Genomic conservation of crop wild relatives: A case study of citrus.

Conservation of crop wild relatives is critical for plant breeding and food security. The lack of clarity on the genetic factors that lead to endangered status or extinction create difficulties when attempting to develop concrete recommendations for conserving a citrus wild relative: the wild relatives of crops. Here, we evaluate the conservation of wild kumquat (Fortunella hindsii) using genomic, geographical, environmental, and phenotypic data, and forward simulations. Genome resequencing data from 73 accessions from the Fortunella genus were combined to investigate population structure, demography, inbreeding, introgression, and genetic load. Population structure was correlated with reproductive type (i.e., sexual and apomictic) and with a significant differentiation within the sexually reproducing population. The effective population size for one of the sexually reproducing subpopulations has recently declined to ~1,000, resulting in high levels of inbreeding. In particular, we found that 58% of the ecological niche overlapped between wild and cultivated populations and that there was extensive introgression into wild samples from cultivated populations. Interestingly, the introgression pattern and accumulation of genetic load may be influenced by the type of reproduction. In wild apomictic samples, the introgressed regions were primarily heterozygous, and genome-wide deleterious variants were hidden in the heterozygous state. In contrast, wild sexually reproducing samples carried a higher recessive deleterious burden. Furthermore, we also found that sexually reproducing samples were self-incompatible, which prevented the reduction of genetic diversity by selfing. Our population genomic analyses provide specific recommendations for distinct reproductive types and monitoring during conservation. This study highlights the genomic landscape of a wild relative of citrus and provides recommendations for the conservation of crop wild relatives.

Pan-mitogenomics reveals the genetic basis of cytonuclear conflicts in citrus hybridization, domestication, and diversification.

Although interactions between the cytoplasmic and nuclear genomes occurred during diversification of many plants, the evolutionary conflicts due to cytonuclear interactions are poorly understood in crop breeding. Here, we constructed a pan-mitogenome and identified chimeric open reading frames (ORFs) generated by extensive structural variations (SVs). Meanwhile, short reads from 184 accessions of citrus species were combined to construct three variation maps for the nuclear, mitochondrial, and chloroplast genomes. The population genomic data showed discordant topologies between the cytoplasmic and nuclear genomes because of differences in mutation rates and levels of heteroplasmy from paternal leakage. An analysis of species-specific SVs indicated that mitochondrial heteroplasmy was common and that chloroplast heteroplasmy was undetectable. Interestingly, we found a prominent divergence in the mitogenomes and the highest genetic load in the, which may provide the basis for cytoplasmic male sterility (CMS) and thus influence the reshuffling of the cytoplasmic and nuclear genomes during hybridization. Using cytoplasmic replacement experiments, we identified a type of species-specific CMS in mandarin related to two chimeric mitochondrial genes. Our analyses indicate that cytoplasmic genomes from mandarin have rarely been maintained in hybrids and that paternal leakage produced very low levels of mitochondrial heteroplasmy in mandarin. A genome-wide association study (GWAS) provided evidence for three nuclear genes that encode pentatricopeptide repeat (PPR) proteins contributing to the cytonuclear interactions in the Citrus genus. Our study demonstrates the occurrence of evolutionary conflicts between cytoplasmic and nuclear genomes in citrus and has important implications for genetics and breeding.

Genomic analyses provide insights into peach local adaptation and responses to climate change.

The environment has constantly shaped plant genomes, but the genetic bases underlying how plants adapt to environmental influences remain largely unknown. We constructed a high-density genomic variation map of 263 geographically representative peach landraces and wild relatives. A combination of whole-genome selection scans and genome-wide environmental association studies (GWEAS) was performed to reveal the genomic bases of peach adaptation to diverse climates. A total of 2092 selective sweeps that underlie local adaptation to both mild and extreme climates were identified, including 339 sweeps conferring genomic pattern of adaptation to high altitudes. Using genome-wide environmental association studies (GWEAS), a total of 2755 genomic loci strongly associated with 51 specific environmental variables were detected. The molecular mechanism underlying adaptive evolution of high drought, strong UVB, cold hardiness, sugar content, flesh color, and bloom date were revealed. Finally, based on 30 yr of observation, a candidate gene associated with bloom date advance, representing peach responses to global warming, was identified. Collectively, our study provides insights into molecular bases of how environments have shaped peach genomes by natural selection and adds candidate genes for future studies on evolutionary genetics, adaptation to climate changes, and breeding.

The transcriptional regulatory module CsHB5-CsbZIP44 positively regulates abscisic acid-mediated carotenoid biosynthesis in citrus (Citrus spp.).

Carotenoids contribute to fruit coloration and are valuable sources of provitamin A in the human diet. Abscisic acid (ABA) plays an essential role in fruit coloration during citrus fruit ripening, but little is known about the underlying mechanisms. Here, we identified a novel bZIP transcription activator called CsbZIP44, which serves as a central regulator of ABA-mediated citrus carotenoid biosynthesis. CsbZIP44 directly binds to the promoters of four carotenoid metabolism-related genes (CsDXR, CsGGPPs, CsBCH1 and CsNCED2) and activates their expression. Furthermore, our research indicates that CsHB5, a positive regulator of ABA and carotenoid-driven processes, activates the expression of CsbZIP44 by binding to its promoter. Additionally, CsHB5 interacts with CsbZIP44 to form a transcriptional regulatory module CsHB5-CsbZIP44, which is responsive to ABA induction and promotes carotenoid accumulation in citrus. Interestingly, we also discover a positive feedback regulation loop between the ABA signal and carotenoid biosynthesis mediated by the CsHB5-CsbZIP44 transcriptional regulatory module. Our findings show that CsHB5-CsbZIP44 precisely modulates ABA signal-mediated carotenoid metabolism, providing an effective strategy for quality improvement of citrus fruit and other crops.

MADS-box protein PpDAM6 regulates chilling requirement-mediated dormancy and bud break in peach.

Bud dormancy is crucial for winter survival and is characterized by the inability of the bud meristem to respond to growth-promotive signals before the chilling requirement (CR) is met. However, our understanding of the genetic mechanism regulating CR and bud dormancy remains limited. This study identified PpDAM6 (DORMANCY-ASSOCIATED MADS-box) as a key gene for CR using a genome-wide association study analysis based on structural variations in 345 peach (Prunus persica (L.) Batsch) accessions. The function of PpDAM6 in CR regulation was demonstrated by transiently silencing the gene in peach buds and stably overexpressing the gene in transgenic apple (Malus × domestica) plants. The results showed an evolutionarily conserved function of PpDAM6 in regulating bud dormancy release, followed by vegetative growth and flowering, in peach and apple. The 30-bp deletion in the PpDAM6 promoter was substantially associated with reducing PpDAM6 expression in low-CR accessions. A PCR marker based on the 30-bp indel was developed to distinguish peach plants with non-low and low CR. Modification of the H3K27me3 marker at the PpDAM6 locus showed no apparent change across the dormancy process in low- and non-low- CR cultivars. Additionally, H3K27me3 modification occurred earlier in low-CR cultivars on a genome-wide scale. PpDAM6 could mediate cell-cell communication by inducing the expression of the downstream genes PpNCED1 (9-cis-epoxycarotenoid dioxygenase 1), encoding a key enzyme for ABA biosynthesis, and CALS (CALLOSE SYNTHASE), encoding callose synthase. We shed light on a gene regulatory network formed by PpDAM6-containing complexes that mediate CR underlying dormancy and bud break in peach. A better understanding of the genetic basis for natural variations of CR can help breeders develop cultivars with different CR for growing in different geographical regions.

miR171-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 regulate somatic embryogenesis in citrus.

Somatic embryogenesis (SE) is a key regeneration pathway in various biotechnology approaches to crop improvement, especially for economically important perennial woody crops like citrus. However, maintenance of SE capability has long been a challenge and becomes a bottleneck in biotechnology-facilitated plant improvement. In the embryogenic callus (EC) of citrus, we identified 2 csi-miR171c-targeted SCARECROW-LIKE genes CsSCL2 and CsSCL3 (CsSCL2/3), which exert positive feedback regulation on csi-miR171c expression. Suppression of CsSCL2 expression by RNA interference (RNAi) enhanced SE in citrus callus. A thioredoxin superfamily protein CsClot was identified as an interactive protein of CsSCL2/3. Overexpression of CsClot disturbed reactive oxygen species (ROS) homeostasis in EC and enhanced SE. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq identified 660 genes directly suppressed by CsSCL2 that were enriched in biological processes including development-related processes, auxin signaling pathway, and cell wall organization. CsSCL2/3 bound to the promoters of regeneration-related genes, such as WUSCHEL-RELATED HOMEOBOX 2 (CsWOX2), CsWOX13, and Lateral Organ Boundaries Domain 40 (LBD40), and repressed their expression. Overall, CsSCL2/3 modulate ROS homeostasis through the interactive protein CsClot and directly suppress the expression of regeneration-related genes, thus regulating SE in citrus. We uncovered a regulatory pathway of miR171c-targeted CsSCL2/3 in SE, which shed light on the mechanism of SE and regeneration capability maintenance in citrus.

Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus.

Polyploidization leads to novel phenotypes and is a major force in evolution. However, the relationship between the evolution of new traits and variations in the post-translational modifications (PTM) of proteins during polyploidization has not been studied. Acetylation of lysine residues is a common protein PTM that plays a critical regulatory role in central metabolism. To test whether changes in metabolism in citrus fruit is associated with the reprogramming of lysine acetylation (Kac) in non-histone proteins during allotetraploidization, we performed a global acetylome analysis of fruits from a synthetic allotetraploid citrus and its diploid parents. A total of 4,175 Kac sites were identified on 1,640 proteins involved in a wide range of fruit traits. In the allotetraploid, parental dominance (i.e. resemblance to one of the two parents) in specific fruit traits, such as fruit acidity and flavonol metabolism, was highly associated with parental Kac level dominance in pertinent enzymes. This association is due to Kac-mediated regulation of enzyme activity. Moreover, protein Kac probably contributes to the discordance between the transcriptomic and proteomic variations during allotetraploidization. The acetylome reprogramming can be partially explained by the expression pattern of several lysine deacetylases (KDACs). Overexpression of silent information regulator 2 (CgSRT2) and histone deacetylase 8 (CgHDA8) diverted metabolic flux from primary metabolism to secondary metabolism and partially restored a metabolic status to the allotetraploid, which expressed attenuated levels of CgSRT2 and CgHDA8. Additionally, KDAC inhibitor treatment greatly altered metabolism in citrus fruit. Collectively, these findings reveal the important role of acetylome reprogramming in trait evolution during polyploidization.

GRAS transcription factor LOSS OF AXILLARY MERISTEMS is essential for stamen and runner formation in wild strawberry.

Axillary bud development is a major factor that impacts plant architecture. A runner is an elongated shoot that develops from axillary bud and is frequently used for clonal propagation of strawberry. However, the genetic control underlying runner production is largely unknown. Here, we identified and characterized loss of axillary meristems (lam), an ethyl methanesulfonate-induced mutant of the diploid woodland strawberry (Fragaria vesca) that lacked stamens in flowers and had reduced numbers of branch crowns and runners. The reduced branch crown and runner phenotypes were caused by a failure of axillary meristem initiation. The causative mutation of lam was located in FvH4_3g41310, which encodes a GRAS transcription factor, and was validated by a complementation test. lamCR mutants generated by CRISPR/Cas9 produced flowers without stamens and had fewer runners than the wild-type. LAM was broadly expressed in meristematic tissues. Gibberellic acid (GA) application induced runner outgrowth from the remaining buds in lam, but failed to do so at the empty axils of lam. In contrast, treatment with the GA biosynthesis inhibitor paclobutrazol converted the runners into branch crowns. Moreover, genetic studies indicated that lam is epistatic to suppressor of runnerless (srl), a mutant of FveRGA1 in the GA pathway, during runner formation. Our results demonstrate that LAM is required for stamen and runner formation and acts sequentially with GA from bud initiation to runner outgrowth, providing insights into the molecular regulation of these economically important organs in strawberry.

miR156 regulates somatic embryogenesis by modulating starch accumulation in citrus.

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.

miR171 modulates induction of somatic embryogenesis in citrus callus.

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.

Reduced expression of a subunit gene of sucrose non-fermenting 1 related kinase, PpSnRK1ßγ, confers flat fruit abortion in peach by regulating sugar and starch metabolism.

BACKGROUND: Fruit abortion is a major limiting factor for fruit production. In flat peach, fruit abortion is present in the whole tree of some accessions during early fruit development. However, the physiological factors and genetic mechanism underlying flat fruit abortion remain largely elusive. RESULTS: In this study, we have revealed that the fertilization process was accomplished and the reduction of sucrose and starch contents might result in flat fruit abortion. By combining association and gene expression analysis, a key candidate gene, PpSnRK1ßγ, was identified. A 1.67-Mb inversion co-segregated with flat fruit shape altered the promoter activity of PpSnRK1ßγ, resulting in much lower expression in aborting flat peach. Ectopic transformation in tomato and transient overexpression in peach fruit have shown that PpSnRK1ßγ could increase sugar and starch contents. Comparative transcriptome analysis further confirmed that PpSnRK1ßγ participated in carbohydrate metabolism. Subcellular localization found that PpSnRK1ßγ was located in nucleus. CONCLUSIONS: This study provides a possible reason for flat fruit abortion and identified a critical candidate gene, PpSnRK1ßγ, that might be responsible for flat fruit abortion in peach. The results will provide great help in peach breeding and facilitate gene identification for fruit abortion in other plant species.

The miR399-CsUBC24 Module Regulates Reproductive Development and Male Fertility in Citrus.

MicroRNA399 (miR399) regulates phosphate homeostasis in plants by down-regulating the expression of PHOSPHATE2 (PHO2, or UBC24 encoding the ubiquitin-conjugating E2 enzyme). We previously identified CsmiR399a.1 in a small RNA sequencing screen of a male-sterile somatic cytoplasmic hybrid (or cybrid) of pummelo (Citrus grandis). Here, we report that miR399 affects reproductive development and male fertility in citrus. Down-regulation of CsmiR399a.1 using a short tandem target mimic (STTM) led to abnormal floral development, inhibition of anther dehiscence, and decreased pollen fertility. When grown in inorganic phosphate (Pi)-sufficient conditions, CsmiR399a.1-STTM plants had lower total phosphorus content in their leaves than the wild type and showed typical symptoms of Pi deficiency. In CsmiR399a.1-STTM plants, the expression of genes involved in starch metabolism and Pi homeostasis was significantly different than in the wild type. Thus, we conclude that miR399-STTM mimicked Pi deficiency, disturbed starch metabolism, and was responsible for pollen grain collapse in the transgenic lines. We identified CsUBC24, a citrus homolog of Arabidopsis (Arabidopsis thaliana) AtUBC24 (PHO2), as a target of CsmiR399a.1 that physically interacts with the floral development regulators SEPALLATA family (CsSEP1.1, CsSEP1.2, and CsSEP3) and the anther dehiscence regulator INDUCER OF CBF EXPRESSION1 (CsICE1). We hypothesize that CsUBC24 downregulates the CsSEPs, which disrupts the floral meristem identity regulatory network and leads to developmental abnormalities in flowers. By interacting with CsICE1, CsUBC24 disturbs stomate function on the anther surface, which inhibits anther dehiscence. These findings indicate that a miR399-based mechanism influences both reproductive development and male fertility in citrus.

Genome-wide analysis of the citrus B3 superfamily and their association with somatic embryogenesis.

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.

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.

miR156-SPL modules regulate induction of somatic embryogenesis in citrus callus.

miR156 is a highly conserved plant miRNA and has been extensively studied because of its versatile roles in plant development. Here, we report a novel role of miR156 in regulating somatic embryogenesis (SE) in citrus, one of the most widely cultivated fruit crops in the world. SE is an important means of in vitro regeneration, but over the course of long-term sub-culturing there is always a decline in the SE potential of the preserved citrus embryogenic callus, and this represents a key obstacle for citrus biotechnology. In this study, the SE competence of citrus callus of wild kumquat (Fortunella hindsii) was significantly enhanced by either overexpression of csi-miR156a or by individual knock-down of the two target genes, CsSPL3 and CsSPL14, indicating that the effect of miR156-SPL modules was established during the initial phases of SE induction. Biological processes that might promote SE in response to miR156 overexpression were explored using RNA-seq, and mainly included hormone signaling pathways, stress responses, DNA methylation, and the cell cycle. CsAKIN10 was identified as interacting protein of CsSPL14. Our results provide insights into the regulatory pathway through which miR156-SPL modules enhance the SE potential of citrus callus, and provide a theoretical basis for improvement of plant SE competence.

An R2R3-MYB transcription factor represses the transformation of α- and ß-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulate.

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and ß-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and ß-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of ß-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and ß-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.

High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis).

BACKGROUND: G1 + HBP is a male sterile cybrid line with nuclear genome from Hirado Buntan pummelo (C. grandis Osbeck) (HBP) and mitochondrial genome from "Guoqing No.1" (G1, Satsuma mandarin), which provides a good opportunity to study male sterility and nuclear-cytoplasmic cross talk in citrus. High-throughput sRNA and degradome sequencing were applied to identify miRNAs and their targets in G1 + HBP and its fertile type HBP during reproductive development. RESULTS: A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2) and transport inhibitor response 1 (TIR1). Eight target genes were confirmed to be sliced by corresponding miRNAs using 5' RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between sterile line G1 + HBP and fertile line HBP were identified. Differential expression of miRNAs and their target genes between two lines was validated by quantitative RT-PCR, and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulatory mechanism of miR167a was investigated by yeast one-hybrid and dual-luciferase assays that one dehydrate responsive element binding (DREB) transcription factor binds to miR167a promoter and transcriptionally repress miR167 expression. CONCLUSION: Our study reveals the altered expression of miRNAs and their target genes in a male sterile line of pummelo and highlights that miRNA regulatory network may be involved in floral bud development and cytoplasmic male sterility in citrus.

Genome-scale mRNA and small RNA transcriptomic insights into initiation of citrus apomixis.

Nucellar embryony (NE) is an adventitious form of apomixis common in citrus, wherein asexual embryos initiate directly from nucellar cells surrounding the embryo sac. NE enables the fixation of desirable agronomic traits and the production of clonal offspring of virus-free rootstock, but impedes progress in hybrid breeding. In spite of the great importance of NE in citrus breeding and commercial production, little is understood about the underlying molecular mechanisms. In this study, the stages of nucellar embryo initiation (NEI) were determined for two polyembryonic citrus cultivars via histological observation. To explore the genes and regulatory pathways involved in NEI, we performed mRNA-seq and sRNA-seq analyses of ovules immediately prior to and at stages during NEI in the two pairs of cultivars. A total of 305 differentially expressed genes (DEGs) were identified between the poly- and monoembryonic ovules. Gene ontology (GO) analysis revealed that several processes are significantly enriched based on DEGs. In particular, response to stress, and especially response to oxidative stress, was over-represented in polyembryonic ovules. Nearly 150 miRNAs, comprising ~90 conserved and ~60 novel miRNAs, were identified in the ovules of either cultivar pair. Only two differentially expressed miRNAs (DEMs) were identified, of which the novel miRN23-5p was repressed whereas the targets accumulated in the polyembryonic ovules. This integrated study on the transcriptional and post-transcriptional regulatory profiles between poly- and monoembryonic citrus ovules provides new insights into the mechanism of NE, which should contribute to revealing the regulatory mechanisms of plant apomixis.

Comparative metabolic and transcriptional analysis of a doubled diploid and its diploid citrus rootstock (C. junos cv. Ziyang xiangcheng) suggests its potential value for stress resistance improvement.

BACKGROUND: Polyploidy has often been considered to confer plants a better adaptation to environmental stresses. Tetraploid citrus rootstocks are expected to have stronger stress tolerance than diploid. Plenty of doubled diploid citrus plants were exploited from diploid species for citrus rootstock improvement. However, limited metabolic and molecular information related to tetraploidization is currently available at a systemic biological level. This study aimed to evaluate the occurrence and extent of metabolic and transcriptional changes induced by tetraploidization in Ziyang xiangcheng (Citrus junos Sieb. ex Tanaka), which is a special citrus germplasm native to China and widely used as an iron deficiency tolerant citrus rootstock. RESULTS: Doubled diploid Ziyang xiangcheng has typical morphological and anatomical features such as shorter plant height, larger and thicker leaves, bigger stomata and lower stomatal density, compared to its diploid parent. GC-MS (Gas chromatography coupled to mass spectrometry) analysis revealed that tetraploidization has an activation effect on the accumulation of primary metabolites in leaves; many stress-related metabolites such as sucrose, proline and γ-aminobutyric acid (GABA) was remarkably up-regulated in doubled diploid. However, LC-QTOF-MS (Liquid chromatography quadrupole time-of-flight mass spectrometry) analysis demonstrated that tetraploidization has an inhibition effect on the accumulation of secondary metabolites in leaves; all the 33 flavones were down-regulated while all the 6 flavanones were up-regulated in 4x. By RNA-seq analysis, only 212 genes (0.8% of detected genes) are found significantly differentially expressed between 2x and 4x leaves. Notably, those genes were highly related to stress-response functions, including responses to salt stress, water and abscisic acid. Interestingly, the transcriptional divergence could not explain the metabolic changes, probably due to post-transcriptional regulation. CONCLUSION: Taken together, tetraploidization induced considerable changes in leaf primary and secondary metabolite accumulation in Ziyang xiangcheng. However, the effect of tetraploidization on transcriptome is limited. Compared to diploid, higher expression level of stress related genes and higher content of stress related metabolites in doubled diploid could be beneficial for its stress tolerance.

Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis.

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 miRNAs/siRNAs 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 miRNAs, 130 miniature inverted-repeat transposable elements (MITEs) derived, 94 other and 235 phased small interfering RNAs (siRNAs), as well as 203 target genes. The majority of the abundantly expressed miRNAs/siRNAs 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, miRNA- and siRNA-mediated silencing of target genes was found under sophisticated regulation in citrus SE system; the enhancement effect of specific conserved miRNAs on SE was discussed, providing new clues for future investigation of mechanisms that control SE.