Comparative physiochemical and transcriptomic analysis reveals the influences of cross-pollination on ovary and fruit development in pummelo (Citrus maxima).

'Shuijingmiyou' pummelo (SJ), one of the most popular fruits in Yunnan province of China, is of relatively low fruit shape (FS) quality. In this study, we compared the FS promoting effects of cross pollinations using pollens from seven pummelo varieties, and found that 'Guanximiyou' pummelo (GX) cross-pollination showed the best FS promoting effects on SJ fruits by shortening its fruit neck. To explore the underlying mechanism, physiochemical and transcriptomic differences between self- and cross-pollinated SJ ovaries (SJO and GXO) were investigated. Higher salicylic acid, gibberellin and indole acetic acid contents and superoxide dismutase, peroxidase and catalase activities, and lower polyphenol oxidase activity were determined in GXO compared with SJO. Enrichment analysis of the identified 578 differentially expressed genes (123 up-regulated and 455 down-regulated) in GXO showed that genes involved in solute transport, RNA biosynthesis, phytohormone action and cell wall organization were significantly enriched. The results obtained in this study will be helpful in understanding the influences of cross-pollination on pummelo ovary and fruit development, and can provide the basis for clarifying the underlying mechanism of cross-pollination improved fruit quality.

Characterization of three tandem-duplicated calcium binding protein (CaBP) genes and promoters reveals their roles in the phytohormone and wounding responses in citrus.

Accumulated evidences have revealed the critical roles of calcium binding protein (CaBP) in growth and stress responses of plants. However, its function in woody plants is poorly understood. In this study, we cloned the CDS, gDNA and promoter sequences of three tandem-duplicated CaBPs (CsCaBP1, CsCaBP2 and CsCaBP3) from Citrus sinensis, analyzed their sequence characteristics, and investigated their gene expression patterns and promoter activities under treatments of CaCl2, several phytohormones and wounding. Results showed that the three CsCaBPs have high sequence similarity. Their expression was strongly induced by CaCl2, ethylene, jasmonic acid, salicylic acid and wounding, and the promoting effect of wounding on their expression was found to be partially ethylene-dependent. Consistently, we identified many phytohormone-related cis-acting elements in their promoters, and their promoter activity could be induced significantly by ethylene, jasmonic acid, salicylic acid and wounding. All the three CsCaBPs can interact with WRKY40, whose encoding gene showed a similar expression pattern to CsCaBPs under phytohormone and wounding treatments. In addition, CsERF14, CsERF21, CsERF3 and CsERF2 could bind to their promoters. The results obtained in this study indicated that the three duplicated CsCaBPs were functionally redundant and played similar roles in the phytohormone and wounding responses of C. sinensis.

Characterization of the complete chloroplast genome of Citrus hongheensis, a key protected wild plant in Yunnan province of China.

Citrus hongheensis is a key protected wild plant endemic to the Honghe river region in southeastern Yunnan, China. In the present study, its chloroplast genome was successfully assembled and annotated based on the Illumina Hiseq-2500 whole genome re-sequencing data. The chloroplast genome is 160,275 bp in size. Its large single copy region, small single copy region and inverted repeat region is 87,886 bp, 18,387 bp and 27,001 bp, respectively. Totally, 114 unique genes, including 80 protein-coding genes, 30 tRNAs and 4 rRNAs, were identified from the C. hongheensis chloroplast genome. According to the phylogenetic analysis result, the relationship between the chloroplast genome of C. hongheensis and C. maxima was found to be the closest.

Re-sequencing and transcriptome analysis reveal rich DNA variations and differential expressions of fertility-related genes in neo-tetraploid rice.

Autotetraploid rice is a useful germplasm for polyploid rice breeding, however, low seed setting is the major barrier in commercial utilization of autotetraploid rice. Our research group has developed neo-tetraploid rice lines, which have the characteristics of high fertility and heterosis when crossed with autotetraploid rice. In the present study, re-sequencing and RNA-seq were employed to detect global DNA variations and differentially expressed genes (DEGs) during meiosis stage in three neo-tetraploid rice lines compared to their parents, respectively. Here, a total of 4109881 SNPs and 640592 InDels were detected in neo-tetraploid lines compared to the reference genome, and 1805 specific presence/absence variations (PAVs) were detected in three lines. Approximately 12% and 0.5% of the total SNPs and InDels identified in three lines were located in genic regions, respectively. A total of 28 genes, harboring at least one of the large-effect SNP and/or InDel which affect the integrity of the encoded protein, were identified in the three lines. Together, 324 specific mutation genes, including 52 meiosis-related genes and 8 epigenetics-related genes were detected in neo-tetraploid rice compared to their parents. Of these 324 genes, five meiosis-related and three epigenetics-related genes displayed differential expressions during meiosis stage. Notably, 498 specific transcripts, 48 differentially expressed transposons and 245 differentially expressed ncRNAs were also detected in neo-tetraploid rice. Our results suggested that genomic structural reprogramming, DNA variations and differential expressions of some important meiosis and epigenetics related genes might be associated with high fertility in neo-tetraploid rice.

Characterization of the complete chloroplast genome of the Hongkong kumquat (Fortunella hindsii Swingle).

Hongkong qumquat (Fortunella hindsii Swingle) is a wild citrus species native to China. In this study, we firstly reporteded its complete chloroplast genome using BGISEQ-500 sequencing. The chloroplast genome is 160,145 bp in size, containing a large single copy region (87,467 bp), a small single copy region (18,730 bp), and a pair of IR regions (26,974 bp). The chloroplast genome contains 112 unique genes, including 79 protein-coding genes, 29 tRNAs, and 4 rRNAs. Phylogenetic maximum-likelihood analysis indicated that F. hindsii is closely related to Citrus species. The complete chloroplast genome would be subsequently used for citrus species researches.

Characterisation of the complete chloroplast genome of Fortunella Crassifolia Swingle and phylogenetic relationships.

In this study, we reported the complete chloroplast genome of Fortunella crassifolia Swingle using the HiSeq-4000 sequencing. The chloroplast genome size is 160,229 bp, which consists of a large single-copy region (87,774 bp), a small single-copy region (18,721 bp), and a pair of IR regions (26,867 bp). The chloroplast genome contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs, and 4 rRNAs. Phylogenetic maximum likelihood analysis showed that F. crassifolia was closest to Hongkong kumquat (F. hindsii). The complete chloroplast genome would be subsequently used for citrus species researches.

Identification of pummelo cultivars by using a panel of 25 selected SNPs and 12 DNA segments.

Pummelo cultivars are usually difficult to identify morphologically, especially when fruits are unavailable. The problem was addressed in this study with the use of two methods: high resolution melting analysis of SNPs and sequencing of DNA segments. In the first method, a set of 25 SNPs with high polymorphic information content were selected from SNPs predicted by analyzing ESTs and sequenced DNA segments. High resolution melting analysis was then used to genotype 260 accessions including 55 from Myanmar, and 178 different genotypes were thus identified. A total of 99 cultivars were assigned to 86 different genotypes since the known somatic mutants were identical to their original genotypes at the analyzed SNP loci. The Myanmar samples were genotypically different from each other and from all other samples, indicating they were derived from sexual propagation. Statistical analysis showed that the set of SNPs was powerful enough for identifying at least 1000 pummelo genotypes, though the discrimination power varied in different pummelo groups and populations. In the second method, 12 genomic DNA segments of 24 representative pummelo accessions were sequenced. Analysis of the sequences revealed the existence of a high haplotype polymorphism in pummelo, and statistical analysis showed that the segments could be used as genetic barcodes that should be informative enough to allow reliable identification of 1200 pummelo cultivars. The high level of haplotype diversity and an apparent population structure shown by DNA segments and by SNP genotypes, respectively, were discussed in relation to the origin and domestication of the pummelo species.