Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

Evolution and arrangement of the <Emphasis Type="Italic">hsp70</Emphasis> gene cluster in two closely related species of the <Emphasis Type="Italic">virilis</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.     

Differential Under-replication of Satellite DNAs during <Emphasis Type="Italic">Drosophila</Emphasis> Development

SATELLITE DNAs are heavily concentrated in the centromeric heterochromatin of metaphase chromosomes^1–3. Satellites and other repeated polynucleotide sequences are under-represented in the polytene, salivary gland cells of Drosophila melanogaster, D. virilis and D. hydei larvae but are fully represented in diploid cells from embryos and imaginal disks^4–6. This under-representation in polytene cells stems from the association of heterochromatin in the chromocentre and the progressive under-replication of the chromocentre during larval development^7,8.     

The polytene dot chromosome of <Emphasis Type="Italic">Drosophila</Emphasis>: <Emphasis Type="Italic">D. melanogaster</Emphasis> and <Emphasis Type="Italic">D. subobscura</Emphasis>

The chromosome arms are assumed to be homologous within the genus Drosophila. Homology at the level of the polytene chromosome banding pattern between non-sibling species is, however, almost impossible to establish as different processes such as inversion, transposition and unequal crossing over, have disturbed it. Even though the band sequences cannot be followed, we may ask whether there is a correlation in the total number of bands between species. The polytene dot chromosome is an excellent starting point for such an approach. Here we present the detailed cytology of polytene chromosome 4 of D. melanogasterand the polytene dot chromosome of D. subobscura using electron microscopy. The results show that the number of bands is about the same, around 30, in both species. We predict that by using thin sections and electron microscopy for the longer polytene chromosome arms, both species will turn out to have approximately equal band numbers.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Gene <Emphasis Type="Italic">RAD31</Emphasis> is identical to gene <Emphasis Type="Italic">MEC1</Emphasis> of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The earlier identified gene RAD31 was mapped on the right arm of chromosome II in the region of gene MEC1 localization. Epistatic analysis demonstrated that the rad31 mutation is an allele of the MEC1 gene, which allows further designation of the rad31 mutation as mec1-212. Mutation mec1-212, similar to deletion alleles of this gene, causes sensitivity to hydroxyurea, disturbs the check-point function, and suppresses UV-induced mutagenesis. However, this mutation significantly increases the frequency of spontaneous canavanine-resistance mutations induced by disturbances in correcting errors of DNA replication and repair, which distinguishes it from all identified alleles of gene MEC1.     

Cytogenetic analysis of the Ethiopian fruit fly <Emphasis Type="Italic">Dacus ciliatus</Emphasis> (Diptera: Tephritidae)

The Ethiopian fruit fly, Dacus ciliatus, is an important pest of cucurbits, which recently invaded the Middle East. The genetics and cytogenetics of D. ciliatus have been scarcely studied. Such information is, however, an essential basis for understanding the biology of insect pests, as well as for the design of modern control strategies. We report here the mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this species. The mitotic metaphase complement consists of six pairs of chromosomes, including one pair of heteromorphic sex (XX/XY) chromosomes. The heterogametic sex is ascribed to the male. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei, and a heterochromatic mass corresponding to the sex chromosomes. Banding patterns, as well as the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between D. ciliatus and Bactrocera oleae are proposed by comparing chromosome banding patterns and by in situ hybridization of the hsp70 gene.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

Exploiting co-linearity among grass species to map the <Emphasis Type="Italic">Aegilops ventricosa-</Emphasis>derived <Emphasis Type="Italic">Pch1</Emphasis> eyespot resistance in wheat and establish its relationship to <Emphasis Type="Italic">Pch2</Emphasis>

Introgressions into wheat from related species have been widely used as a source of agronomically beneficial traits. One such example is the introduction of the potent eyespot resistance gene Pch1 from the wild relative Aegilops ventricosa onto chromosome 7DL of wheat. In common with genes carried on many other such introgressions, the use of Pch1 in commercial wheat varieties has been hindered by linkage drag with yield-limiting traits. Attempts to break this linkage have been frustrated by a lack of co-dominant PCR markers suitable for identifying heterozygotes in F₂ populations. We developed conserved orthologous sequence (COS) markers, utilising the Brachypodium distachyon (Brachypodium) genome sequence, to provide co-dominant markers in the Pch1 region. These were supplemented with previously developed sequence-tagged site (STS) markers and simple sequence repeat (SSR) markers. Markers were applied to a panel of varieties and to a BC₆ F₂ population, segregating between wheat and Ae. ventricosa over the distal portion of 7DL, to identify recombinants in the region of Pch1. By exploiting co-linearity between wheat chromosome 7D, Brachypodium chromosome 1, rice chromosome 6 and sorghum chromosome 10, Pch1 was located to an interval between the flanking markers Xwg7S and Xcos7-9. Furthermore candidate gene regions were identified in Brachypodium (364 Kb), rice (178 Kb) and sorghum (315 Kb) as a prelude to the map-based cloning of the gene. In addition, using homoeologue transferable markers, we obtained evidence that the eyespot resistances Pch1 and Pch2 on chromosomes 7D and 7A, respectively, are potentially homoeoloci. It is anticipated that the COS marker methodology could be used for the identification of recombinants in other introgressions into wheat from wild relatives. This would assist the mapping of genes of interest and the breaking of deleterious linkages to enable greater use of these introgressions in commercial varieties.     

A <Emphasis Type="Italic">Pseudo-Response Regulator</Emphasis> is misexpressed in the photoperiod insensitive <Emphasis Type="Italic">Ppd-D1a</Emphasis> mutant of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the “green revolution” converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a ‘Chinese Spring’ wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Beales and A. Turner contributed equally to the work.     

In vivo analysis of <Emphasis Type="Italic">Drosophila</Emphasis> SU(<Emphasis Type="Italic">Z</Emphasis>)12 function

Polycomb group (PcG) proteins are required to maintain a stable repression of the homeotic genes during Drosophila development. Mutants in the PcG gene Supressor of zeste 12 (Su(z)12) exhibit strong homeotic transformations caused by widespread misexpression of several homeotic genes in embryos and larvae. Su(z)12 has also been suggested to be involved in position effect variegation and in regulation of the white gene expression in combination with zeste. To elucidate whether SU(Z)12 has any such direct functions we investigated the binding pattern to polytene chromosomes and compared the localization to other proteins. We found that SU(Z)12 binds to about 90 specific eukaryotic sites, however, not the white locus. We also find staining at the chromocenter and the nucleolus. The binding along chromosome arms is mostly in interbands and these sites correlate precisely with those of Enhancer-of-zeste and other components of the PRC2 silencing complex. This implies that SU(Z)12 mainly exists in complex with PRC2. Comparisons with other PcG protein-binding patterns reveal extensive overlap. However, SU(Z)12 binding sites and histone 3 trimethylated lysine 27 residues (3meK27 H3) do not correlate that well. Still, we show that Su(z)12 is essential for tri-methylation of the lysine 27 residue of histone H3 in vivo, and that overexpression of SU(Z)12 in somatic clones results in higher levels of histone methylation, indicating that SU(Z)12 is rate limiting for the enzymatic activity of PRC2. In addition, we analyzed the binding pattern of Heterochromatin Protein 1 (HP1) and found that SU(Z)12 and HP1 do not co-localize.     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Inheritance and mapping of powdery mildew resistance gene <Emphasis Type="Italic">Pm43</Emphasis> introgressed from <Emphasis Type="Italic">Thinopyrum</Emphasis><Emphasis Type="Italic">intermedium</Emphasis> into wheat

Powdery mildew resistance from Thinopyrum intermedium was introgressed into common wheat (Triticum aestivum L.). Genetic analysis of the F₁, F₂, F₃ and BC₁ populations from powdery mildew resistant line CH5025 revealed that resistance was controlled by a single dominant allele. The gene responsible for powdery mildew resistance was mapped by the linkage analysis of a segregating F₂ population. The resistance gene was linked to five co-dominant genomic SSR markers (Xcfd233, Xwmc41, Xbarc11, Xgwm539 and Xwmc175) and their most likely order was Xcfd233–Xwmc41–Pm43–Xbarc11–Xgwm539–Xwmc175 at 2.6, 2.3, 4.2, 3.5 and 7.0 cM, respectively. Using the Chinese Spring nullisomic-tetrasomic and ditelosomic lines, the polymorphic markers and the resistance gene were assigned to chromosome 2DL. As no powdery mildew resistance gene was previously assigned to chromosome 2DL, this new resistance gene was designated Pm43. Pm43, together with the identified closely linked markers, could be useful in marker-assisted selection for pyramiding powdery mildew resistance genes. Runli He and Zhijian Chang contributed equally to this work.     

The mouse mutants <Emphasis Type="Italic">recoil wobbler</Emphasis> and <Emphasis Type="Italic">nmf373</Emphasis> represent a series of <Emphasis Type="Italic">Grm1</Emphasis> mutations

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP₃). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Functional conservation of the human <Emphasis Type="Italic">EXT1</Emphasis> tumor suppressor gene and its <Emphasis Type="Italic">Drosophila</Emphasis> homolog <Emphasis Type="Italic">tout</Emphasis><Emphasis Type="Italic">velu</Emphasis>

Heparan sulfate proteoglycans play a vital role in signaling of various growth factors in both Drosophila and vertebrates. In Drosophila, mutations in the tout velu (ttv) gene, a homolog of the mammalian EXT1 tumor suppressor gene, leads to abrogation of glycosaminoglycan (GAG) biosynthesis. This impairs distribution and signaling activities of various morphogens such as Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp). Mutations in members of the exostosin (EXT) gene family lead to hereditary multiple exostosis in humans leading to bone outgrowths and tumors. In this study, we provide genetic and biochemical evidence that the human EXT1 (hEXT1) gene is conserved through species and can functionally complement the ttv mutation in Drosophila. The hEXT1 gene was able to rescue a ttv null mutant to adulthood and restore GAG biosynthesis.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Organization of phenylalanine ammonia lyase (<Emphasis Type="Italic">PAL</Emphasis>), acidic <Emphasis Type="Italic">PR-5</Emphasis> and osmotin-like (<Emphasis Type="Italic">OSM</Emphasis>) defence-response gene families in the potato genome

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens. The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC) library containing ~50,000 clones was constructed from high-molecular weight genomic DNA of the diploid potato clone PD59, a hybrid between Solanum tuberosum and S. phureja. BAC clones carrying one or more copies of the DR genes were identified and characterized by Southern hybridization, sequence analysis and genetic mapping. PAL, acidic PR-5 and OSM (basic PR-5) genes were all organized into gene families of varying complexity. The PAL gene family consisted of at least 16 members, several of which were physically linked. Four acidic PR-5 homologous were localized to a 45-kb segment on potato chromosome XII. One of these, PR-5/319, codes for the acidic thaumatin-like protein C found in intercellular fluids of potato. Nine OSM genes were organized at two loci: eight form a 90-kb cluster on chromosome VIII, and a single gene was found on chromosome XI. The topology of a phylogenetic tree based on PR-5 and OSM protein sequences from Solanaceae suggests a mode of evolution for these gene families. The results will form the basis for further studies on the potential role of these defence-related loci in quantitative resistance to pathogens.