Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee)

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L⁻¹) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L⁻¹ sodium nitrophenol and 0.01 ~ 0.2 mg· L⁻¹ of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L⁻¹ sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L⁻¹ significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

     

Successful application of microspore culture in pakchoi (Brassica campestris L. ssp. chinensis Makino var. communis Tsen et Lee) for hybrid breeding

Protoplasma - Microspore embryogenesis is an effective method of obtaining double haploid (DH) lines in only 1 year. However, the microspore embryogenesis protocol was not efficient in...     

Identification of a genomic region linked with sex determination of Undaria pinnatifida (Alariaceae) through genomic resequencing and genetic linkage analyses of a segregating gametophyte family

Different from the traditional knowledge about kelp, three sexual phenotypes (female, male, and monoecious) exist in the haploid gametophytes of Undaria pinnatifida. However, the sex-determining mechanisms remain unknown. Genetic linkage mapping is an efficient tool to identify sex-linked regions. In the present study, we resequenced a segregating gametophyte family based on the male genome of U. pinnatifida. A high-density genetic linkage map was constructed using 9887 SNPs, with an average distance of 0.41 cM between adjacent SNPs. On the basis of this genetic map and using the composite interval mapping method, we identified 62 SNPs significantly linked with the sexual phenotype. They were located at a position of 67.67 cM on the linkage group 23, corresponding to a physical range of 14.67 Mbp on the HiC_Scaffold_23 of the genome. Reanalysis of the previous specific length amplified fragment sequencing data according to the reference genome led to the identification of a sex-linked genomic region that encompassed the above-mentioned 14.67 Mbp region. Hence, this overlapped genomic range was likely the sex-determining region. Within this region, 129 genes were retrieved and 39 of them were annotated with explicit function, including the potential male sex-determining gene-encoding high mobility group (HMG) domain protein. Relative expression analysis of the HMG gene showed that its expression was higher in male gametophytes during the vegetative phase and monoecious gametophytes during both the vegetative and gametogenesis phases, but significantly lower in male gametophytes during the gametogenesis phase. These results provide a foundation for deciphering the sex-determining mechanism of U. pinnatifida.     

ploidyfrost: Reference-free estimation of ploidy level from whole genome sequencing data based on de Bruijn graphs

Polyploidy is ubiquitous and its consequences are complex and variable. A change of ploidy level generally influences genetic diversity and results in morphological, physiological and ecological differences between cells or organisms with different ploidy levels. To avoid cumbersome experiments and take advantage of the less biased information provided by the vast amounts of genome sequencing data, computational tools for ploidy estimation are urgently needed. Until now, although a few such tools have been developed, many aspects of this estimation, such as the requirement of a reference genome, the lack of informative results and objective inferences, and the influence of false positives from errors and repeats, need further improvement. We have developed ploidyfrost , a de Bruijn graph-based method, to estimate ploidy levels from whole genome sequencing data sets without a reference genome. ploidyfrost provides a visual representation of allele frequency distribution generated using the ggplot2 package as well as quantitative results using the Gaussian mixture model. In addition, it takes advantage of colouring information encoded in coloured de Bruijn graphs to analyse multiple samples simultaneously and to flexibly filter putative false positives. We evaluated the performance of ploidyfrost by analysing highly heterozygous or repetitive samples of Cyclocarya paliurus and a complex allooctoploid sample of Fragaria × ananassa. Moreover, we demonstrated that the accuracy of analysis results can be improved by constraining a threshold such as Cramér's V coefficient on variant features, which may significantly reduce the side effects of sequencing errors and annoying repeats on the graphical structure constructed.     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

一种模拟高原低氧复合冷冻的简易动物实验舱

以氮稀释法使舱内氧含量维持实验模拟高度,以普通冰箱制冷系统维持舱内恒温,以多功能低温浴槽作为致冻装置。该舱可于２０ｍｉｎ内升至模拟６０００ｍ高度,每次可供１６只大鼠作高原冻伤实验。实验期间高度恒定于６０００±２００ｍ,舱温２１±１℃。相对湿度５５％±１５％,冷冻液－２５．０±０．５℃。该舱结构简单、使用方便,可在实验室中模拟高原条件进行冷冻实验研究。由于不使用人体减压舱,不仅避免了减压缺氧对实验人员的不利影响,也减少了实验消耗。