Second Division Restitution Research Articles

Heterozygosity analysis of spontaneous 2n female gametes and centromere mapping of the diploid Hevea brasiliensis based on full-sib triploid populations.

Unreduced megagametophytes via second-division restitution were confirmed through heterozygosity analysis, and four candidate physical centromeres of rubber were located for the first time. The evaluation of maternal heterozygosity restitution (MHR) is vital in identifying the mechanism of 2n gametogenesis and assessing the utilization value of 2n gametes. In this study, three full-sib triploid populations were employed to evaluate the MHR of 2n female gametes of rubber tree clone GT1 and to confirm their genetic derivation. The 2n female gametes of GT1 were derived from second-division restitution (SDR) and transmitted more than half of the parental heterozygosity. In addition, low recombination frequency markers were developed, and four candidate physical centromeres of rubber tree were located for the first time. The confirmation that 2n female gametes of rubber tree clone GT1 are derived from SDR provides insights into the molecular mechanisms of 2n gametogenesis. In addition, the identified centromere location will aid in the development of centromeric markers for the rapid identification of the 2n gametogenesis mechanism.

Genome sequence analysis provides insights into the mode of 2n egg formation in Solanum malmeanum.

Our genomic investigation confirms the mechanism of 2n eggs formation in S. malmeanum and aid in optimizing the use of wild germplasm. Wild potatoes are a valuable source of agronomic traits. However, substantial reproductive barriers limit gene flow into cultivated species. 2n gametes are instrumental in preventing endosperm abortion caused by genetic imbalances in the endosperm. However, little is known about the molecular mechanisms underlying the formation of 2n gametes. Here, the wild species Solanum malmeanum Bitter (2x, 1EBN, endosperm balance number) was used in inter- and intrapoloid crosses with other Solanum species, with viable seeds being produced only when S. malmeanum was used as the female parent to cross the 2EBN Solanum genus and with the likely involvement of 2n gametes. Subsequently, we substantiated the formation of 2n eggs in S. malmeanum using fluorescence in situ hybridization (FISH) and genomic sequencing technology. Additionally, the transmission rate of maternal heterozygous polymorphism sites was assessed from a genomic perspective to analyze the mode of 2n egg formation in S. malmeanum × S. tuberosum and S. malmeanum × S. chacoense crosses; each cross acquired an average of 31.12% and 22.79% maternal sites, respectively. This confirmed that 2n egg formation in S. malmeanum attributed to second-division restitution (SDR) coupled with the occurrence of exchange events. The high-throughput sequencing technology used in this study has strong advantages over traditional cytological analyses. Furthermore, S. malmeanum, which has a variety of excellent traits not available from present cultivated potato genepool, has received little research attention and has successfully achieved gene flow in cultivated species in the current study. These findings will facilitate the understanding and optimization of wild germplasm utilization in potatoes.

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.

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.

Identification of Parental Genome Construction and Inherited Morphological Characteristics in Triploid and AneuploidIntergeneric Hybrids from a Diploid−Diploid Cross between Citrus and Fortunella

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst; H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E; Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.

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.

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.

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.

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.

Cold-Induced Male Meiotic Restitution in Arabidopsis thaliana Is Not Mediated by GA-DELLA Signaling.

Short periods of cold stress induce male meiotic restitution and diploid pollen formation in Arabidopsis thaliana by specifically interfering with male meiotic cytokinesis. Similar alterations in male meiotic cell division and gametophytic ploidy stability occur when gibberellic acid (GA) signaling is perturbed in developing anthers. In this study, we found that exogenous application of GA primarily induces second division restitution (SDR)-type pollen in Arabidopsis, similar to what cold does. Driven by the close similarity in cellular defects, we tested the hypothesis that cold-induced meiotic restitution is mediated by GA-DELLA signaling. Using a combination of chemical, genetic and cytological approaches, however, we found that both exogenously and endogenously altered GA signaling do not affect the cold sensitivity of male meiotic cytokinesis. Moreover, in vivo localization study using a GFP-tagged version of RGA protein revealed that cold does not affect the expression pattern and abundance of DELLA in Arabidopsis anthers at tetrad stage. Expression study found that transcript of RGA appears enhanced in cold-stressed young flower buds. Since our previous work demonstrated that loss of function of DELLA causes irregular male meiotic cytokinesis, we here conclude that cold-induced meiotic restitution is not mediated by DELLA-dependent GA signaling.

Unreduced Megagametophyte Production in Lemon Occurs via Three Meiotic Mechanisms, Predominantly Second-Division Restitution.

Unreduced (2n) gametes have played a pivotal role in polyploid plant evolution and are useful for sexual polyploid breeding in various species, particularly for developing new seedless citrus varieties. The underlying mechanisms of 2n gamete formation were recently revealed for Citrus reticulata but remain poorly understood for other citrus species, including lemon (C. limon [L.] Burm. f.). Here, we investigated the frequency and causal meiotic mechanisms of 2n megagametophyte production in lemon. We genotyped 48progeny plants of two lemon genotypes, “Eureka Frost” and “Fino”, using 16 Simple Sequence Repeat (SSR) and 18 Single Nucleotide Polymorphism (SNP) markers to determine the genetic origin of the progenies and the underlying mechanisms for 2n gamete formation. We utilized a maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric markers and analysis of PHR patterns along the chromosome. The frequency of 2n gamete production was 4.9% for “Eureka Frost” and 8.3% for “Fino”, with three meiotic mechanisms leading to 2n gamete formation. We performed the maximum-likelihood method at the individual level via centromeric marker analysis, finding that 88% of the hybrids arose from second-division restitution (SDR), 7% from first-division restitution (FDR) or pre-meiotic doubling (PRD), and 5% from post-meiotic genome doubling (PMD). The pattern of PHR along LG1 confirmed that SDR is the main mechanism for 2n gamete production. Recombination analysis between markers in this LG revealed partial chiasma interference on both arms. We discuss the implications of these restitution mechanisms for citrus breeding and lemon genetics.

Tetraploid citrus progenies arising from FDR and SDR unreduced pollen in 4x X 2x hybridizations

Polyploid citrus arise by somatic and sexual polyploidization, and both have been used in triploid breeding programs. Sexual polyploidization is mainly achieved by first-division restitution (FDR) or second-division restitution (SDR) meiotic restitution mechanisms. In citrus, mostly SDR producing 2n ovule has been described. However, we obtained 72 tetraploid hybrids from 4x X 2x sexual hybridizations using two doubled-diploid mandarins as female parents (‘Moncada’ mandarin and ‘Fina’ clementine) and a diploid hybrid tangor as male parent (clementine X sweet orange—‘CSO’) suggesting 2n pollen formation. This material was used to confirm the existence of 2n pollen in Citrus and to analyze its origin. SSR and SNP molecular marker analyses revealed that 64 out of the 72 recovered tetraploid plants resulted from the fertilization of a reduced diploid female gamete by unreduced (diploid) pollen from ‘CSO’, whereas eight tetraploid plants arose from self-pollination of the tetraploid parent. The maximum-likelihood method based on parental heterozygosity restitution (PHR) of centromeric loci identified both FDR and SDR as the mechanisms leading to unreduced male gamete formation. From the 64 unreduced gametes produced by diploid ‘CSO’ tangor, 41 (64.1%) were originated by FDR, whereas 12 (18.8%) were significant for SDR. Non-conclusive results were obtained for 11gametes (17.2%). The pattern of PHR variation of markers located along the linkage group 2 confirmed our results at population level. To our knowledge, this is the first report of tetraploid citrus progenies arising from unreduced pollen and the first description of the coexistence of two meiotic restitution mechanisms (SDR and FDR) producing unreduced pollen in citrus.

Male Parent Identification of Triploid Rubber Trees (Hevea brasiliensis) and the Mechanism of 2n Gametes Formation

Eight triploids were screened among offspring of the rubber tree clone GT1 × different clones by flow cytometry and chromosome counting. Twenty-five simple sequence repeat (SSR) markers were screened to identify the origin of 2n gametes, to determine the male parents of these triploids, and to evaluate the mechanism of 2n gamete formation using band configurations and microsatellite DNA allele counting peak ratios (MAC-PR). The results showed that 2n gametes originated from the maternal rubber tree clone GT1, contributing the extra genome copy present in the triploids. It was confirmed that GT1 is able to produce a 2n megagametophyte spontaneously. Many male parents were shown to provide pollen for formation of triploid rubber trees, including clones RRIC 103, Yunyan 277-5, and three other clones. The second division restitution (SDR) was likely the main mechanism involved in formation of megagametophytes in GT1, as the rate of maternal heterozygosity restitution (HR) of all eight triploids varied from 27.78% to 75.00%, with a mean of 51.46%, and all 25 markers varied from 0% to 100%, with a mean of 51.69%. Elucidation of the origin and formation of 2n gametes will help optimize further sexual hybridization of polyploid rubber trees.

Transcriptomic changes following synthesis of a Populus full-sib diploid and allotriploid population with different heterozygosities driven by three types of 2n female gamete.

Diploid gametes are usually applied to produce triploids of Populus [originating from first-division restitution (FDR), second-division restitution (SDR), and postmeiotic restitution (PMR) 2n eggs]. Three types of 2n gametes transmitted different parental heterozygosities in Populus. Failed spindle formation and no chromosomal separation to opposite poles during meiosis I mean that FDR 2n gametes carry nonsister chromatids that are potentially heterozygous. By contrast, SDR 2n gametes result from failed sister chromatid separation in meiosis II, and therefore, they carry sister chromatid that are potentially homozygous. Completely homozygous 2n gametes can arise from the PMR mechanism. The alteration of gene expression resulting from allopolyploidization is a prominent feature in plants. We compared gene expression in the full-sib progeny of three allotriploid Populus populations (triploid-F, triploid-S, and triploid-P) with that in its parent species, and their full-sib diploid F1 hybrid. Genome-wide expression level dominance was biased toward the maternal in the diploid F1 hybrid and three allotriploid populations, whereas our data indicated important, but different, effects of the transmission of different heterozygosity by 2n female gametes in the expression patterns of allopolyploids. Because of the higher level of heterozygosity, the triploids had higher rates of non-additive and transgressive expression patterns in the triploid-F than in triploid-S and triploid-P. Compared with diploid F1, about 30-fold more genes (251) were differently expressed in the triploid-F than in the triploid-S (9) and triploid-P (8), respectively. These findings indicate that hybridization and polyploidization have immediate and distinct effects on the large-scale patterns of gene expression, and different effects on the transmission of heterozygosity by three 2n female gametes.

Inheritance and variation of Cytosine methylation in three populus allotriploid populations with different heterozygosity.

DNA methylation is an epigenetic mechanism with the potential to regulate gene expression and affect plant phenotypes. Both hybridization and genome doubling may affect the DNA methylation status of newly formed allopolyploid plants. Previous studies demonstrated that changes in cytosine methylation levels and patterns were different among individual hybrid plant, therefore, studies investigating the characteristics of variation in cytosine methylation status must be conducted at the population level to avoid sampling error. In the present study, an F1 hybrid diploid population and three allotriploid populations with different heterozygosity [originating from first-division restitution (FDR), second-division restitution (SDR), and post-meiotic restitution (PMR) 2n eggs of the same female parent] were used to investigate cytosine methylation inheritance and variation relative to their common parents using methylation-sensitive amplification polymorphism (MSAP). The variation in cytosine methylation in individuals in each population exhibited substantial differences, confirming the necessity of population epigenetics. The total methylation levels of the diploid population were significantly higher than in the parents, but those of the three allotriploid populations were significantly lower than in the parents, indicating that both hybridization and polyploidization contributed to cytosine methylation variation. The vast majority of methylated status could be inherited from the parents, and the average percentages of non-additive variation were 6.29, 3.27, 5.49 and 5.07% in the diploid, FDR, SDR and PMR progeny populations, respectively. This study lays a foundation for further research on population epigenetics in allopolyploids.

Maximum-likelihood method identifies meiotic restitution mechanism from heterozygosity transmission of centromeric loci: application in citrus.

Polyploidisation is a key source of diversification and speciation in plants. Most researchers consider sexual polyploidisation leading to unreduced gamete as its main origin. Unreduced gametes are useful in several crop breeding schemes. Their formation mechanism, i.e., First-Division Restitution (FDR) or Second-Division Restitution (SDR), greatly impacts the gametic and population structures and, therefore, the breeding efficiency. Previous methods to identify the underlying mechanism required the analysis of a large set of markers over large progeny. This work develops a new maximum-likelihood method to identify the unreduced gamete formation mechanism both at the population and individual levels using independent centromeric markers. Knowledge of marker-centromere distances greatly improves the statistical power of the comparison between the SDR and FDR hypotheses. Simulating data demonstrated the importance of selecting markers very close to the centromere to obtain significant conclusions at individual level. This new method was used to identify the meiotic restitution mechanism in nineteen mandarin genotypes used as female parents in triploid citrus breeding. SDR was identified for 85.3% of 543 triploid hybrids and FDR for 0.6%. No significant conclusions were obtained for 14.1% of the hybrids. At population level SDR was the predominant mechanisms for the 19 parental mandarins.

Differential transcriptome analysis between Populus and its synthesized allotriploids driven by second-division restitution.

In this report, we compared transcriptomic differences between a synthetic Populus section Tacamahaca triploid driven by second-division restitution and its parents using a high-throughput RNA-seq method. A total of 4,080 genes were differentially expressed between the high-growth vigor allotriploids (SDR-H) and their parents, and 719 genes were non-additively expressed in SDR-H. Differences in gene expression between the allotriploid and male parent were more significant than those between the allotriploid and female parent, which may be caused by maternal effects. We observed 3,559 differentially expressed genes (DEGs) between the SDR-H and male parent. Notably, the genes were mainly involved in metabolic process, cell proliferation, DNA methylation, cell division, and meristem and developmental growth. Among the 1,056 DEGs between SDR-H and female parent, many genes were associated with metabolic process and carbon utilization. In addition, 1,789 DEGs between high- and low-growth vigor allotriploid were mainly associated with metabolic process, auxin poplar transport, and regulation of meristem growth. Our results indicated that the higher poplar ploidy level can generate extensive transcriptomic diversity compared with its parents. Overall, these results increased our understanding of the driving force for phenotypic variation and adaptation in allopolyploids driven by second-division restitution.

Genetic mapping of centromeres in the nine Citrus clementina chromosomes using half-tetrad analysis and recombination patterns in unreduced and haploid gametes.

BackgroundMapping centromere locations in plant species provides essential information for the analysis of genetic structures and population dynamics. The centromere’s position affects the distribution of crossovers along a chromosome and the parental heterozygosity restitution by 2n gametes is a direct function of the genetic distance to the centromere. Sexual polyploidisation is relatively frequent in Citrus species and is widely used to develop new seedless triploid cultivars. The study’s objectives were to (i) map the positions of the centromeres of the nine Citrus clementina chromosomes; (ii) analyse the crossover interference in unreduced gametes; and (iii) establish the pattern of genetic recombination in haploid clementine gametes along each chromosome and its relationship with the centromere location and distribution of genic sequences.ResultsTriploid progenies were derived from unreduced megagametophytes produced by second-division restitution. Centromere positions were mapped genetically for all linkage groups using half-tetrad analysis. Inference of the physical locations of centromeres revealed one acrocentric, four metacentric and four submetacentric chromosomes. Crossover interference was observed in unreduced gametes, with variation seen between chromosome arms. For haploid gametes, a strong decrease in the recombination rate occurred in centromeric and pericentromeric regions, which contained a low density of genic sequences. In chromosomes VIII and IX, these low recombination rates extended beyond the pericentromeric regions. The genomic region corresponding to a genetic distance < 5cM from a centromere represented 47% of the genome and 23% of the genic sequences.ConclusionsThe centromere positions of the nine citrus chromosomes were genetically mapped. Their physical locations, inferred from the genetic ones, were consistent with the sequence constitution and recombination pattern along each chromosome. However, regions with low recombination rates extended beyond the pericentromeric regions of some chromosomes into areas richer in genic sequences. The persistence of strong linkage disequilibrium between large numbers of genes promotes the stability of epistatic interactions and multilocus-controlled traits over successive generations but also maintains multi-trait associations. Identification of the centromere positions will allow the development of simple methods to analyse unreduced gamete formation mechanisms in a large range of genotypes and further modelling of genetic inheritance in sexual polyploidisation breeding schemes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0464-y) contains supplementary material, which is available to authorized users.

DIFFERENCES IN THE GENETIC STRUCTURE OF CITRUS TRIPLOID HYBRIDS RECOVERED FROM 2X × 2X AND 4X × 2X SEXUAL HYBRIDIZATIONS

Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as autotetraploid. The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends on the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNP and SSR markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one displays a tendency for disomy. The PHR in the 2n gametes is 2/3 of that obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x X 2x hybridizations are more efficient than 2x X 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x X 2x hybridisations provides a greater opportunity to select innovative products. (Resume)