Zhao Shihui, Huang Liangjun, Zhang Qing, Zhou Ying, Yang Meicui, Shi Haoran, Li Yun, Yang Jin, Li Chao, Ge Xianhong, Gong Wanzhuo, Wang Jisheng, Zou Qiong, Tao Lanrong, Kang Zeming, Li Zhuang, Xiao Chaowen, Hu Qiong, Fu Shaohong
Chengdu Academy of Agriculture and Forestry Sciences, Chengdu Research Branch, National Rapeseed Genetic Improvement Center, Chengdu, China.
Agricultural College, Sichuan Agricultural University, Chengdu, China.
Front Plant Sci. 2023 Oct 30;14:1256338. doi: 10.3389/fpls.2023.1256338. eCollection 2023.
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- exogenous gene used as a marker into hexaploid Y3380- 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 in the embryo was detected by PCR, and at 13-23 d after pollination, the probability of F embryos containing gene was up to 97.27%, but then declined gradually to 0% at 23-33 d. At the same time, the expression of was observed by immunofluorescence in the 3rd to 29th day embryo. As the embryos developed, marker genes were constantly lost, accompanied by embryonic death. After 30 d, the presence of 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.
一种人工合成的八倍体油菜Y3380，当其作为花粉供体对植株进行授粉时，可诱导产生母本双单倍体。然而，双单倍体形成的潜在机制仍不清楚。我们推测，双单倍体诱导是在诱导系染色体传递至母本卵细胞时发生的，随后通过受精形成合子。在合子有丝分裂过程中，父本染色体被特异性消除。在消除过程中，部分父本基因可能通过同源交换渗入母本基因组。然后，合子的单倍体基因组加倍（早期单倍体加倍，即EH现象），加倍后的合子继续发育成完整胚胎，最终形成双单倍体后代。为验证我们的假设，在本研究中，将八倍体Y3380品系与用作标记的4122-外源基因回交，培育出六倍体Y3380-作为父本材料，对三种不同的母本材料进行授粉。授粉48小时后观察诱导系与母本亲本杂交的受精过程，受精率分别达到97.92%和98.72%。授粉12天后，通过PCR检测胚胎中是否存在 ，在授粉后13 - 23天，含有 基因的F胚胎的概率高达97.27%，但在23 - 33天逐渐下降至0%。同时，在授粉后第3天至第29天的胚胎中通过免疫荧光观察 的表达情况。随着胚胎发育， 标记基因不断丢失，同时伴随着胚胎死亡。30天后，在存活胚胎中未检测到 的存在。同时，对诱导后代进行SNP检测证实了双单倍体的存在，进一步表明诱导过程是由父本染色体特异性丢失导致的。四倍体诱导后代表现出诱导系基因位点的渗入，存在杂合性和纯合性。结果表明，诱导系染色体在胚胎发育过程中被消除，母本单倍体染色体在胚胎中同步加倍。这些发现支持了我们的假设，为进一步定位或克隆油菜中参与双单倍体诱导的功能基因奠定了理论基础。
