一个萝卜绿皮新基因GST1的遗传定位

Genetic Mapping of a Novel Gene GST1 for Green-Skinned Taproot in Radish(Raphanus sativus L.)

为深入理解萝卜皮色形成机制,开发萝卜皮色标记进行辅助育种,本研究利用绿皮萝卜G-2和白皮萝卜W-1构建了遗传群体,明确了绿皮表型的遗传规律;结合混合分离群体分析和RNA-Seq技术(BSA-seq)确定了绿皮调控位点所在染色体,接着开发分子标记定位了绿皮基因。结果显示,G-2和W-1的F_(1)代植株肉质根表现为介于亲本之间的中间色(浅绿色),其F_(2)代群体单株分离出绿色、中间色和白色肉质根,大致符合1∶2∶1的分离比例(χ^(2)=3.21,P>0.05),表明绿皮表型在遗传后代中呈不完全显性,并由1个主效基因控制,暂将其命名为Green-Skinned Taproot 1(GST1)。BSA-Seq结果显示,有1827个萝卜单核苷酸多态性(SNP)位点在G-2×W-1 F_(2)代群体绿皮池和白皮池间具有多态性,其中31.86%位于R01染色体,并且主要分布在短臂端部0~5 Mb的物理区间内。在此区段附近开发分子标记,最终将GST1定位到标记sxau30和sxau34之间,遗传距离分别为6.4和8.3 cM,对应萝卜品种Radicula参考基因组R01:2.93~8.99 Mb的物理区间。此区间共有210个高置信注释基因可在肉质根表皮中表达,其中有44个在绿皮池和白皮池之间表达差异显著,对可能参与叶绿素合成或代谢途径的4个候选基因进行了实时荧光定量PCR(qRT-PCR)验证,其表达水平与BSA-Seq结果一致。本研究结果为萝卜绿皮成色机制解析奠定了理论基础,也为皮色分子育种提供了新标记。

To well understand of the formation mechanism of radish skin color and to develop related molecular markers for radish breeding,genetic populations were created by crossing the green-skinned radish G-2 with the white-skinned radish W-1,and the genetic rule of green skin was clarified.By combining bulked segregant analysis with RNA-seq technology(BSA-seq),the chromosome that carrys the locus controlling green skin was identified,then the green skin gene was mapped using the developed molecular markers.The results showed that the taproots of F_(1) plants derived from G-2 and W-1 exhibited an intermediate color(light green)compared to their parent plants.The F_(2) population appeared taproots with green-skinned,intermediate color-skinned,and white-skinned,approximately in a ratio of 1∶2∶1(χ^(2)=3.21,P>0.05),indicating that the green skin phenotype demonstrated incomplete dominance in the genetic offspring and was controlled by one single major locus,provisionally designated as GST1(Green-Skinned Taproot 1).BSA-seq results showed that 1827 single nucleotide polymorphisms(SNPs)exhibited polymorphism between the green-and white�skinned radish bulks from the G-2×W-1 F_(2) population.Of these SNPs,31.86%were located on chromosome R01,primarily distributed within the physical region of 0-5 Mb at the terminal of the short arm.Molecular markers were developed in proximity to this section,ultimately leading to the mapping of GST1 within the genetic interval between the flanking markers sxau30 and sxau34,with the genetic distances of 6.4 and 8.3 cM,respectively.This corresponds to the physical section R01:2.93-8.99 Mbp of the reference genome for the radish variety Radicula.A total of 210 annotated high-confidence genes expressed in taproot skin were identified within this interval,of which 44 exhibited significantly different expression between the green-and white-skinned bulks.Quantitative real time-PCR(qRT-PCR)validation was performed on four candidate genes that may be involved in the chlorophyll synthesis or metabolism pathway,confirming that their expression levels aligh with the RNA-seq results.These results lay a foundation for elucidating the mechanisms behind radish green skin and provide new markers for molecular breeding of skin color.