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BACKGROUND: Artificial synthesis of octoploid rapeseed double haploid (DH) induction lines Y3380 and Y3560 was made possible by interspecific hybridization and genome doubling techniques. Production of pure lines by DH induction provides a new way to achieve homozygosity earlier in B.napus. Previously, the mechanism of induction, and whether the induction has obvious maternal genotypic differences or not, are not known so far. RESULTS: In this study, different karyogene and cytoplasmic genotype of B.napus were pollinated with the previously reported DH inducers e.g. Y3380 and Y3560. Our study presents a fine comparison of different cytoplasmic genotypes hybridization to unravel the mechanism of DH induction. Ploidy identification, fertility and SSR marker analysis of induced F1 generation, revealed that ploidy and phenotype of the induced F1 plants were consistent with that type of maternal, rather than paternal parent. The SNP chip analysis revealed that induction efficiency of DH inducers were affected by the karyogene when the maternal cytoplasmic genotypes were the same. However, DH induction efficiency was also affected by cytoplasmic genotype when the karyogenes were same, and the offspring of the ogura cytoplasm showed high frequency inducer gene hybridization or low-frequency infiltration. CONCLUSION: The induction effect is influenced by the interaction between maternal karyogene and cytoplasmic genotype, and the results from the partial hybridization of progeny chromosomes indicate that the induction process may be attributed to the selective elimination of paternal chromosome. This study provides a basis for exploring the mechanism of DH inducer in B.napus, and provides new insights for utilization of inducers in molecular breeding.
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Brassica napus/genética , Cromossomos de Plantas/genética , Embaralhamento de DNA/métodos , Hibridização Genética , Núcleo Celular/genética , Citoplasma/genética , Genótipo , Haploidia , Fenótipo , Melhoramento VegetalRESUMO
BACKGROUND: Our recently reported doubled haploid (DH) induction lines e.g., Y3380 and Y3560 are allo-octoploid (AAAACCCC, 2n = 8× ≈ 76), which can induce the maternal parent to produce DH individuals. Whether this induction process is related to the production of aneuploid gametes form male parent and genetic characteristics of the male parent has not been reported yet. RESULTS: Somatic chromosome counts of DH inducer parents, female wax-less parent (W1A) and their F1 hybrid individuals revealed the reliability of flow cytometry analysis. Y3560 has normal chromosome behavior in metaphase I and anaphase I, but chromosome division was not synchronized in the tetrad period. Individual phenotypic identification and flow cytometric fluorescence measurement of F1 individual and parents revealed that DH individuals can be distinguished on the basis of waxiness trait. The results of phenotypic identification and flow cytometry can identify the homozygotes or heterozygotes of F1 generation individuals. The data of SNP genotyping coupled with phenotypic waxiness trait revealed that the genetic distance between W1A and F1 homozygotes were smaller as compared to their heterozygotes. It was found that compared with allo-octoploids, aneuploidy from allo-octoploid segregation did not significantly increase the DH induction rate, but reduced male infiltration rate and heterozygous site rate of induced F1 generation. The ploidy, SNP genotyping and flow cytometry results cumulatively shows that DH induction is attributed to the key genes regulation from the parents of Y3560 and Y3380, which significantly increase the induction efficiency as compared to ploidy. CONCLUSION: Based on our findings, we hypothesize that genetic characteristics and aneuploidy play an important role in the induction of DH individuals in Brassca napus, and the induction process has been explored. It provides an important insight for us to locate and clone the genes that regulate the inducibility in the later stage.
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Brassica napus/genética , Cromossomos de Plantas/genética , Diploide , Citometria de Fluxo , Haploidia , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos TestesRESUMO
MAIN CONCLUSION: We found a new in vivo route to produce maternal doubled haploid of Brassica napus . The pollen donor, an allooctaploid rapeseed, acts as a DH inducer. Inbred line has a powerful advantage in cultivar breeding and genetic analysis. Compared to the traditional breeding methods, doubled haploid production can save years off the breeding process. Though genotype-dependent tissue culture methods are widely used in the Brassica crops, seed-based in vivo doubled haploid developing systems are rare in nature and in the laboratory. As interspecific cross and interploid hybridization play an important role in genome evolution and plant speciation, we created a new Brassica artificial hybrid, a Brassica allooctaploid (AAAACCCC, 2n = 8× = 76), by interspecific crossing and genome doubling. A homozygous line was observed at the third self-generation of a synthesized Brassica allohexaploid (AAAACC, 2n = 6× = 58). Crosses between B. napus as female and Brassica allooctaploid as pollen donor were conducted, which yielded maternal doubled haploid B. napus that were identified based on phenotype, ploidy, and molecular analysis. The Brassica octaploid acted as a maternal doubled haploid inducer and had a relatively high induction rate. Our research provides a new insight for generation of homozygous lines in vivo using a single-step approach, as well as promotes the understanding in breeding programs and genetic studies involving the Brassicas.
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Brassica napus/genética , Brassica/genética , Hibridização Genética , Brassica rapa/genética , Cruzamento , Genótipo , Haploidia , Fenótipo , Pólen/genética , Poliploidia , Sementes/genéticaRESUMO
Haploids are individuals with gametophytic chromosome numbers. Homozygous doubled haploids can be quickly gained by genome doubling. Haploids and doubled haploids play an important role on crop breeding, mutant screening, gene functional analysis, cytological studies, construction of genetic population and so on, and have been a research hotspot of plants in recent years. In this review, we summarize the production means, identification, the genetic mechanism and the applications of haploids and doubled haploids. We hope to provide a reference for study and application of haploids and doubled haploids.
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Haploidia , Plantas/genética , Cruzamento , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Plantas/metabolismoRESUMO
A synthetic octoploid rapeseed, Y3380, induces maternal doubled haploids when used as a pollen donor to pollinate plant. However, the mechanism underlying doubled haploid formation remains elusive. We speculated that double haploid induction occurs as the inducer line's chromosomes pass to the maternal egg cell, and the zygote is formed through fertilization. In the process of zygotic mitosis, the paternal chromosome is specifically eliminated. Part of the paternal gene might have infiltrated the maternal genome through homologous exchange during the elimination process. Then, the zygote haploid genome doubles (early haploid doubling, EH phenomenon), and the doubled zygote continues to develop into a complete embryo, finally forming doubled haploid offspring. To test our hypothesis, in the current study, the octoploid Y3380 line was back bred with the 4122-cp4-EPSPS exogenous gene used as a marker into hexaploid Y3380-cp4-EPSPS as paternal material to pollinate three different maternal materials. The fertilization process of crossing between the inducer line and the maternal parent was observed 48 h after pollination, and the fertilization rate reached 97.92% and 98.72%. After 12 d of pollination, the presence of cp4-EPSPS in the embryo was detected by in situ PCR, and at 13-23 d after pollination, the probability of F1 embryos containing cp4-EPSPS gene was up to 97.27%, but then declined gradually to 0% at 23-33 d. At the same time, the expression of cp4-EPSPS was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, cp4-EPSPS marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of cp4-EPSPS was not detected in surviving embryos. Meanwhile, SNP detection of induced offspring confirmed the existence of double haploids, further indicating that the induction process was caused by the loss of specificity of the paternal chromosome. The tetraploid-induced offspring showed infiltration of the induced line gene loci, with heterozygosity and homozygosity. Results indicated that the induced line chromosomes were eliminated during embryonic development, and the maternal haploid chromosomes were synchronously doubled in the embryo. These findings support our hypothesis and lay a theoretical foundation for further localization or cloning of functional genes involved in double haploid induction in rapeseed.
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Interspecific hybridization of rapeseed is an important way to innovate breeding resources. This research used Brassica napus and Brassica rapa for artificial synthesis interspecific hybridization of F1. The F1 self-fruiting rate was particularly low. By comparing the fertilization rate and seed setting rate of nine crosses and selfing combinations of interspecific hybrid progeny F1 and control B. napus, the results proved that the genetic stability of egg cells was greater than that of sperm cells, so the F1 could get seed by artificial pollination with other normal pollen. Based on these results, interspecific maternal inbred offspring (induced F1) from egg cells was obtained by emasculation and pollination with the pollen of DH inducer Y3380. It was found through morphological analysis, flow cytometry identification, and meiotic observation of induced F1, the plants had most normal fertile tetraploid and the meiosis was normal. The FISH results showed that the induced F1 were B. napus (2n = 4x = 38, AACC), 20 A and 19 C chromosomes. The results of SNP chip detection and genetic cluster analysis found that the genetic variation between interspecies could be preserved or broadened in the induced F1. The use of DH inducer created special breeding resources for interspecific hybridization and distant hybridization of rapeseed while shortening time, improving efficiency, and providing a new insight into innovate breeding resources.
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When homozygously fertile plants were induced using doubled haploid (DH) induction lines Y3380 and Y3560, the morphology of the induced F1 generation was basically consistent with the female parent, but the fertility was separated, showing characteristics similar to cytoplasmic male sterile (CMS) and maintainer lines. In this study, the morphology, fertility, ploidy, and cytoplasm genotype of the induced progeny were identified, and the results showed that the sterile progeny was polima cytoplasm sterile (pol CMS) and the fertile progeny was nap cytoplasm. The molecular marker and test-cross experimental results showed that the fertile progeny did not carry the restorer gene of pol CMS and the genetic distance between the female parent and the offspring was 0.002. This suggested that those inductions which produced sterile and fertile progeny were coordinated to CMS and maintainer lines. Through the co-linearity analysis of the mitochondrial DNA (mtDNA), it was found that the rearrangement of mtDNA by DH induction was the key factor that caused the transformation of fertility (nap) into sterility (pol). Also, when heterozygous females were induced with DH induction lines, the induction F2 generation also showed the segregation of fertile and sterile lines, and the genetic distance between sterile and fertile lines was approximately 0.075. Therefore, the induction line can induce different types of female parents, and the breeding of the sterile line and the maintainer line can be achieved through the rapid synchronization of sister crosses and self-crosses. The induction of DH inducer in B. napus can provide a new model for the innovation of germplasm resources and open up a new way for its application.
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Skin aging is an inevitable physiological process and periorbital wrinkling is an active sign of the process. Laser therapy is an effective method for improving periorbital wrinkles and wound care after laser therapy can accelerate the wound healing process. This case report describes a typical case of a 47-year-old male that presented with a 10-year history of gradually-worsening bilateral periorbital wrinkles. These were treated using a 2940 nm erbium (Er):YAG lattice laser combined with recombinant bovine basic fibroblast growth factor (bFGF) gel and hydrogel (HG) treatment on the left side of his face compared with laser therapy and bFGF gel on the right side of his face. HG combined with bFGF gel treatment after 2940 nm Er:YAG lattice laser therapy improved postoperative swelling and pigmentation compared with bFGF gel alone; and it promoted periorbital wrinkle improvement and wound healing. In conclusion, HG combined with GFs after laser therapy could be an alternative therapy for periorbital wrinkles.
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Lasers de Estado Sólido , Envelhecimento da Pele , Animais , Bovinos , Érbio , Humanos , Hidrogéis , Lasers de Estado Sólido/uso terapêutico , Masculino , Pessoa de Meia-Idade , Rejuvenescimento , Resultado do Tratamento , CicatrizaçãoRESUMO
Polyploidization plays a crucial role in the evolution of angiosperm species. Almost all newly formed polyploids encounter genetic or epigenetic instabilities. However, the molecular mechanisms contributing to genomic instability in synthetic polyploids have not been clearly elucidated. Here, we performed a comprehensive transcriptomic and methylomic analysis of natural and synthetic polyploid rapeseeds (Brassica napus). Our results showed that the CHG methylation levels of synthetic rapeseed in different genomic contexts (genes, transposon regions, and repeat regions) were significantly lower than those of natural rapeseed. The total number and length of CHG-DMRs between natural and synthetic polyploids were much greater than those of CG-DMRs and CHH-DMRs, and the genes overlapping with these CHG-DMRs were significantly enriched in DNA damage repair and nucleotide metabolism pathways. These results indicated that CHG methylation may be more sensitive than CG and CHH methylation in regulating the stability of the polyploid genome of B. napus. In addition, many genes involved in DNA damage repair, nucleotide metabolism, and cell cycle control were significantly differentially expressed between natural and synthetic rapeseeds. Our results highlight that the genes related to DNA repair and nucleotide metabolism display differential CHG methylation patterns between natural and synthetic polyploids and reveal the potential connection between the genomic instability of polyploid plants with DNA methylation defects and dysregulation of the DNA repair system. In addition, it was found that the maintenance of CHG methylation in B. napus might be partially regulated by MET1. Our study provides novel insights into the establishment and evolution of polyploid plants and offers a potential idea for improving the genomic stability of newly formed Brassica polyploids.
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Whole genome duplications (WGDs) have played a major role in angiosperm species evolution. Polyploid plants have undergone multiple cycles of ancient WGD events during their evolutionary history. However, little attention has been paid to the additional WGD of the existing allopolyploids. In this study, we explored the influences of additional WGD on the allopolyploid Brassica napus Compared to tetraploid B. napus, octoploid B. napus (AAAACCCC, 2n = 8x =76) showed significant differences in phenotype, reproductive ability and the ploidy of self-pollinated progeny. Genome duplication also altered a key reproductive organ feature in B. napus, that is, increased the number of pollen apertures. Unlike autopolyploids produced from the diploid Brassica species, the octoploid B. napus produced from allotetraploid B. napus had a relatively stable meiotic process, high pollen viability and moderate fertility under self-pollination conditions, indicating that sub-genomic interactions may be important for the successful establishment of higher-order polyploids. Doubling the genome of B. napus provided us with an opportunity to gain insight into the flexibility of the Brassica genomes. The genome size of self-pollinated progeny of octoploid B. napus varied greatly, and was accompanied by extensive genomic instability, such as aneuploidy, mixed-ploidy and mitotic abnormality. The octoploid B. napus could go through any of genome reduction, equilibrium or expansion in the short-term, thus providing a novel karyotype library for the Brassica genus. Our results reveal the short-term evolutionary consequences of recurrent polyploidization events, and help to deepen our understanding of polyploid plant evolution.
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Brassica napus , Brassica , Brassica/genética , Brassica napus/genética , Genoma de Planta , Ploidias , PoliploidiaRESUMO
Lead-zinc deposits are often difficult to classify because clear criteria are lacking. In recent years, new tools, such as Cd and Zn isotopes, have been used to better understand the ore-formation processes and to classify Pb-Zn deposits. Herein, we investigate Cd concentrations, Cd isotope systematics and Zn/Cd ratios in sphalerite from nine Pb-Zn deposits divided into high-temperature systems (e.g., porphyry), low-temperature systems (e.g., Mississippi Valley type [MVT]) and exhalative systems (e.g., sedimentary exhalative [SEDEX]). Our results showed little evidence of fractionation in the high-temperature systems. In the low-temperature systems, Cd concentrations were the highest, but were also highly variable, a result consistent with the higher fractionation of Cd at low temperatures. The δ(114/110)Cd values in low-temperature systems were enriched in heavier isotopes (mean of 0.32 ± 0.31). Exhalative systems had the lowest Cd concentrations, with a mean δ(114/110)Cd value of 0.12 ± 0.50. We thus conclude that different ore-formation systems result in different characteristic Cd concentrations and fraction levels and that low-temperature processes lead to the most significant fractionation of Cd. Therefore, Cd distribution and isotopic studies can support better understanding of the geochemistry of ore-formation processes and the classification of Pb-Zn deposits.
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Ten-maize inbred lines of maize (Zea mays L.) with high-induction rate and proliferation ability of embryonic calli were selected from 70-maize inbred lines by immature embryo culturing. Some of the embryonic calli were transferred onto regeneration medium to examine the ability of regeneration, some were transformed via Agrobacterium tumifaciens C58 carrying intron-beta-glucuronidase (gus) gene, and GV3301 carrying the green fluorescent protein (gfp) gene to study the susceptibility of different genotypes in maize to A. tumifaciens. All embryonic calli initiated from 10-maize inbred lines were able to regenerate into plantlets, and the regeneration frequencies of inbred lines 6010, 6038, 6015, 6051, and 6060 were 61.11%, 31.94%, 45%, 33.33%, and 56.94%, respectively, which were higher than that of other lines. Analysis of variance indicated that the susceptibility of the various genotypes in maize to A. tumifacien C58 showed a significant difference among each other, and the inbred lines 6010, 6015, 6051, 6050, 6058, 6060, 6069, 6077 were susceptible to A. tumifacien C58, of which frequency of gus expression were over 70%. Expression of GFP was observed in six-inbred lines (6050, 6015, 6051, 6058, 6069, 6077). The inbred lines 6051, 6010, 6015, 6060, and 6050 had the high regeneration and the susceptibility to A. tumifaciens C58; and the inbred lines 6051, 6015, and 6060 had the high regeneration and the susceptibility to Agrobacterium tumifaciens GV3301.