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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 two 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. 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.

Cytoplasmic male sterility (CMS) has long been used to produce seedless fruits in perennial woody crops like citrus. A male sterile somatic cybrid citrus (G1+HBP) was generated from protoplast fusion between a CMS callus parent ‘Guoqing No. 1’ Satsuma mandarin (Citrus unshiu, G1) and a fertile mesophyll parent Hirado Buntan pummelo (Citrus grandis, HBP). To uncover the male sterile mechanism of G1+HBP, we compared the transcriptome profiles of stamen organ and cell types at five stages between G1+HBP and HBP, including the initial stamen primordia, enlarged stamen primordia, pollen mother cells, tetrads and microspores captured by laser microdissection (LMD). The stamen organ and cell types showed distinct gene expression profiles. A majority of genes involved in stamen development were differentially expressed, especially CgAP3.2 that was downregulated in enlarged stamen primordia and upregulated in tetrads of G1+HBP compared with HBP. The jasmonic acid (JA) and auxin related biological processes were enriched among the differential expression genes (DEGs) of stamen primordia, and the content of JA biosynthesis metabolites was higher in flower buds and anthers of G1+HBP. In contrast, the content of auxin biosynthesis metabolites was lower in G1+HBP. The mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) processes were enriched among the DEGs in stamen primordia, meiocytes and microspores, indicating the dysfunction of mitochondria in stamen organ and cell types of G1+HBP. Taken together, the malfunction of mitochondria-nuclear interaction might cause disorder in stamen development, and thus lead to male sterility in the citrus cybrid.

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.

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.

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.

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.

Cytoplasmic male sterility (CMS) and seedlessness are highly desirable traits for citrus. However, a high-quality reference mitochondrial genome of citrus is unavailable, and the mechanisms of CMS remain to be elucidated in citrus. We previously generated a male sterile somatic cybrid of pummelo (G1+HBP) by fusing protoplasts from CMS ‘Guoqing No. 1’ Satsuma mandarin (G1) and fertile ‘Hirado Buntan’ pummelo (HBP). Here, we sequenced and assembled the mitochondrial genome of G1, G1+HBP, and HBP into circular molecules of 521.6 kb, 538.4 kb, and 518.3 kb, respectively. Mitochondrial genome of citrus encodes 35–36 proteins, three ribosomal RNAs, and 21 tRNAs. Comparative analysis indicated that nuclear genome and chloroplast genome of the cybrid are from HBP, whereas the mitochondrial genome sequences of the cybrid and G1 have 96.9% synteny, and that the cybrid has an extra recombinant mitochondrial DNA fragment of only 16.8 kb. Compared with the fertile HBP mitochondrial genome, 21 and 20 CMS-specific mitotype-specific sequences (MSSs), and 89 and 70 CMS-specific ORFs were identified in the cybrid and G1 mitochondrial genomes, respectively. Among them, 57 ORFs that co-transcribed with the same mitochondrial genes in the cybrid and G1 were identified as the CMS-specific ORFs. The assembly of mitochondrial genomes and the identification of the CMS-specific ORFs provide basis for determination of the CMS-associated gene and elucidation of CMS mechanisms 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.

The phenotypic variations that follow polyploidization are expected to improve agricultural productivity and efficiency [ 1 ]. However, the effect of polyploidization on plant metabolism has rarely been studied. This study evaluated the metabolic alterations that followed autotetraploidization in the fruit of Ponkan mandarin (C. reticulata Blanco) for three consecutive years and explored the underlying changes to the transcriptome. The autotetraploid (4x) Ponkan fruit had higher levels of total acids, ascorbic acid and total phenolic compounds than the diploid (2x). The primary metabolites especially the organic acids tended to accumulate at higher levels in the 4x fruit. Conversely, two major groups of secondary metabolites (i.e. flavonoids and carotenoids) tended to accumulate at lower levels. The expression levels of citric acid biosynthesis-related genes were unaltered in 4x fruit compared to the 2x fruit. Additionally, genes associated with the transport and utilization of citric acid were significantly down-regulated during ripening, which might induce increases in the levels of citric acid in the 4x fruit. Lower levels of flavonoids and carotenoids in the 4x fruit are potentially associated with decreases in the transport and utilization of citric acid, which is an important metabolite. Citric acid contributes to respiration by serving as an intermediated in the tricarboxylic acid cycle (TCA) and also provides carbon for the production of secondary metabolites. This study demonstrates that polyploidization can influence metabolism in plants.

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.

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.

Citrus allotetraploid somatic hybrids obtained via protoplast fusion have been employed as pollen/seed parents in interploidy crosses to produce triploid plants aiming to develop seedless citrus. However, their meiotic behavior, which directs the parental heterozygosity transmission, has been rarely explored. In this study, the meiotic inheritance pattern was evaluated in a citrus allotetraploid somatic hybrid between ‘Nova’ tangelo and ‘HB’ (Hirado Buntan) pummelo through cytogenetic observation of meiosis and segregation analysis using 18 simple sequence repeat (SSR) markers. Meiotic analysis of pollen mother cells (PMCs) showed a high rate of quadrivalents (31.6 %) in this allotetraploid although bivalents were predominant (57.6 %). For segregation analysis, the meiotic chromosome-pairing behavior was analyzed by genotyping a triploid population derived from a cross between diploid ‘Nadorcott’ tangor and this allotetraploid using the microsatellite DNA allele counting-peak ratios (MAC-PR) method, from which the genotypes of diploid male gametes were inferred. In agreement with the expectation of cytogenetic analysis, a mixture of diploid, tetraploid, and intermediated inheritance patterns was deduced due to the co-existence of preferential, non-preferential, and intermediate pairing models among all analyzed loci. Parental heterozygosity transmission was further analyzed among 14 loci where each chromosome in the allotetraploid could be clearly tracked. It showed that the mean value of 23.8 % intra-parental and 76.2 % inter-parental heterozygosity was transmitted. The preferential pairing pattern at the loci displaying an intermediate inheritance pattern was also discussed.

2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement. 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 2x × 4x 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 2n 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 2n megagametophytes. Based on the genotypes of 2n 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 2n megagametophyte formation in ‘Nadorcott’ tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.