Lijun Chai

Transcriptome analysis of a spontaneous mutant in sweet orange [Citrus sinensis (L.) Osbeck] during fruit development

Bud mutations often arise in citrus. The selection of mutants is one of the most important breeding channels in citrus. However, the molecular basis of bud mutation has rarely been studied. To identify differentially expressed genes in a spontaneous sweet orange [C. sinensis (L.) Osbeck] bud mutation which causes lycopene accumulation, low citric acid, and high sucrose in fruit, suppression subtractive hybridization and microarray analysis were performed to decipher this bud mutation during fruit development. After sequencing of the differentially expressed clones, a total of 267 non-redundant transcripts were obtained and 182 (68.2%) of them shared homology (E-value ≤1×10−10) with known gene products. Few genes were constitutively up- or down-regulated (fold change ≥2) in the bud mutation during fruit development. Self-organizing tree algorithm analysis results showed that 95.1% of the differentially expressed genes were extensively coordinated with the initiation of lycopene accumulation. Metabolic process, cellular process, establishment of localization, response to stimulus, and biological regulation-related transcripts were among the most regulated genes. These genes were involved in many biological processes such as organic acid metabolism, lipid metabolism, transport, and pyruvate metabolism, etc. Moreover, 13 genes which were differentially regulated at 170 d after flowering shared homology with previously described signal transduction or transcription factors. The information generated in this study provides new clues to aid in the understanding of bud mutation in citrus.

An integrative analysis of the transcriptome and proteome of the pulp of a spontaneous late-ripening sweet orange mutant and its wild type improves our understanding of fruit ripening in citrus

Summary: ABA, ethylene, sucrose, and their related genes and pathways are involved in fruit ripening of citrus, and ABA may play a central role during the ripening process.

Transcriptional profiling of genes involved in embryogenic, non-embryogenic calluses and somatic embryogenesis of Valencia sweet orange by SSH-based microarray

Somatic embryogenesis (SE) is a most promising technology that is used for in vitro germplasm conservation and genetic improvement via biotechnological approaches in citrus. Herein, three suppression subtractive hybridization (SSH) libraries were constructed using calluses of Citrus sinensis cv. ‘Valencia’ to explore the molecular mechanisms that underlie the SE in citrus. A total of 880 unisequences were identified by microarray screening based on these three SSH libraries. Gene ontology analysis of the differentially expressed genes indicated that nucleolus associated regulation and biogenesis processes, hormone signal transduction, and stress factors might be involved in SE. Transcription factors might also play an important role. LEC1/B3 domain regulatory network genes (LEC1, L1L, FUS3, ABI3, and ABI5) were isolated in citrus SE. Some new transcription factors associated with citrus SE, like a B3 domain containing gene and HB4, were identified. To understand the influence of these isolated genes on SE competence, their expression profiles were compared among callus lines of seven citrus cultivars with different SE competence. The expression dynamics suggested that these genes could be necessary for the SE initiation and might play a role in embryogenic competence maintenance in different cultivars. On the basis of gene expression profiles, an overview of major physiological and biosynthesis processes at different developmental stages during citrus SE is presented. For the first time, these data provide a global resource for transcriptional events important for SE in citrus, and the specific genes offer new information for further investigation on citrus SE maintenance and development.

Selection and validation of suitable reference genes for mRNA qRT-PCR analysis using somatic embryogenic cultures, floral and vegetative tissues in citrus

Accuracy of the quantitative real-time reverse transcription-PCR (qRT-PCR) depends on the stability of the reference gene(s), i.e. housekeeping gene(s) used for data normalization. Recent studies have shown that the expression of common reference genes can vary considerably in certain experimental conditions. However, reference genes of qRT-PCR in fruit trees have not been well identified. In this study, stability of expression of ten potential reference genes in citrus, including CitACT7, CiteIF-1A, CiteIF4α, CitHistone H3, CitHistone H4, CitTUA3, CitTUB8, CitUBL5, CitUBQ1 and CitUBQ14 was assessed. Furthermore, this was validated by qRT-PCR in diverse sets of biological samples, including embryonic callus at seven stages, embryos maintained at three different temperatures, five distinct plant organs, four floral tissues and four stages of flower development. Three distinct statistical algorithms, geNorm, NormFinder and Bestkeeper, were used to evaluate the expression stability of the candidate reference genes. The three outputs were merged by means of a non-weighted unsupervised rank aggregation method. GeNorm was also used to determine both the optimal number and the best combination of reference genes for each experimental set. The expression of CitUBQ1 was the most stable one across the set of all samples, flower developmental stages and somatic embryogenesis process under two conditions i.e. different temperature treatment and normal temperature treatment. CitUBQ14 presented more stable expression in different plant organs and floral tissues. CitHistone H3 was the most unsuitable reference gene in many citrus sample sets. In addition, the relative gene expression profile of citrus receptor-like kinase gene CitSERK1-like was conducted to confirm the validity of the reference genes in this study. Taken together, this study identified the reference genes that are most suitable for normalizing the gene expression data in citrus. These results provide guidelines for the selection of reference gene(s) under different experimental conditions, and will benefit future research on more accurate gene expression studies in a wide variety of samples in citrus.

The Papaver Self-Incompatibility Pollen S-Determinant, PrpS, Functions in Arabidopsis thaliana

Summary Many angiosperms use specific interactions between pollen and pistil proteins as “self” recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by a multiallelic S locus, comprising pollen and pistil S-determinants [1, 2]. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein [3, 4], interact to trigger a Ca2+-dependent signaling network [5–10], resulting in inhibition of pollen tube growth, cytoskeletal alterations [11–13], and programmed cell death (PCD) [14, 15] in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI [11–15]. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ∼140 million years ago [16]. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.

Self-sterility in the mutant ‘Zigui shatian’ pummelo (Citrus grandis Osbeck) is due to abnormal post-zygotic embryo development and not self-incompatibility

Abstract‘Shatian’ pummelo (Citrus grandis Osbeck), one of the main citrus cultivars in China, is self-incompatible, and its pollen tubes are believed to be arrested in style after self-pollination.We have characterized one ‘Shatian’ pummelo mutant, named ‘Zigui shatian’ pummelo. The mutant pummelo had identical DNA ploidy level, morphology (leaf shape, stoma size and density, pollen shape and size) and developmental progress of pistil and male organs to that of the common ‘Shatian’ pummelo. However, unlike the common ‘Shatian’ pummelo, ‘Zigui shatian’ is self-compatible since its pollen tubes can self-pollinate allowing for successful fertilization. Histological analyses of ‘Shatian’ pummelo further verified abnormal post-zygotic development which led to seed abortion. Simple sequence repeats (SSR) analysis revealed polymorphism in 1 of the 120 primers screened showing that ‘Zigui shatian’ and ‘Shatian’ pummelo are different at the DNA level. Taken together, these data suggested mutant ‘Zigui shatian’ pummelo might be derived from ‘Shatian’ pummelo with self-sterility by self-incongruity after self-fertilization.

Comparative transcriptome analyses between a spontaneous late-ripening sweet orange mutant and its wild type suggest the functions of ABA, sucrose and JA during citrus fruit ripening.

A spontaneous late-ripening mutant of ‘Jincheng’ (C. sinensis L. Osbeck) sweet orange exhibited a delay of fruit pigmentation and harvesting. In this work, we studied the processes of orange fruit ripening through the comparative analysis between the Jincheng mutant and its wild type. This study revealed that the fruit quality began to differ on 166th days after anthesis. At this stage, fruits were subjected to transcriptome analysis by RNA sequencing. 13,412 differentially expressed unigenes (DEGs) were found. Of these unigenes, 75.8% were down-regulated in the wild type, suggesting that the transcription level of wild type was lower than that of the mutant during this stage. These DEGs were mainly clustered into five pathways: metabolic pathways, plant-pathogen interaction, spliceosome, biosynthesis of plant hormones and biosynthesis of phenylpropanoids. Therefore, the expression profiles of the genes that are involved in abscisic acid, sucrose, and jasmonic acid metabolism and signal transduction pathways were analyzed during the six fruit ripening stages. The results revealed the regulation mechanism of sweet orange fruit ripening metabolism in the following four aspects: First, the more mature orange fruits were, the lower the transcription levels were. Second, the expression level of PME boosted with the maturity of the citrus fruit. Therefore, the expression level of PME might represent the degree of the orange fruit ripeness. Third, the interaction of PP2C, PYR/PYL, and SnRK2 was peculiar to the orange fruit ripening process. Fourth, abscisic acid, sucrose, and jasmonic acid all took part in orange fruit ripening process and might interact with each other. These findings provide an insight into the intricate process of sweet orange fruit ripening.

Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray

BackgroundSeedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored.ResultsAn integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated.ConclusionOur research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.

De novo transcriptome assembly of pummelo and molecular marker development.

Pummelo (Citrus grandis) is an important fruit crop worldwide because of its nutritional value. To accelerate the pummelo breeding program, it is essential to obtain extensive genetic information and develop relative molecular markers. Here, we obtained a 12-Gb transcriptome dataset of pummelo through a mixture of RNA from seven tissues using Illumina pair-end sequencing, assembled into 57,212 unigenes with an average length of 1010 bp. The annotation and classification results showed that a total of 39,584 unigenes had similar hits to the known proteins of four public databases, and 31,501 were classified into 55 Gene Ontology (GO) functional sub-categories. The search for putative molecular markers among 57,212 unigenes identified 10,276 simple sequence repeats (SSRs) and 64,720 single nucleotide polymorphisms (SNPs). High-quality primers of 1174 SSR loci were designed, of which 88.16% were localized to nine chromosomes of sweet orange. Of 100 SSR primers that were randomly selected for testing, 87 successfully amplified clear banding patterns. Of these primers, 29 with a mean PIC (polymorphic information content) value of 0.52 were effectively applied for phylogenetic analysis. Of the 20 SNP primers, 14 primers, including 54 potential SNPs, yielded target amplifications, and 46 loci were verified via Sanger sequencing. This new dataset will be a valuable resource for molecular biology studies of pummelo and provides reliable information regarding SNP and SSR marker development, thus expediting the breeding program of pummelo.

Generation, functional analysis and utility of Citrus grandis EST from a flower-derived cDNA library

Pummelo (Citrus grandis) is one of the most important species found in the genus Citrus and one of the ancestors of sweet oranges. We used flower buds at different developmental stages to construct the first cDNA library for this species. A total of 3,758 EST sequences were generated from the cDNA library and clustered into 2,228 unigenes, comprising 451 contigs and 1,777 singletons. Among these unigene sequences, 1,266 have significant homology to the non-redundant protein database, from which 891 were assigned to one or more gene ontology categories. Functional categorization of the annotated unigenes showed that 760 genes were involved in molecular function, 1,189 in biological processes and 1,154 in cellular component categorization. Homologs of genes regulating many aspects of flower development were also identified, including those for organ development, cell-cycle control and cell and tissue differentiation. The majority of these genes (e.g., embryo relatives, YABBY-like, MAD Box, SKP-like and SRNAs) are the first representatives in Citrus, providing an opportunity to explore the cause of self incompatibility and embryo development in Citrus. Patterns of transcript accumulation were characterized by real-time qPCR for 13 of these genes. Many potential molecular markers were also identified in this EST data set; 212 Simple Sequences Repeats (SSRs), 717 transposon elements and 115 candidate single nucleotide polymorphisms (SNPs) were found. An assessment of a set of 212 SSR primer pairs on 16 citrus genotypes showed polymorphism with 122 (57.82%) markers. Similarly, a set of eight contigs were used to confirm in silico predicated SNPs in a set of five genotypes using wet lab experiments, three contigs were generated as scorable and sequenceable amplicons and no PCR amplicons were obtained from five contigs. The outcome of this study could aid in the discovery of genes involved in reproductive developments. Identified candidate genes can be experimentally tested for their functions in various important processes. SSR, SNP and transposon element-containing data sets may facilitate marker development and can be used for citrus molecular breeding, linkage map construction, evolutionary, phylogenetic and population genetic studies.