Molecular cytogenetic map visualizes the heterozygotic genome and identifies translocation chromosomes in Citrus sinensis.

Citrus sinensis is the most cultivated and economically valuable Citrus species in the world, whose genome has been assembled by three generation sequencings. However, chromosome recognition remains a problem due to the small size of chromosomes, and difficulty in differentiating between pseudo and real chromosomes because of a highly heterozygous genome. Here, we employ fluorescence in situ hybridization (FISH) with 9 chromosome painting probes, 30 oligo pools, and 8 repetitive sequences to visualize 18 chromosomes. Then, we develop an approach to identify each chromosome in one cell through single experiment of oligo-FISH and Chromoycin A3 (CMA) staining. By this approach, we construct a high-resolution molecular cytogenetic map containing the physical positions of CMA banding and 38 sequences of FISH including centromere regions, which enables us to visualize significant differences between homologous chromosomes. Based on the map, we locate several highly repetitive sequences on chromosomes and estimate sizes and copy numbers of each site. In particular, we discover the translocation regions of chromosomes 4 and 9 in C. sinensis "Valencia." The high-resolution molecular cytogenetic map will help improve understanding of sweet orange genome assembly and also provide a fundamental reference for investigating chromosome evolution and chromosome engineering for genetic improvement in Citrus.

Localization and characterization of Citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling.

KEY MESSAGE: The physical locations of citrus centromere are revealed by combining genetic and immunological assays for the first time and nine citrus centromere-specific markers for cytogenetics are mined. Centromere localization is challenging, because highly redundant repetitive sequences in centromeric regions make sequence assembly difficult. Although several citrus genomes have been released, the centromeric regions and their characteristics remain to be elucidated. Here, we mapped citrus centromeres through half-tetrad analysis (HTA) that included the genotyping of 54 tetraploid hybrids derived from 2n megagametophytes of Nadorcott tangor with 212 single nucleotide polymorphism (SNP) markers. The sizes of centromeric regions, which estimated based on the heterozygosity restitution rate pattern along the chromosomes, ranged from 1.12 to 18.19 Mb. We also profiled the binding sequences with the centromere-specific histone variant CenH3 by chromatin immunoprecipitation sequencing (ChIP-seq). Based on the positions of the top ten CenH3-enriched contigs, the sizes of centromeric regions were estimated to range from 0.01 to 7.60 Mb and were either adjacent to or included in the centromeric regions identified by HTA. We used DNA probes from two repeats selected from the centromeric regions and seven CenH3-binding centromeric repeats to verify centromeric locations by fluorescence in situ hybridization (FISH). Centromere localization in citrus will contribute to the mining of centromeric/pericentromeric markers, thus to facilitate the rapid identification of mechanisms underlying 2n gamete formation and serve the polyploidy breeding.

Genome-wide identification, characterization, interaction network and expression profile of GAPDH gene family in sweet orange (Citrus sinensis).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that plays important roles in multiple cellular processes including phytohormone signaling, plant development, and transcriptional regulation. Although GAPDH genes have been well characterized in various plant species such as Arabidopsis, tobacco, wheat, rice, and watermelon, comprehensive analysis has yet to be completed at the whole genome level in sweet orange (Citrus sinensis). In this study, six GAPDH genes distributed across four chromosomes were identified within the sweet orange genome. Their gene structures, conserved subunits, and subcellular localization were also characterized. Cis-element analysis of CsGAPDHs' promoter regions and the results of dark treatments indicate that CsGAPDH may be involved in photosynthesis. CsGAPDH genes expressed either in a tissue-specific manner or constitutively were ultimately identified along with their expression response to phosphorus deficiency treatments. In addition, a dual-luciferase transient assay was performed to reveal the transcriptional activation of CsGAPDH proteins. Gene Ontology (GO) analysis for proteins interacting with CsGAPDHs helped to uncover the roles these CsGAPDHs play in other plant processes such as citrus seed germination. This study provides a systematic analysis of the CsGAPDH gene family in the sweet orange genome, which can serve as a strong foundation for further research into the biochemical properties and physiological functions of CsGAPDHs.