Division Restitution Research Articles

Intergeneric chromosome-specific painting reveals differential chromosomal transmission from Tripidium arundinaceum in sugarcane progeny

Sugarcane has recently attracted increasing attention for its potential as a source of sugar and bioethanol, so increasing its yield is essential to ensure the sugar security and bioenergy production. Intergeneric hybridization is a highly efficient method to produce new genetic variants of crop plants, particularly those species with high ploidy such as sugarcane (Saccharum spp.). Tripidium arundinaceum exhibits many desirable agronomic traits, and has been widely studied to produce hybrids with improved stress tolerance and other characteristics in sugarcane breeding. However, the genetic relationship between T. arundinaceum and Saccharum species, and the individual T. arundinaceum chromosomal compositions in sugarcane hybrids are still elusive. Here we used whole-genome single-nucleotide polymorphisms (SNPs) to ascertain the phylogenetic relationships between these species and found that T. arundinaceum is more closely related to Saccharum than Sorghum, in contrast to the previous narrow genetic analyses using chloroplast DNA. Additionally, oligonucleotide (oligo)-based chromosome-specific painting derived from Saccharum officinarum was able to distinctly identify the chromosomes of T. arundinaceum. We developed the oligo-genomic in situ hybridization (GISH) system for the first time, to unveil the novel chromosome translocations and the transmission of individual T. arundinaceum chromosomes in sugarcane progeny. Notably, we discovered that the chromosomal transmission of T. arundinaceum exhibited several different inheritance modes, including n, 2n, and over 2n in the BC1 progenies. Such inheritance patterns may have resulted from first division restitution (FDR) or FDR+nondisjunction of a chromosome with the sister chromatids in the second meiosis division/second division restitution (FDR+NSC/SDR) model during meiosis. These results will be of substantial benefit for the further selection of T. arundinaceum chromosomes for sugarcane genetic improvement.

Cytological origination of the first found allotriploid tree peony, Paeonia × lemoinei ‘Oukan’ (AAB), reveled by molecular karyotype comparation

Polyploid breeding is one of the most important approaches to improve the traits of cultivated plants, but there has been no feasible method to get polyploid tree peony. In this study, we revealed a sexual triploidization approach from a diploid intersubsectional hybrid tree peony, Paeonia × lemoinei ‘High Noon’, to an allotriploid one, P. × lemoinei ‘Oukan’, by accurate karyotyping based on genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). The ancestor genomes from P. suffruticosa and P. delavayi, which were defined as A-genome and B-genome respectively, were clearly distinguished by GISH in ‘High Noon’ and ‘Oukan’. Nonhomologous chromosomes were successfully recognized by means of 5S and 45S rDNA FISH. Karyotype comparation based on GISH and FISH indicated that the B-genome and one of the two A-genomes in ‘Oukan’ (2n=3x=15, AAB) were highly consistent to the corresponding genomes in ‘High Noon’ (2n=2x=10, AB), implying the triploid origination via 2n gametes produced by first division restitution (FDR). In addition, it was demonstrated for the first time that the dual in series sites of 5S rDNA located on the short arm of chromosome 3 in both A and B-genomes. Besides the familiar strong 45S rDNA signals observed at terminal region of short arms, single weak 45S rDNA signal was newly detected at terminal region in the short arm of certain chromosome 1 in A-genome, and at interstitial-distal region in the long arm of chromosome 5 in both genomes. These findings provide a proof and theoretical support for tree peony polyploid breeding, and an accurate karyotyping method for genetic research of plants in Paeonia.

Asynapsis and meiotic restitution in tomato male meiosis induced by heat stress.

Susceptibility of the reproductive system to temperature fluctuations is a recurrent problem for crop production under a changing climate. The damage is complex as multiple processes in male and female gamete formation are affected, but in general, particularly pollen production is impaired. Here, the impact of short periods of elevated temperature on male meiosis of tomato (Solanum lycopersicon L.) is reported. Meiocytes in early stage flower buds exposed to heat stress (>35°C) exhibit impaired homolog synapsis resulting in partial to complete omission of chiasmata formation. In the absence of chiasmata, univalents segregate randomly developing unbalanced tetrads and polyads resulting in aneuploid spores. However, most heat-stressed meiotic buds primarily contain balanced dyads, indicating a propensity to execute meiotic restitution. With most meiocytes exhibiting a complete loss of chiasma formation and concomitantly showing a mitotic-like division, heat stress triggers first division restitution resulting in clonal spores. These findings corroborate with the plasticity of male meiosis under heat and establish a natural route for the induction of sexual polyploidization in plants and the engineering of clonal seed.

Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollen formation in a woody plant.

The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats(SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

Occurrence of triploids in oil palm and their origin.

Oil palms showing exceptional vigour and dubbed as 'giant palms' were identified in some progeny during breeding. A panel of phenotypical traits were studied to characterize these trees. The hypothesis that gigantism and other anomalies might be linked to polyploidy was investigated. Twenty sib pairs of palms from different crosses, each comprising a giant and a normal oil palm, were studied by flow cytometry with rice 'Nipponbare' as standard reference. In parallel, palms were assessed in the field using 11 phenotypic traits. A principal component analysis (PCA) was conducted to define relationships between these phenotypical traits, and a linear discriminant analysis (LDA) to predict ploidy level and giant classification. Finally, a co-dominant molecular marker study was implemented to highlight the sexual process leading to the formation of 2n gametes. The first group of oil palms presented an oil palm/rice peak ratio of around 4.8 corresponding to diploid oil palms, whereas the second group presented a ratio of around 7, classifying these plants as triploid. The PCA enabled the classification of the plants in three classes: 21 were normal diploid palms; ten were giant diploid palms; while 11 were giant triploid palms. The LDA revealed three predictors for ploidy classification: phyllotaxy, petiole size and circumference of the plant, but surprisingly not height. The molecular study revealed that triploid palms arose from 2n gametes resulting from the second division restitution of meiosis in parents. This study confirms and details the process of sexual polyploidization in oil palm. It also identifies three phenotypical traits to assess the ploidy level of the giant oil palms in the field. In practical terms, our results provide a cheap scientific method to identify polyploid palms in the field.

Cytological Study of 2n Pollen Formation in Musa

Current Musa breeding strategies are complex and time consuming involving the selection of tetraploids from 3x - 2x crosses. Secondary triploids are then obtained by crossing these tetraploids with diploids. Considering the very low hybrid seed set, routine embryo rescue procedures of hybrid seeds and the long growth cycle of banana, it takes approximately 10 - 12 years to produce an acceptable banana hybrid. The banana breeding process could benefit tremendously if triploid bananas could be obtained directly from 2x - 2x crosses through the process of unilateral sexual polyploidization. There are few reports on the mechanisms through which Musa species produce 2n pollen. This study investigated the type of meiotic irregularities that lead to 2n pollen formation in diploid, triploid and tetraploid Musa accessions using cytological analyses. The results showed that aberrations in cytokinesis and karyokinesis during microsporogenesis are possible mechanisms for 2n pollen formation in Musa. The meiotic aberrations described in this study have implications for Musa breeding. It appears that 2n pollen formation in Musa occurs via both first division restitution (FDR) and second division restitution (SDR). FDR is said to be more promising in transferring more heterozygosity from parents to offspring.

Behavior of Centromeres during Restitution of the First Meiotic Division in a Wheat-Rye Hybrid.

In first division restitution (FDR)-type meiosis, univalents congregate on the metaphase I plate and separate sister chromatids in an orderly fashion, producing dyads with somatic chromosome numbers. The second meiotic division is abandoned. The separation of sister chromatids requires separation of otherwise fused sister centromeres and a bipolar attachment to the karyokinetic spindle. This study analyzed packaging of sister centromeres in pollen mother cells (PMCs) in a wheat–rye F1 hybrid with a mixture of standard reductional meiosis and FDR. No indication of sister centromere separation before MI was observed; such separation was clearly only visible in univalents placed on the metaphase plate itself, and only in PMCs undergoing FDR. Even in the FDR, PMCs univalents off the plate retained fused centromeres. Both the orientation and configuration of univalents suggest that some mechanism other than standard interactions with the karyokinetic spindle may be responsible for placing univalents on the plate, at which point sister centromeres are separated and normal amphitelic interaction with the spindle is established. At this point it is not clear at all what univalent delivery mechanism may be at play in the FDR.

Variation of Chromosome Composition in a Full-Sib Population Derived From 2x × 3x Interploidy Cross of Populus.

Interploidy cross commonly results in complex chromosome number and structural variations. In our previous study, a progeny with segregated ploidy levels was produced by an interploidy cross between diploid female parent Populus tomentosa × Populus bolleana clone TB03 and triploid male parent Populus alba × Populus berolinensis ‘Yinzhong’. However, the chromosome compositions of aneuploid genotypes in the progeny were still unclear. In the present study, a microsatellite DNA allele counting–peak ratios (MAC-PR) method was employed to analyze allelic configurations of each genotype to clarify their chromosome compositions, while 45S rDNA fluorescence in situ hybridization (FISH) analysis was used to reveal the mechanism of chromosome number variation. Based on the MAC-PR analysis of 47 polymorphic simple sequence repeat (SSR) markers distributed across all 19 chromosomes of Populus, both chromosomal number and structural variations were detected for the progeny. In the progeny, 26 hypo-triploids, 1 hyper-triploid, 16 hypo-tetraploids, 10 tetraploids, and 5 hyper-tetraploids were found. A total of 13 putative structural variation events (duplications and/or deletions) were detected in 12 genotypes, involved in chromosomes 3, 6, 7, 14, 15, 16, and 18. The 46.2% (six events) structural variation events occurred on chromosome 6, suggesting that there probably is a chromosome breakpoint near the SSR loci of chromosome 6. Based on calculation of the allelic information, the transmission of paternal heterozygosity in the hypo-triploids, hyper-triploid, hypo-tetraploids, tetraploids, and hyper-tetraploids were 0.748, 0.887, 0.830, 0.833, and 0.836, respectively, indicating that the viable pollen gains of the male parent ‘Yinzhong’ were able to transmit high heterozygosity to progeny. Furthermore, 45S rDNA–FISH analysis showed that specific-chromosome segregation feature during meiosis and chromosome appointment in normal and fused daughter nuclei of telophase II of ‘Yinzhong,’ which explained that the formation of aneuploids and tetraploids in the progeny could be attributed to imbalanced meiotic chromosomal segregation and division restitution of ‘Yinzhong,’ The data of chromosomal composition and structural variation of each aneuploid in the full-sib progeny of TB03 × ‘Yinzhong’ lays a foundation for analyzing mechanisms of trait variation relying on chromosome or gene dosages in Populus.

Integrated transcriptome and miRNA sequencing approaches provide insights into salt tolerance in allotriploid Populus cathayana.

Some salt-stress responsive DEGs, mainly involved in ion transmembrane transport, hormone regulation, antioxidant system, osmotic regulation, and some miRNA jointly regulated the salt response process in allotriploid Populus cathayana. The molecular mechanism of plant polyploid stress resistance has been a hot topic in biological research. In this study, Populus diploids and first division restitution (FDR) and second division restitution (SDR) triploids were selected as research materials. All materials were treated with 70mM NaCl solutions for 30days in the same pot environment. We observed the growth state of triploids and diploids and determined the ratio of potassium and sodium ions, peroxidase (POD) activity, proline content, and ABA and jasmonic acid (JA) hormone content in leaves in the same culture environment with the same concentration of NaCl solution treatment. In addition, RNA-seq technology was used to study the differential expression of mRNA and miRNA. The results showed that triploid Populus grew well and the K+ content and the K+/Na+ ratio in the salt treatment were significantly lower than those in the control. The contents of ABA, JA, POD, and proline were increased compared with contents in diploid under salt stress. The salt-stress responsive DEGs were mainly involved in ion transport, cell homeostasis, the MAPK signaling pathway, peroxisome, citric acid cycle, and other salt response and growth pathways. The transcription factors mainly included NAC, MYB, MYB_related and AP2/ERF. Moreover, the differentially expressed miRNAs involved 32 families, including 743 miRNAs related to predicted target genes, among which 22 miRNAs were significantly correlated with salt-stress response genes and related to the regulation of hormones, ion transport, reactive oxygen species (ROS) and other biological processes. Our results provided insights into the physiological and molecular aspects for further research into the response mechanisms of allotriploid Populus cathayana to salt stress. This study provided valuable information for the salt tolerance mechanism of allopolyploids.

Genomics-based discrimination of 2n gamete formation mechanisms in polyploids: a case study in nonaploid Diospyros kaki 'Akiou'.

Unreduced gametes (2n gametes), possessing double the haploid genome, whatever ploidy that happens to be, are a common source of ploidy variation in plant populations. First and second division restitution (FDR and SDR) are the dominant mechanisms of 2n gamete production; all else being equal, FDR gametes have a higher degree of heterozygosity, thus they are advantageous in breeding. The discrimination of these mechanisms from the consequence of hybridization is challenging, especially in higher polyploids, and usually requires information on centromere location. In this study, we propose a genotyping-based strategy to uncover the mechanisms of 2n gamete formation in progeny that has a higher ploidy than its parents. Simulation of 2n gamete production revealed that FDR and SDR pathways can be discriminated based on allele transmission patterns alone without information on centromere location. We applied this strategy to study the formation mechanism of a nonaploid Diospyros kaki ‘Akiou', which was bred via hybridization between D. kaki hexaploid cultivars. The result demonstrated that ‘Akiou' was derived from the fertilization of a normal female gamete by a 2n male gamete and that this 2n gamete was produced through FDR. Consequently, the distinct duplex transmission pattern in the FDR gamete enabled us to infer the genomic characteristics of polyploid persimmon. The method could be tested only for the plant being polypoid, which allows for the ability to discriminate causes of 2n gamete formation using allele dosage in progeny, and will be useful in future studies of polyploid genomics.

UNREDUCED MEGAGAMETOPHYTE FORMATION VIA SECOND DIVISION RESTITUTION CONTRIBUTES TO TETRAPLOID PRODUCTION IN INTERPLOIDY CROSSES WITH 'ORAH' MANDARIN (CITRUSRETICULATA)

<List> <ListItem><ItemContent> • In addition to triploid progeny, tetraploid hybrids derived from the fertilization of 2n megagametophytes are frequently regenerated from 2x × 4x crosses that utilize ‘Orah’ mandarin as the female parent. </ItemContent></ListItem> <ListItem><ItemContent> • Data here indicate that ‘Orah’ mandarin is a cultivar that readily produces 2n megagametophytes. </ItemContent></ListItem> <ListItem><ItemContent> • Second division restitution is the mechanism underlying 2n megagametophyte formation in ‘Orah’ mandarin. </ItemContent></ListItem></List> Seedless fruits are desirable in the citrus fresh fruit market. Triploid production via diploid × tetraploid interploidy crosses is thought to be the most efficient and widely-used strategy for the breeding of seedless citrus. Although ‘Orah’ mandarin has desirable organoleptic qualities, seeds in the fruits weaken its market competitiveness. To produce new seedless cultivars that are similar to ‘Orah’ mandarin, we performed three 2x × 4x crosses using ‘Orah’ mandarin as the seed parent to regenerate triploid plantlets. A total of 182 triploid and 36 tetraploid plantlets were obtained. By analyzing their genetic origins using nine novel single nucleotide polymorphism (SNP) markers, all of the triploids and tetraploids derived from these three crosses were proven to be hybrids. Also, we demonstrated that 2n megagametophyte formation in ‘Orah’ mandarin result in tetraploid production in these three interploidy crosses. These tetraploid plantlets were genotyped using eight pericentromeric SNP markers and nine centromere distal SNP markers. Based on the genotypes of the 2n megagametophytes, the parental heterozygosity rates in 16 SNP loci and all 2n megagametophytes were less than 50%, indicating that second division restitution was the mechanism underlying 2n megagametophyte formation at both the population and individual levels. These triploid hybrids enrich the germplasm available for seedless breeding. Moreover, the tetraploid hybrids are valuable as parents for ploidy breeding for the production of seedless citrus fruits.

Whether Gametophytes are Reduced or Unreduced in Angiosperms Might Be Determined Metabolically.

In angiosperms, meiotic failure coupled with the formation of genetically unreduced gametophytes in ovules (apomeiosis) constitute major components of gametophytic apomixis. These aberrant developmental events are generally thought to be caused by mutation. However, efforts to locate the responsible mutations have failed. Herein, we tested a fundamentally different hypothesis: apomeiosis is a polyphenism of meiosis, with meiosis and apomeiosis being maintained by different states of metabolic homeostasis. Microarray analyses of ovules and pistils were used to differentiate meiotic from apomeiotic processes in Boechera (Brassicaceae). Genes associated with translation, cell division, epigenetic silencing, flowering, and meiosis characterized sexual Boechera (meiotic). In contrast, genes associated with stress responses, abscisic acid signaling, reactive oxygen species production, and stress attenuation mechanisms characterized apomictic Boechera (apomeiotic). We next tested whether these metabolic differences regulate reproductive mode. Apomeiosis switched to meiosis when premeiotic ovules of apomicts were cultured on media that increased oxidative stress. These treatments included drought, starvation, and H2O2 applications. In contrast, meiosis switched to apomeiosis when premeiotic pistils of sexual plants were cultured on media that relieved oxidative stress. These treatments included antioxidants, glucose, abscisic acid, fluridone, and 5-azacytidine. High-frequency apomeiosis was initiated in all sexual species tested: Brassicaceae, Boechera stricta, Boechera exilis, and Arabidopsis thaliana; Fabaceae, Vigna unguiculata; Asteraceae, Antennaria dioica. Unreduced gametophytes formed from ameiotic female and male sporocytes, first division restitution dyads, and nucellar cells. These results are consistent with modes of reproduction and types of apomixis, in natural apomicts, being regulated metabolically.

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species.

In F1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer–protector model.

High temperatures alter cross-over distribution and induce male meiotic restitution in Arabidopsis thaliana

Plant fertility is highly sensitive to elevated temperature. Here, we report that hot spells induce the formation of dyads and triads by disrupting the biogenesis or stability of the radial microtubule arrays (RMAs) at telophase II. Heat-induced meiotic restitution in Arabidopsis is predominantly SDR-type (Second Division Restitution) indicating specific interference with RMAs formed between separated sister chromatids. In addition, elevated temperatures caused distinct deviations in cross-over formation in male meiosis. Synapsis at pachytene was impaired and the obligate cross-over per chromosome was discarded, resulting in partial univalency in meiosis I (MI). At diakinesis, interconnections between non-homologous chromosomes tied separate bivalents together, suggesting heat induces ectopic events of non-homologous recombination. Summarized, heat interferes with male meiotic cross-over designation and cell wall formation, providing a mechanistic basis for plant karyotype change and genome evolution under high temperature conditions.

Sub-Genome Polyploidization Effects on Metabolomic Signatures in Triploid Hybrids of Populus

Allopolyploids are known to have superior advantages such as high growth speed. Triploids have even greater heterozygosity, explaining more phenotypic variance than 2n hybrid F1 and have therefore become new resources in breeding. To date, the metabolomic basis underlying polyploidization vigor remains unclear. Here, we identified and compared 235 metabolites in the shoot apical buds between multiple allo-triploid populations and parental 2n hybrid F1 in Populus via metabolome profiling using liquid chromatography–mass spectrometry (LC–MS) assays. Associations with growth vigor in three types of allo-triploid populations, namely first division restitution (FDR), second division restitution (SDR) and postmeiotic restitution (PMR) generated from doubled 2n female gametes and male gametes of 2n hybrid, were also investigated. Each allo-triploid population has different sub-genome duplicated. Major metabolomes were amino acids, secondary metabolism associated, and carbohydrates. We mapped 181 metabolites into known metabolism pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Ten compounds, i.e., fructose 1,6-diphosphate and xylulose, were more abundant in all allo-triploids than the 2n hybrid. Principal component analysis revealed the abundance of metabolites fell into distinct clusters corresponding to ploidy composition. Heterozygosity in triploids mainly effected the contents of carbohydrates and secondary metabolites rather than lipids. Comparisons between subgroups with different growth rates revealed some carbohydrates and secondary metabolites of flavonoids were positively associated with gene expression and the high growth vigor. The results provided an informative metabolomic basis for factors conferring growth vigor in polyploid Populus.

The different origins of artificially-induced unreduced female gametes and their effect on transmitted parental heterozygosity in Populus

The induction of unreduced gametes via chromosome doubling is an effective way to produce triploids. During this process, the transmitted parental heterozygosity varies by different origins of unreduced gametes. In this study, a total of 110 allotriploid individuals from two cross combinations who shared only one male parent were obtained by chromosome doubling of megaspore via high temperature treatment in Populus tomentosa. Twenty-six SSR loci, at which the allelic configurations of the female parents ‘MC1’ and ‘MC2’ were heterozygous and different from male parent ‘YX1’, were screened. Among them, five pairs of pericentromeric SSR primers were selected to identify the origins of unreduced female gametes. Our results showed that 30 individuals from 110 allotriploids derived from the first division restitution (FDR), and 80 allotriploids from the second division restitution (SDR) in these two cross combinations. The transmitted parental heterozygosity of unreduced female gametes via FDR were 0.824 and 0.769 in two cross combinations, respectively, which was significantly higher than that of SDR (0.395 and 0.396, respectively). The FDR type unreduced gametes with higher heterozygosity transmitted from parents may be more desirable in triploid breeding. However, no significant difference in transmitted parental heterozygosity was found between the two FDR groups and two SDR groups. Correct elucidation of the origins of unreduced gametes may be conducive to their effective utilization for further sexual hybridization in triploid poplar breeding.

Callose is integral to meiotic diplospory of the Taraxacum type: new evidence from ovules of Chondrilla brevirostris (Asteraceae-Cichorioideae)

ABSTRACTAn essential, but still not fully understood, aspect of angiosperms’ reproduction is the issue of callose deposition during early female reproductive processes in sexual and apomictic taxa. It was believed that callose may be a marker for an early identification of the reproduction mode and lack of callose was suggested as characteristic for apomicts. However, our findings clearly prove that meiotic diplospory of the Taraxacum type is accompanied by callose deposition and show that lack of callose is not an obligatory feature of the apomictic reproduction mode. In young ovules of C. brevirostris, callose was a marker of the cell entering the first meiotic division restitution. Synthesis and degradation of callose followed a strictly defined pattern, which differed from that described in the majority of analyzed angiosperms. Callose deposition began at the micropylar pole of the diplosporous megaspore mother cell and, finally, this cell wall polysaccharide showed a bipolar location. This pattern dynamically changed in the later stages of meiotic diplospory and, like in sexual species, it was related to the selection of the functional megaspore. Thus, it seems that callose can play a similar regulatory function in cell-cell communication between somatic tissues and the reproductive precursor cell in the ovules of both sexual and diplosporous taxa.

Mechanism underlying 2n male and female gamete formation in lemon via cytological and molecular marker analysis

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.

Low temperature increases the frequency of 2n female gametes in the diploid rubber tree (Hevea brasiliensis (Willd. ex A.Juss.) Müll. Arg.)

Triploid breeding via the fusion of 2n gametes and n gametes is an efficient method for breeding new varieties in woody plants. Triploid plants are frequently observed in the seedlings of the diploid rubber clone GT1. However, the factors affecting the occurrence and source of triploid progeny in GT1 are still unknown. In this study, the correlation between temperature and triploid progeny frequency in GT1 in different environments over 6 years was analyzed to evaluate the influence of temperature on the frequency of 2n female gametes. A total of 76 triploid progenies were analyzed using 19 simple sequence repeat markers, with the aim of revealing their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that low temperature increased the frequency of triploid progeny in GT1, and the determination of allelic status revealed that all triploids were derived from the 2n female gametes, thereby confirming that low temperature increased the frequency of 2n female gametes. The rate of maternal heterozygosity restitution (HR) of seven loci varied from 2.70 to 67.65% with a mean value of 40.56%, and the rate of maternal HR of 37 triploids varied from 14.29 to 66.67% with a mean value of 41.38%, indicating that the production of 2n eggs in the triploid seedlings of GT1 was by second division restitution. The results of this study are valuable for the polyploid breeding of rubber trees.

Triploid production via heat-induced diploidisation of megaspores in Populus pseudo-simonii

Triploid breeding in Populus enhances the commercial properties of cultivated trees. Sexual polyploidization through the production of diploid gametes produces elite triploids with heterosis and ploidy vigour. Populus pseudo-simonii is a species indigenous to China. It is highly adaptable and resistant drought, cold, and saline–alkaline stresses. To improve the properties in growth and pulpwood traits of P. pseudo-simonii, we used heat treatments to induce chromosome doubling in the megaspores as a first step in the production of triploid saplings. The heat treatments were applied during development stages of the female flower buds. We produced 117 triploids via the diploidisation of megaspores. The highest proportion of triploids among the offspring (51.43%) was obtained by applying a heat treatment of 40 °C over 4 h to flower buds in which one-third of the catkins were outside the bract scales. The proportion of pachytene was positively correlated with the average rate of triploid production in each bud developmental stage. Thus, among the phases of meiosis, the pachytene stage may be most susceptible to heat treatments aiming to diploidise the megaspores in P. pseudo-simonii. The megasporocytes in these treated flower buds were mostly in the first meiotic division stages, suggesting that the first meiotic division restitution (FDR) type 2n megaspores may have been induced by our experimental procedures. These triploid hybrids derived from FDR type 2n megaspores will probably contribute significantly to programmes aimed at improving commercial properties of Populus congeners in the section Tacamahaca.