination maps of Hunt and Solignac. This work is in progress and begun with discrepancies between chromosomes 1 and 12 in the physical and cytogenetic maps. The work
supported by this grant was presented at workshops on the honey bee genome at the International Congress of Genetics in Melborne AU 2003 and at the International Congress of Entomology in Brisbane 2004.
Impacts
The effort to date has anchored the cytological and genetic maps to the physical map from the completed honey bee genome project, an essential part of any complete genome effort. The next phase will resolve differences between the two major honey bee maps and the assembled honey bee physical map. For example, two chromosomes number 1 and 12 may contain pieces of each in the existing assembly. These conflicts need to be resolved in the final asssembly by the use of BAC-FISH.
Publications
- Aquino Pered, G. and J. S. Jphnston. 2005. Improved chromsomal preparations for BAC-FISH of Honey bee chromosomes. Submitted for internal review Aquino Pered, G. and J. S. Jphnston. 2005. BAC-FISH of Honey Bee chromosmes. Submitted for internal review
|
Progress 01/01/03 to 12/31/03
Outputs
The PI hired in August 2003 a skilled cytogeneticist, Guildardo Aquino-Perez who is trained and available for these studies. In September and November of 2003, the honey bee queen that produced the drones for the sequencing project was still alive and still producing drones. Since this would probably be the last chance to do so, we prepared an additional 500 slides from her drones. In the process, we developed a much-improved method for pachytene spreads that uses younger drones and combines portions of seven different protocols to produce exceptionally clear, well-spread chromosomes that are 5-8 times larger than those at metaphase. All 500 slides met the criteria we set up for excellent FISH preparations, which is that one pass across the preparation at 400X has at least 10 complete pachytene spreads with no chromosome overlap and no debris. Since good and plentiful spreads of the appropriate material are the single most essential step for BAC FISH, we consider the
slides made by the improved methods a breakthrough for this effort. The method and pachytene karyotype of the sequenced female with initial BAC/FSIH results are being written for submission to Chromosoma. With Co-PI, N. Islam-Faridi, we worked out the few problems we encountered when we first attempted BAC FISH in honey bees and have begun BAC FISH to anchor at least one mapped BAC onto each chromosome arm (as proposed), and expect to complete that work by the end of the current granting period and before the proposed starting date of this proposal. Three additional students from genetics/molecular biology are available in the laboratory of the PI to do BAC end sequencing and BAC DNA preparations as needed for this project.
Impacts
The effort to date has anchored the cytological and genetic maps to the physical map from the completed honey bee genome project, an essential part of any complete genome effort.
Publications
- Johnston, J. S. $ G. Aquino-Perez (2004) Improved cytological preparations for high resolution BAC FISH in the Honey bee,in preparation for Chromosoma
|