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.

Temperature-sensitive male sterility in rice determined by the roles of AGO1d in reproductive phasiRNA biogenesis and function.

Phased secondary siRNAs (phasiRNAs) are broadly present in the reproductive tissues of flowering plants, with spatial-temporal specificity. However, the ARGONAUTE (AGO) proteins associated with phasiRNAs and their miRNA triggers remain elusive. Here, through histological and high-throughput sequencing analyses, we show that rice AGO1d, which is specifically expressed in anther wall cells before and during meiosis, associates with both miR2118 and miR2275 to mediate phasiRNA biogenesis. AGO1d preferentially binds to miR2118-triggered 21-nucleotide (nt) phasiRNAs with a 5'-terminal uridine, suggesting a dual role in phasiRNA biogenesis and function. Depletion of AGO1d causes a reduction of 21- and 24-nt phasiRNAs and temperature-sensitive male sterility. At lower temperatures, anthers of the ago1d mutant predominantly show excessive tapetal cells with little starch accumulation during pollen formation, possibly caused by the dysregulation of cell metabolism. These results uncover an essential role of AGO1d in rice anther development at lower temperatures and demonstrate coordinative roles of AGO proteins during reproductive phasiRNA biogenesis and function.

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

GRAS genes are suggested to be grouped into plant-specific transcriptional regulatory families that have been reported to participate in multiple processes, including plant development, phytohormone signaling, the formation of symbiotic relationships, and response to environmental signals. GRAS genes have been characterized in a number of plant species, but little is known about this gene family in Citrus sinensis. In this study, we identified a total of 50 GRAS genes and characterized the gene structures, conserved motifs, genome localizations and cis-elements within their promoter regions. According to their structural and phylogenetic features, the identified sweet orange GRAS members were divided into 11 subgroups, of which subfamily CsGRAS34 was sweet orange-specific. Based on publicly available RNA-seq data generated from callus, flower, leaf and fruit in sweet orange, we found that some sweet orange GRAS genes exhibited tissue-specific expression patterning. Three of the six members of subfamily AtSHR, particularly CsGRAS9, and two of the six members of subfamily AtPAT1 were preferentially expressed in leaf. Moreover, protein-protein interactions with CsGRAS were predicted. Gene expression analysis was performed under conditions of phosphate deficiency, and GA3 and NaCl treatment to identify the potential functions of GRAS members in regulating stress and hormone responses. This study provides the first comprehensive understanding of the GRAS gene family in the sweet orange genome. As such, the study generates valuable information for further gene function analysis and identifying candidate genes to improve abiotic stress tolerance in citrus plants.

Fragile Sites of 'Valencia' Sweet Orange (Citrus sinensis) Chromosomes Are Related with Active 45s rDNA.

Citrus sinensis chromosomes present a morphological differentiation of bands after staining by the fluorochromes CMA and DAPI, but there is still little information on its chromosomal characteristics. In this study, the chromosomes in 'Valencia' C. sinensis were analyzed by fluorescence in situ hybridization (FISH) using telomere DNA and the 45S rDNA gene as probes combining CMA/DAPI staining, which showed that there were two fragile sites in sweet orange chromosomes co-localizing at distended 45S rDNA regions, one proximally locating on B-type chromosome and the other subterminally locating on D-type chromosome. While the chromosomal CMA banding and 45S rDNA FISH mapping in the doubled haploid line of 'Valencia' C. sinensis indicated six 45S rDNA regions, four were identified as fragile sites as doubled comparing its parental line, which confirmed the cytological heterozygosity and chromosomal heteromorphisms in sweet orange. Furthermore, Ag-NOR identified two distended 45S rDNA regions to be active nucleolar organizing regions (NORs) in diploid 'Valencia' C. sinensis. The occurrence of quadrivalent in meiosis of pollen mother cells (PMCs) in 'Valencia' sweet orange further confirmed it was a chromosomal reciprocal translocation line. We speculated this chromosome translocation was probably related to fragile sites. Our data provide insights into the chromosomal characteristics of the fragile sites in 'Valencia' sweet orange and are expected to facilitate the further investigation of the possible functions of fragile sites.