New Cytogenetic Photomap and Molecular Diagnostics for the Cryptic Species of the Malaria Mosquitoes Anopheles messeae and Anopheles daciae from Eurasia

The Eurasian malaria vector Anopheles messeae is a widely spread and genetically diverse species. Five widespread polymorphic chromosomal inversions were found in natural populations of this mosquito. A cryptic species, Anopheles daciae, was differentiated from An. messeae by the presence of several nucleotide substitutions in the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA. However, because of the absence of a high-quality reference cytogenetic map, the inversion polymorphisms in An. daciae and An. messeae remain poorly understood. Moreover, a recently determined heterogeneity in ITS2 in An. daciae questioned the accuracy of the previously used Restriction Fragment Length Polymorphism (RFLP) assay for species diagnostics. In this study, a standard-universal cytogenetic map was constructed based on orcein stained images of chromosomes from salivary glands for population studies of the chromosomal inversions that can be used for both An. messeae and An. daciae. In addition, a new ITS2-RFLP approach for species diagnostics was developed. Both methods were applied to characterize inversion polymorphism in populations of An. messeae and An. daciae from a single location in Western Siberia in Russia. The analysis demonstrates that cryptic species are remarkably different in their frequencies of chromosomal inversion variants. Our study supports previous observations that An. messeae has higher inversion polymorphism in all autosomes than the cryptic species An. daciae.

Chromosome Identification and Cytogenetic Map Construction of Zhikong Scallop (Chlamys farreri) Based on Fluorescence in situ Hybridization

Zhikong scallop (Chlamys farreri) is a bivalve species with broad economic and biological value, and an essential species of aquaculture in North China. Recently, efforts have been made to improve knowledge of genome, genetics, and cytogenetics, which is devoted to develop the molecular breeding project for the scallop. In this study, we constructed a cytogenetic map and identified all chromosomes of C. farreri using fluorescence in situ hybridization (FISH). A total of 100 Bacterial Artificial Chromosome (BAC) clones and 27 fosmid clones, including 58 microsatellite marker-anchored BAC clones, 4 genes-anchored BAC clones, 38 random BAC clones, 22 repetitive sequences-anchored fosmid clones, and 5 gene-anchored fosmid clones, were tested as probes, and 69 of them produced specific and stable signal on one pair of chromosomes. Then, multiple co-hybridizations were conducted to distinguish all the submetacentric and subtelocentric chromosomes with similar morphology by the abovementioned chromosome-specific markers. On this basis, a cytogenetic map of C. farreri containing 69 clones was constructed by co-hybridization and karyotype analysis. The markers covered all 19 pairs of chromosomes, and the average number of markers on each chromosome was 3.6. The cytogenetic map provides a platform for genetic and genomic analysis of C. farreri, which facilitates the molecular breeding project of C. farreri and promotes the comparative studies of chromosome evolution in scallops and even bivalves.

Expression of Mst89B and CG31287 is Needed for Effective Sperm Storage and Egg Fertilization in Drosophila

In Drosophila, male reproductive fitness can be affected by any number of processes, ranging from development of gametes, transfer to and storage of mature sperm within the female sperm storage organs, and utilization of sperm for fertilization. We have previously identified the 89B cytogenetic map position of D. melanogaster as a hub for genes that effect male paternity success when disturbed. Here, we used RNA interference to test 11 genes that are highly expressed in the testes and located within the 89B region for their role in sperm competition and male fecundity when their expression is perturbed. Testes-specific knockdown (KD) of bor and CSN5 resulted in complete sterility, whereas KD of CG31287, Manf and Mst89B, showed a breakdown in sperm competitive success when second to mate (P2 < 0.5) and reduced fecundity in single matings. The low fecundity of Manf KD is explained by a significant reduction in the amount of mature sperm produced. KD of Mst89B and CG31287 does not affect sperm production, sperm transfer into the female bursa or storage within 30 min after mating. Instead, a significant reduction of sperm in female storage is observed 24 h after mating. Egg hatchability 24 h after mating is also drastically reduced for females mated to Mst89B or CG31287 KD males, and this reduction parallels the decrease in fecundity. We show that normal germ-line expression of Mst89B and CG31287 is needed for effective sperm usage and egg fertilization.

A Comprehensive Integrated Genetic Map of the Complete Karyotype of Solea senegalensis (Kaup 1858)

Solea senegalensis aquaculture production has experienced a great increase in the last decade and, consequently, the genome knowledge of the species is gaining attention. In this sense, obtaining a high-density genome mapping of the species could offer clues to the aquaculture improvement in those aspects not resolved so far. In the present article, a review and new processed data have allowed to obtain a high-density BAC-based cytogenetic map of S. senegalensis beside the analysis of the sequences of such BAC clones to achieve integrative data. A total of 93 BAC clones were used to localize the chromosome complement of the species and 588 genes were annotated, thus almost reaching the 2.5% of the S. senegalensis genome sequences. As a result, important data about its genome organization and evolution were obtained, such as the lesser gene density of the large metacentric pair compared with the other metacentric chromosomes, which supports the theory of a sex proto-chromosome pair. In addition, chromosomes with a high number of linked genes that are conserved, even in distant species, were detected. This kind of result widens the knowledge of this species’ chromosome dynamics and evolution.

Integration of mandarin (Citrus reticulata) cytogenetic map with its genome sequence

Citrus is an extremely important genus in terms of world fruit production. Despite its economic importance and the small genome sizes of its species (2n = 18, 1C = 430 ± 68 Mbp), entire genomic assemblies have only recently become available for some of its representatives. Together with the previous CMA/DAPI banding and fluorescence in situ hybridization (FISH) in the group, these data are important for understanding the complex relationships between its species and for assisting breeding programs. To anchor genomic data with the cytogenetic map of mandarin (Citrus reticulata), the parental species of several economically important hybrids such as sweet orange and clementine, 18 BAC (bacterial artificial chromosome) clones were used. Eleven clementine BACs were positioned by BAC-FISH, doubling the number of chromosome markers so far available for BAC-FISH in citrus. Additionally, six previously mapped BACs were end-sequenced, allowing, together with one BAC previously sequenced, their assignment to scaffolds and the subsequent integration of chromosomes and the genome assembly. This study therefore established correlations between mandarin scaffolds and chromosomes, allowing further structural genomic and comparative study with the sweet orange genome, as well as insights into the chromosomal evolution of the group.

Field cress genome mapping: Integrating linkage and comparative maps with cytogenetic analysis for rDNA carrying chromosomes

Field cress (Lepidium campestre L.), despite its potential as a sustainable alternative oilseed plant, has been underutilized, and no prior attempts to characterize the genome at the genetic or molecular cytogenetic level have been conducted. Genetic maps are the foundation for anchoring and orienting annotated genome assemblies and positional cloning of candidate genes. Our principal goal was to construct a genetic map using integrated approaches of genetic, comparative and cytogenetic map analyses. In total, 503 F2 interspecific hybrid individuals were genotyped using 7,624 single nucleotide polymorphism markers. Comparative analysis demonstrated that ~57% of the sequenced loci in L. campestre were congruent with Arabidopsis thaliana (L.) genome and suggested a novel karyotype, which predates the ancestral crucifer karyotype. Aceto-orcein chromosome staining and fluorescence in situ hybridization (FISH) analyses confirmed that L. campestre, L. heterophyllum Benth. and their hybrids had a chromosome number of 2n = 2x = 16. Flow cytometric analysis revealed that both species possess 2C roughly 0.4 picogram DNA. Integrating linkage and comparative maps with cytogenetic map analyses assigned two linkage groups to their particular chromosomes. Future work could incorporate FISH utilizing A. thaliana mapped BAC clones to allow the chromosomes of field cress to be identified reliably.

Cytogenetic Map of Pummelo and Chromosome Evolution of True Citrus Species and the Hybrid Sweet Orange

Pummelo (Citrus maxima) is considered as one of the true citrus species. Together with mandarin (C. reticulata), it gave rise to the hybrid sweet orange (C. sinensis) and other important citrus crops. Although these species have 2n = 18, each has a unique heterochromatin distribution. The aims of this study were to identify chromosome homoeologies between pummelo and other true citrus species, to investigate the karyotypic changes involved in the chromosomal evolution between true citrus and to shed light into the origin of sweet orange hybrid karyotype. Mitotic metaphase chromosomes of pummelo and sweet orange were double stained with the fluorochromes CMA/DAPI (Chromomycin A3/4&rsquo;-6-diamidino-2-phenylindole), and identified by FISH (Fluorescence in Situ Hybridization) with chromosome-specific BAC (Bacterial Artificial Chromosome) markers. The results were compared to previously established cytogenetic maps of mandarin, C. medica and Poncirus trifoliata. Only chromosomes 1, 4 and 8 were maintained unaltered among species, with chromosomes 2 and 3 being among the least conserved in heterochromatin distribution. BACs were conserved in position among homoeologs and the markers mapped to chromosomes 2 and 3 indicated that sweet orange karyotype largely conserved one chromosome from pummelo and one from mandarin. Despite conserved synteny, expansion and contraction of heterochromatic blocks accounted for the differences between karyotypes, even between the hybrid sweet orange and pummelo.

Fluorescence In Situ Hybridization (FISH) Analysis of the Locations of the Oligonucleotides 5S rDNA, (AGGGTTT)3, and (TTG)6 in Three Genera of Oleaceae and Their Phylogenetic Framework

We report the cytogenetic map for a collection of species in the Oleaceae, and test similarities among the karyotypes relative to their known species phylogeny. The oligonucleotides 5S ribosomal DNA (rDNA), (AGGGTTT)3, and (TTG)6 were used as fluorescence in situ hybridization (FISH) probes to locate the corresponding chromosomes in three Oleaceae genera: Fraxinus pennsylvanica, Syringa oblata, Ligustrum lucidum, and Ligustrum × vicaryi. Forty-six small chromosomes were identified in four species. (AGGGTTT)3 signals were observed on almost all chromosome ends of four species, but (AGGGTTT)3 played no role in distinguishing the chromosomes but displayed intact chromosomes and could thus be used as a guide for finding chromosome counts. (TTG)6 and 5S rDNA signals discerned several chromosomes located at subterminal or central regions. Based on the similarity of the signal pattern (mainly in number and location and less in intensity) of the four species, the variations in the 5S rDNA and (TTG)6 distribution can be ordered as L. lucidum < L. × vicaryi < F. pennsylvanica < S. oblata. Variations have observed in the three genera. The molecular cytogenetic data presented here might serve as a starting point for further larger-scale elucidation of the structure of the Oleaceae genome, and comparison with the known phylogeny of Oleaceae family.