Gene Mapping And Transcriptome Analysis Of Thermosensitive Albino Mutant Btal In Barley

The leaf blade is one of the six major organs of plants and maintaining its leaf colour properly is essential for plant photosynthesis.Chloroplasts contain a variety of chlorophylls that affect leaf colour and are essential semi-autonomous organelles for plants.Chloroplasts are able to carry out photosynthesis and convert light energy into chemical energy to provide energy for their own growth.Abnormal chloroplast development will lead to changes in leaf colour and reduced photosynthetic capacity,which will further affect the normal growth of the plant.Most barley chloroplast mutations result in a loss of green colour,reduced light capture and reduced photosynthesis,which in turn inhibits the growth of barley and reduces its yield.Therefore,it is important to study the molecular mechanism of leaf colour change in barley in order to breed new high-yielding barley varieties and increase barley yield.In this study,we used the barley temperature-sensitive albino leaf mutant btal（Barley temperature-sensitive albino leaf）as a research object,and conducted phenotypic identification and candidate gene screening using physiological and biochemical analysis,map cloning and transcriptome analysis.The main findings are as follows:1.Under field conditions,the mutant btal showed different degrees of albino phenotypes at the seedling,tillering and grain-filling stages.At the seedling stage the leaves of btal were white striped,and the albino phenomenon was obvious at the tillering stage.However,the albino leaf phenotype of btal was weakened and gradually turned green at the grain-filling stage.The determination of the photosynthetic pigment content showed that the photosynthetic pigment content of the mutant btal at the seedling stage and tillering stage was significantly lower than that of the wild type,and at the grain-filling stage,except carotenoid content,other photosynthetic pigment contents were significantly lower than wild type.2.Under field conditions,POD activity,SOD activity,and MDA content were measured at the seedling,tillering and grain-filling stages,respectively.The results showed that compared with the wild type,the mutant btal accumulated more ROS;The excessive accumulation of internal ROS resulted in severe oxidative damage to the membrane system,and the damage failed to return to normal with increasing temperature.3.The phenotypic evolution of the panicle showed that compared with the wild type,the albino phenotype of the mutant btal panicle was obvious;the determination of POD activity,SOD activity and MDA content in the panicle showed that the mutant btal panicle had more ROS content,it caused damage to the antioxidant system of the spike,which further led to impaired lipid oxidation of the cell membrane.Therefore,the mutation had a certain effect on the phenotypic changes of the panicle.The analysis of agronomic traits under field conditions showed that compared with the wild type,the mutant btal showed shorter plant,shorter awn length and panicle length,fewer grains per panicle,lighter 100-grain weight,reduced grain length and grain width narrowing.4.The temperature conversion experiment showed that the leaf phenotype of the two-leaf stage mutant btal was similar to that of the wild type at normal temperature（22℃）,and its leaves were normal green;while under the condition of low temperature（6℃）,the leaf phenotype was albino.The content of photosynthetic pigments in leaves under temperature conversion and transmission electron microscope observation and analysis showed that the content of photosynthetic pigments in the two-leaf stage mutant btal at low temperature was significantly lower than that of the wild type,and compared with the wild type,its chloroplast development was incomplete;The content of photosynthetic pigments in the mutant btal was significantly lower than that of the wild type,but the chloroplasts were fully developed,with tightly packed grana and stroma sheets.The photosynthetic pigment content of the mutant btal at low temperature was lower than that at normal temperature,indicating that low temperature affects the development of chloroplasts.The POD and SOD activities,H₂O₂ and MDA contents were determined.The POD,SOD and H₂O₂ of the mutant btal at two temperatures were extremely significantly higher than those of the wild type,and more obvious at low temperature,indicating that the mutant btal at the second leaf stage at low temperature leaves accumulated more ROS than normal temperature.The MDA content of the mutant btal was also significantly higher than that of the wild type at low temperature and normal temperature,but the MDA content of the mutant btal was higher at low temperature,indicating that the low temperature caused the membrane system of the mutant btal leaves to be severely damaged.5.Genetic analysis showed that the albino phenotype of mutant btal was controlled by a single recessive nuclpanicle gene.Using BSA-seq technology and map-based cloning method,the target gene of mutant btal was located in the 2.24Mb interval on the 5H long arm of barley,and 117 candidate barley genes were screened,which laid the foundation for the next step of gene cloning.6.Transcriptome analysis showed that a total of 6280 differentially expressed genes were obtained in the comparison group between btal and ZJU3 at low temperature（6℃）,of which 3354 were up-regulated and 2926 were down-regulated.There were 2118 differentially expressed genes,were obtained in the comparison group between btal and ZJU3 at normal temperature（22℃）,and the number of up-regulated and down-regulated differentially expressed genes were both 1059.GO enrichment and KEGG Pathway enrichment analysis showed that most of the differentially expressed genes were related to chloroplast development at low temperature.The significantly differentially expressed genes related to chloroplast development and photosynthesis were further screened,and 440 significantly differentially expressed genes were screened at low temperature,of which 180 were up-regulated and 260 were down-regulated;while only 8 significantly up-regulated genes were screened at normal temperature.The results showed that low temperature affected the normal development of chloroplasts.In addition,the significantly differentially expressed genes related to the mutant btal localization interval were also screened.8 were screened at low temperature,of which 5 were up-regulated and 3were down-regulated;4 were screened at normal temperature,of which 3 were up-regulated and 1 was down-regulated.The expression of HORVU.MOREX.r3.5HG0520950 was down-regulated at low temperature and up-regulated at normal temperature,which was greatly affected by temperature.It was speculated that the gene was likely to be the target gene of mutant btal.