水稻温度敏感型黄叶突变体yl2(t)的表型分析和基因定位

Characterization and gene mapping of a thermo-sensitive yellow leaf mutant yl2(t) in rice

[目的]本试验旨在对水稻温度敏感型黄叶突变体yl2(t)进行表型分析和基因定位,为图位克隆该基因奠定基础。[方法]在连续20℃温度处理下测定突变体yl2(t)叶绿素含量和观察其叶绿体结构;构建yl2(t)与籼稻品种‘南京11’的杂交F2群体,借助SSR和In Del分子标记,选取隐性极端个体进行基因定位;利用qRT-PCR测定叶绿素合成相关基因表达水平。[结果]20℃持续处理15 d,yl2(t)幼苗表现黄叶;若处理时间延长至20 d,yl2(t)幼苗则不能继续生长最后死亡,处理15 d后转移至30℃或在30℃连续生长时叶色正常。田间生长条件下,yl2(t)的农艺性状与野生型相比,没有显著差异。叶绿素含量测定结果表明,20℃时yl2(t)叶绿素含量较野生型显著降低,而30℃时yl2(t)中仅叶绿素b的含量显著降低。叶绿体超微结构观察显示,yl2(t)幼苗中叶绿体发育正常。遗传分析表明,yl2(t)突变表型受一对隐性核基因控制,利用图位克隆的方法将该突变基因定位在第8染色体长臂末端标记N8-36与P16之间,物理距离为647 kb。qRT-PCR结果表明,20℃和30℃两种温度条件下,与野生型相比,yl2(t)幼苗中叶绿素合成相关基因的表达出现不同的变化。[结论]水稻温敏型黄叶突变体yl2(t)突变表型受一对隐性核基因控制,该基因被定位在第8染色体长臂末端,是一个控制水稻叶色的新基因。本研究结果为YL2(T)基因的克隆及在水稻育种中的应用提供了依据。

[Objectives]We analyzed the phenotype of a rice thermo-sensitive yellow leaf mutant yl2（ t） and mapped the mutant locus to lay the foundation for map-based cloning of this gene. [Methods]Determination of the chlorophyll contents and observation of the ultra-structures of chloroplasts in wild type and the yl2（ t） mutant seedlings under continuous 20 ℃. The yl2（ t） mutant was crossed with an indica cultivar‘Nanjing 11＇to generate the hybrid F2 population. Recessive individuals with the same phenotype of the yl2（ t） mutant were selected and applied with SSR and In Del markers to map the locus. qRT- PCR was conducted to analyze the expression levels of genes controlling chlorophyll biosynthesis. [Results]The yl2（ t） mutant seedlings showed yellow leaf at 2-leaf stage when growing under continuous 20 ℃ for 15 days and were lethal when extending time of low temperature treatment to 20 days,but turned to normal green color when transferred to 30 ℃ or growing under continuous 30 ℃. However,agronomic traits such as plant height of yl2（ t） were not affected by the mutation in the field. Compared with wild type,chlorophyll contents were significantly decreased in yl2（ t） seedlings at 2-leaf stage growing under continuous 20 ℃,but only the chlorophyll b content significantly dropped in the yl2（ t） seedlings when growing under continuous 30 ℃. The morphology and structure of chloroplasts were normally developed in yl2（ t） mutant seedlings under continuous 20 ℃. Genetic analysis suggested that the yellow leaf phenotype was controlled by a pair of recessive nuclear genes and finally the locus was mapped to a 647 kb region between the markers N8-36 and P16 at the terminus of long arm of rice chromosome 8. qRT-PCR showed that the expression levels of genes involved in chlorophyll synthesis in yl2（ t）seedlings had different changes under the two temperatures. [Conclusions]The thermo-sensitive yellow leaf phenotype of rice yl2（ t）mutant was controlled by a pair of recessive nuclear genes and the locus was mapped to the end of long arm of chromosome 8,which was not reported. This study provides references for the map-based cloning of the YL2（ T） locus and application in rice breeding.