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Highlights: [•] More than forty leaf-parent-type cybrids or putative cybrids were produced in citrus. [•] Somatic cybridization aiming to produce male sterile and seedless citrus cybrids was detailed. [•] Agronomic performance and omics-based studies of some selected cybrids were reviewed. [ABSTRACT FROM AUTHOR]

Abstract: More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo (Citrus reticulata Blanco× C. paradisi Macf) and mesophyll protoplasts of rough lemon (C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed. [Copyright &y& Elsevier]

Abstract: Previous experience with citrus somatic fusion suggests that hybrid cells are generally more vigorous and have higher capacity for embryogenesis, compared to unfused cells from the embryogenic callus parent. However, this observation has never been validated due to the unavailability of an appropriate experimental design. Citrus mesophyll protoplasts never divide and regenerate into plants. Herein, mesophyll protoplasts of transgenic Valencia orange (Citrus sinensis) which expressed the green fluorescent protein (GFP) gene were fused with protoplasts isolated from an embryogenic callus line of Shekwasha mandarin (C. reticulata) to visually screen somatic hybrids. At the multi-cell cluster stage, the regenerated callus lines expressing GFP were morphologically identical to the callus parent regenerate. All hybrid callus lines expressing the GFP developed into embryoids, while most callus parent regenerates still remained in a callus stage. Embryoids with strong or weak GFP expression were verified to be diploid cybrid and tetraploid somatic hybrid embryoids, respectively, by flow cytometry, simple sequence repeat (SSR) and chloroplast SSR analyses. Diploid cybrid globular embryoids appeared light green in color and had a tendency to enter subsequent developmental stages earlier than the dark green tetraploids; however, GFP plants were regenerated from both sources. Thus, expression of the GFP marker gene was effectively used to visually screen somatic hybrids, and confirmed our previous observation that somatic hybrid tissue has a regeneration advantage. The intensity of GFP fluorescence and the color difference of regenerated embryoids was also an early indicator of ploidy level (based on visual GFP observation and confirmed by flow cytometry analysis), and could serve as a tool to study the regeneration mechanism of diploid cybrids via symmetric fusion in citrus and other higher plants. [Copyright &y& Elsevier]

Key Message: Genome-wide identification of C2H2-ZF gene family in the poly- and mono-embryonic citrus species and validation of the positive role of CsZFP7 in sporophytic apomixis. The C2H2 zinc finger (C2H2-ZF) gene family is involved in plant vegetative and reproductive development. Although a large number of C2H2 zinc-finger proteins (C2H2-ZFPs) have been well characterized in some horticultural plants, little is known about the C2H2-ZFPs and their function in citrus. In this work, we performed a genome-wide sequence analysis and identified 97 and 101 putative C2H2-ZF gene family members in the genomes of sweet orange (C. sinensis, poly-embryonic) and pummelo (C. grandis, mono-embryonic), respectively. Phylogenetic analysis categorized citrus C2H2-ZF gene family into four clades, and their possible functions were inferred. According to the numerous regulatory elements on promoter, citrus C2H2-ZFPs can be divided into five different regulatory function types that indicate functional differentiation. RNA-seq data revealed 20 differentially expressed C2H2-ZF genes between poly-embryonic and mono-embryonic ovules at two stages of citrus nucellar embryogenesis, among them CsZFP52 specifically expressed in mono-embryonic pummelo ovules, while CsZFP7, 37, 44, 45, 67 and 68 specifically expressed in poly-embryonic sweet orange ovules. RT-qPCR further validated that CsZFP7 specifically expressed at higher levels in poly-embryonic ovules, and down-regulation of CsZFP7 in the poly-embryonic mini citrus (Fortunella hindsii) increased rate of mono-embryonic seeds compared with the wild type, indicating the regulatory potential of CsZFP7 in nucellar embryogenesis of citrus. This work provided a comprehensive analysis of C2H2-ZF gene family in citrus, including genome organization and gene structure, phylogenetic relationships, gene duplications, possible cis-elements on promoter regions and expression profiles, especially in the poly- and mono-embryogenic ovules, and suggested that CsZFP7 is involved in nucellar embryogenesis. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) is a major regeneration approach for in vitro cultured tissues of plants, including citrus. However, SE capability is difficult to maintain, and recalcitrance to SE has become a major obstacle to plant biotechnology. We previously reported that miR156- SPL modules regulate SE in citrus callus. However, the downstream regulatory pathway of the miR156- SPL module in SE remains unclear. In this study, we found that transcription factors CsAGL15 and CsFUS3 bind to the CsMIR156A promoter and activate its expression. Suppression of csi-miR156a function leads to up-regulation of four target genes, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (CsSPL) genes, and reduction of SE efficiency. In the short tandem target mimic (STTM)-miR156a overexpression callus (MIM156), the number of amyloplasts and starch content were significantly reduced, and genes involved in starch synthesis and transport were down-regulated. csi-miR172d was down-regulated, whereas the target genes, CsTOE1.1 and CsTOE1.2 , which inhibit the expression of starch biosynthesis genes, were up-regulated. In our working model, CsAGL15 and CsFUS3 activate csi-miR156a, which represses CsSPLs and further regulates csi-miR172d and CsTOEs , thus altering starch accumulation in callus cells and regulating SE in citrus. This study elucidates the pathway of miR156- SPLs and miR172- TOEs -mediated regulation of SE, and provides new insights into enhancing SE capability in citrus. [ABSTRACT FROM AUTHOR]

Key message: Overexpression of miR171 restored SE competence in the recalcitrant citrus callus, and inhibition of miR171 function weakened SE competence in the strongly embryogenic citrus callus. Somatic embryogenesis (SE) is an important way of in vitro regeneration for plants. For perennial woody crops such as citrus, embryogenic callus is usually induced from unfertilized aborted ovules and widely used in biotechnology aided breeding. However, SE capacity always declines in callus during subculture, which makes regeneration difficult and hinders the application of biotechnology. We previously found that miR171 may be a regulator of SE in citrus, based on the abundant expression of csi-miR171c in the embryogenic callus and during SE of citrus. Here, we report that miR171 promotes SE and is required for SE in citrus. Overexpression of miR171 restored SE competence in the recalcitrant callus of 'Guoqing No.1' Satsuma mandarin (G1), whereas inhibition of miR171 function by Short Tandem Target Mimic (STTM) weakened SE competence in the strongly embryogenic callus of 'Valencia' sweet orange (V). The comparative transcriptomic analysis in miR171 overexpressed callus line (OE) and the wild type callus (WT) indicated that overexpression of miR171 decreased the expression level of its SCARECROW-LIKE (CsSCL) targets, and activated stress response related biological processes and metabolic processes that are required for cell differentiation. However, CsSCLs were up-regulated in the OE callus during SE induction process, which activated the cell division and developmental processes that are required for embryogenesis progress. Our results validate the function of miR171 in regulation of SE and reveal the biological responses provoked by miR171 in citrus that may promote SE. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: In citrus, genetic improvement via biotechnology is hindered by the obstacle of in vitro regeneration via somatic embryogenesis (SE). Although a few B3 transcription factors are reported to regulate embryogenesis, little is known about the B3 superfamily in citrus, and which members might be involved in SE. Results: Genome-wide sequence analysis identified 72 (CsB3) and 69 (CgB3) putative B3 superfamily members in the genomes of sweet orange (Citrus sinensis, polyembryonic) and pummelo (C. grandis, monoembryonic), respectively. Genome duplication analysis indicated that segmental and tandem duplication events contributed to the expansion of the B3 superfamily in citrus, and that the B3 superfamily evolved under the effect of purifying selection. Phylogenetic relationships were well supported by conserved gene structure and motifs outside the B3 domain, which allowed possible functions to be inferred by comparison with homologous genes from Arabidopsis. Expression analysis identified 23 B3 superfamily members that were expressed during SE in citrus and 17 that may play functional roles at late SE stages. Eight B3 genes were identified that were specific to the genome of polyembryonic sweet orange compared to monoembryonic pummelo. Of these eight B3 genes, CsARF19 was found to be specifically expressed at higher levels in embryogenic callus (EC), implying its possible involvement in EC initiation. Conclusions: This study provides a genome-wide analysis of the citrus B3 superfamily, including its genome organization, evolutionary features and expression profiles, and identifies specific family members that may be associated with SE. [ABSTRACT FROM AUTHOR]

Highlights • Four 2x × 4x crosses were conducted using three diploid seedy cultivars as seed parents. • High germination rate and shoot regeneration was achieved via an optimized embryo rescue approach. • Numerous triploid and tetraploid progenies were obtained as determined by flow cytometry analysis. • Hybrid nature of the selected triploids and two different origins of the selected tetraploids were confirmed. Abstract Seedlessness is a primary breeding objective for citrus fresh fruit market, and triploids have been proven to have great value to produce seedless fruits. In this study, aiming to produce triploid plants for developing some seedless cultivars, four 2x × 4x interploid crosses were conducted using three elite but seedy cultivars as seed parents and one newly flowered doubled diploid and two allotetraploid somatic hybrids as pollen parents. As a result, a total of 1454 developed and 3409 undeveloped seeds from 341 fruits were obtained. Using an optimized embryo rescue approach, 669 developed and 1301 undeveloped seeds germinated in vitro, with an average germination rate as 52.5% for the crosses using 'Nadorcott' tangor and 'Bendizao' tangerine (polyembryonic) as seed parents and 31.0% for the crosses using 'Orah' mandarin (monoembryonic) as seed parent. Then by shoot and root induction, totally 1354 plantlets were regenerated, among which 401 and 54 plants were proved to be triploids and tetraploids by flow cytometry (FCM) analysis and chromosome counting. Hybrid nature of the selected triploid progenies, as well as two different origins (doubled diploid and hybrid origins) for the selected tetraploid progenies was further confirmed by single nucleotide polymorphism (SNP) markers. [ABSTRACT FROM AUTHOR]

The unreduced (2n) gametes have long been used in triploid breeding of citrus. In lemon, the previously reported mechanisms for 2n megagametophyte formation were controversial, whereas that for 2n pollen production is still unknown. Herein, the frequency of and mechanism underlying 2n megagametophyte and 2n pollen formation in 'Eureka' lemon were investigated based on cytological observation and genotyping of the triploid hybrids between 'Eureka' lemon and 'Early gold' sweet orange. As a result, 4.79% of the viable pollens of 'Eureka' lemon were identified as the 2n pollen with a larger diameter (70.16 ± 3.92 µm). The 2n pollen might be resulted from the formation of parallel spindles at meiosis stage II. Among the 204 plantlets regenerated from embryo rescue following the sexual cross, 12 were triploids as identified by flow cytometry. According to the analysis of heterozygosity transmission using 13 pericentromeric single nucleotide polymorphism (SNP) markers and 20 randomly distributed simple sequence repeat (SSR) markers, 11 triploids were identified to be originated from the fertilization of 2n megagametophytes of 'Eureka' lemon, with a frequency of 5.39%. Among them, nine 2n megagametophytes were supposed to be arisen from the second division restitution (SDR), whereas the other two were from post-meiotic genome doubling (PMD). These results to understand the mechanism underlying 2n gamete formation in lemon are valuable for its efficient polyploid breeding. [ABSTRACT FROM AUTHOR]

Highlights • The expression of CsFUS 3 is positively related with the capacity of citrus callus for somatic embryogenesis (SE). • Overexpression of CsFUS 3 promotes SE and restores normal late embryogenesis in fus 3. • Overexpression of CsFUS 3 has effects on the expression of SE-related transcription factors and hormone pathways, especially the ABA and GA pathways. Abstract In citrus, genetic improvement via biotechnology is challenging due to insufficient understanding of molecular barriers that prevent regeneration by somatic embryogenesis (SE). Our previous study indicated that LEC genes were involved in SE in citrus, but their regulatory roles remain to be elucidated. Here, we cloned one of the LEC genes, CsFUS3 , and show that it is preferentially expressed during SE and in the embryogenic callus (EC) derived from citrus varieties with strong embryogenic competence. The overexpression of CsFUS3 in recalcitrant citrus callus restored embryogenic competence. Complementation of the loss-of-function Arabidopsis fus3 mutant with the CsFUS3 gene restored normal late embryogenesis, which is consistent with the CsFUS3 and AtFUS3 proteins contributing to the same regulatory network in Arabidopsis. Transcriptome profiling revealed that the expression of particular TFs that promote SE was up-regulated in the citrus overexpression (OE) line. The 104 differentially expressed genes associated with hormone biosynthesis, catabolism, and signaling are particularly noteworthy. The dynamic change in the ratio of ABA to GA during SE in wild-type callus mirrored the expression pattern of CsFUS3. In contrast, in the OE line, the ratio of ABA to GA was higher and the capacity for SE was greater when the OE line was separately treated with ABA and GA biosynthesis inhibitors. Taken together, our results demonstrate that the overexpression of CsFUS3 appears to establish a cellular environment favorable to SE, at least in part by promoting a high ABA to GA ratio and by regulating the expression of TFs that promote SE. [ABSTRACT FROM AUTHOR]

• Autotetraploid of citrus junos shows higher salt tolerance than the corresponding diploid. • Transcriptomes of diploid and autotetraploid differ greatly under salt stress. • Plant chitin signaling is the key pathway responding to salt stress in autotetraploid citrus junos. Polyploidization is a widespread phenomenon in flowering plants, and it often enhances abiotic stress tolerance. However, the related molecular mechanism remains largely unknown, especially for perennial woody plants. Here, we found that autotetraploid citrus (Citrus junos Sieb. ex Tanaka) had greater salt tolerance than their diploid progenitors. Transcriptome analysis identified a large number of differentially expressed genes (DEGs) between autotetraploid and diploid roots under salt stress. GO analysis showed that chitin response-related genes were enriched in roots of autotetraploid. Consistently, the autotetraploids exhibited higher chitinases activity than the diploids under salt stress. The top five transcription factor (TF) families with the largest number of TFs were bHLH, ERF, MYB, NAC, and WRKY in autotetraploids and diploids, and 25 TFs were higher expressed in autotetraploids than in diploids under salt stress. Our results revealed that salt stress induced the activation of chitin pathway, thus reducing in ROS accumulation, eventually enhancing salt tolerance of autotetraploid citrus. Our research provides the theoretical basis for citrus breeding aiming at abiotic stress tolerance enhancement. [ABSTRACT FROM AUTHOR]

Introduction: Polyploidy is a widespread phenomenon in nature and is thought to play a major role in the evolution of flowering plants. Additionally, polyploidization produces novel phenotypes that through plant breeding have enhanced the production of biomass and improved the stress tolerance of major economic crops. However, the effect of polyploidization on plant metabolism is still unclear. Objectives: In order to test whether there are common metabolic responses following genome doubling, we performed a comparative metabolomic analysis of mature leaves from doubled diploids and the corresponding diploids of red tangerine ( Citrus reticulata Blanco), trifoliate orange ( Poncirus trifoliata L. Raf.) and precocious trifoliate orange ( P. trifoliata). Methods: Non-targeted and targeted metabolic profiling of mature leaves from three doubled diploids and their diploid controls were performed by using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). Results: About 11-34% of the detected metabolic features differentially accumulated in the doubled diploids, mostly by less than fivefold. The levels of primary metabolites tended to increase in the doubled diploids. Concentrations of tricarboxylic acid cycle intermediates-citric acid, malic acid, fumaric acid and succinic acid, enhanced in all of the doubled diploids. The levels of secondary metabolites, including phenylpropanoids and terpenoids, tended to decrease in the doubled diploids. This is consistent with the lower C/N ratios in the doubled diploids. Conclusions: Polyploidization had a significant but relatively limited influence on the accumulation of metabolites in these citrus species. We conclude that primary metabolism takes priority over secondary metabolism in doubled diploid plants to relieve the 'genomic stress' encountered during the early stages of genome doubling, probably to promote vitality and growth. [ABSTRACT FROM AUTHOR]

Homozygous genotypes have great potential in breeding and genomic researches in higher plants. Induction of parthenogenesis via pollination with gamma-irradiated pollens is an efficient method to obtain haploid plants that can overcome the biological limitations of fruit trees. In this study, we investigated the induction of haploid plants in ‘Hirado Buntan’ pummlo ( Citrus grandis ) through in-situ gynogenesis by pollination with irradiated pollens of trifoliate orange (255, 325 and 500 Gy) and ‘Tongshui 72-1 Jincheng’ sweet orange (255, 395 and 500 Gy). The pollen stainability, pollen tube behavior after pollination were evaluated. The seeds extracted from the fruits were rescued by in vitro embryo culture, and the ploidy level of the obtained plantlets was determined by flow cytometry. It showed that two haploid plants and one triploid plant, were obtained from the cross-pollination of ‘Hirado Buntan’ pummelo with irradiated pollens of trifoliate orange; two haploid plants were obtained from the cross-pollination of ‘Hirado Buntan’ pummlo with irradiated pollens of ‘Tongshui 72-1 Jincheng’ sweet orange. The parthenogenic origin of the haploid plants was further confirmed by chromosomal cytogenetic and SSR analysis. The ‘Hirado Buntan’ haploid plants obtained here hold great potential in citrus genetic and genomic studies in the future. [ABSTRACT FROM AUTHOR]

The homeodomain–leucine zipper (HD-Zip) transcription factors, which belong to a class of Homeobox proteins, has been reported to be involved in different biological processes of plants, including growth and development, photomorphogenesis, flowering, fruit ripening and adaptation responses to environmental stresses. In this study, 27 HD-Zip genes ( CsHBs ) were identified in Citrus . Based on the phylogenetic analysis and characteristics of individual gene or protein, the HD-Zip gene family in Citrus can be classified into 4 subfamilies, i.e. HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV containing 16, 2, 4, and 5 members respectively. The digital expression patterns of 27 HD-Zip genes were analyzed in the callus, flower, leaf and fruit of Citrus sinensis . The qRT-PCR and RT-PCR analyses of six selected HD-Zip genes were performed in six citrus cultivars with different embryogenic competence and in the embryo induction stages, which revealed that these genes were differentially expressed and might be involved in citrus somatic embryogenesis (SE). The results exhibited that the expression of CsHB1 was up-regulated in somatic embryo induction process, and its expression was higher in citrus cultivars with high embryogenic capacity than in cultivars recalcitrant to form somatic embryos. Moreover, a microsatellite site of three nucleotide repeats was found in CsHB1 gene among eighteen citrus genotypes, indicating the possible association of CsHB1 gene to the capacity of callus induction. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis ( SE) is a process of somatic cells becoming dedifferentiated and generating embryos. SE has been widely used in biotechnology as a powerful way of regeneration and a model system for studying plant embryogenesis, but the controlling mechanisms of SE are far from clear. Here, we show the genomewide profiles of mi RNAs/si RNAs and their target genes in nonembryogenic and embryogenic tissues of 'Valencia' sweet orange. By high-throughput sequencing ( HTS) of small RNAs and RNA degradome tags, we identified 50 known and 45 novel mi RNAs, 130 miniature inverted-repeat transposable elements ( MITEs) derived, 94 other and 235 phased small interfering RNAs (si RNAs), as well as 203 target genes. The majority of the abundantly expressed mi RNAs/si RNAs exhibit lower expression levels in embryogenic callus ( EC) or during SE process than in nonembryogenic callus ( NEC), which is supposed to derepress the target genes that are involved in development and stress response, thus to activate the biological processes required for cell differentiation. However, the conserved csi-miR156a/b, miR164b and 171c directed suppression of specific transcription factors ( TFs) are supposed to inactivate the postembryonic growth thus to maintain normal SE. In this study, mi RNA- and si RNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved mi RNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE. [ABSTRACT FROM AUTHOR]

Key Message: 2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement. Abstract: Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2 x × 4 x crosses using polyembryonic 'Nadorcott' tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2 n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2 n megagametophytes. Based on the genotypes of 2 n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2 n megagametophyte formation in 'Nadorcott' tangor. The elucidation of the mechanism of 2 n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus. [ABSTRACT FROM AUTHOR]

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30 °C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14 + U6 for SE tissues and snoR14 + U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14 + U6, snoR14 + U5 were identified. [ABSTRACT FROM AUTHOR]

Abstract: Most of what we know about the molecular genetics of flowering time regulation comes from studies in the model plants. However, little is known about the regulation of flowering transition in perennial species or in species with particular growth habits compared with model plants. Here comparative transcriptome and proteome profiling of spring shoots was performed on an early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata) and its wild-type. A total of 19,215 read-mapped genes were observed in two genotypes by RNA-Seq. Of these, 1450 and 1159 genes were specifically observed in the mutant and wild-type libraries, respectively. There were 355 genes that were expressed differently in the two genotypes. A total of 1664 proteins were identified by the iTRAQ technique, and transcript and protein profiles were parallel across the time course for 50% of the comparisons made, but divergent patterns were also observed, indicative of post-transcriptional events. In addition, a global survey of messenger RNA splicing events identified 16,343 splice junctions among 12,688 genes and showed that alternative 3′ splice is the most prevalent form of alternative splicing. We further identify 5698 novel transcribed regions that are not overlapping with annotated citrus transcriptome in two genotypes. Understanding of the regulation of flowering transition in citrus can help in the development of new genetic or management strategies to improve fruit production. [Copyright &y& Elsevier]

Background: Seedlessness 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. Results: An 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. Conclusion: Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future. [ABSTRACT FROM AUTHOR]

Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor ( Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange ( C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy level conversion can affect transgene expression and citrus diploid cybrid and allotetraploid somatic hybrid represents another example of gene regulation coupled to ploidy. [ABSTRACT FROM AUTHOR]

Abstract: The present study was to assess informativeness and efficiency of three different molecular markers for genetic diversity among 24 Citrus and its relative species. Sixty one SSR, 33 SRAP and 24 CAPS-SNP markers were used to evaluate the level of polymorphism and discriminating capacity. A total of 596, 656 and 135 polymorphic amplicons were observed in SSR, SRAP and CAPS-SNP markers with average polymorphism information content (PIC) of 0.97, 0.98 and 0.89, respectively. High levels of polymorphism were recorded for SSR and SRAP compared with CAPS-SNP markers. The highest correlations (r =0.930) were obtained between SSR and SRAP markers, whereas SSR and CAPS-SNP were poorly correlated (r =0.833). Cluster analysis was performed to construct dendrograms using UPGMA. And the dendrogram from SSR data was most congruent with the general dendrogram. These findings provide basis for future efficient use of these molecular markers in the genetic analysis of Citrus and its relatives. [Copyright &y& Elsevier]