Cold Tolerance Evaluation And Transcriptome Analysis Of Winter Rapeseed(Brassica Napus L.) In Northern China In Response To Freezing Stress

The area of rapeseed production in China was mainly divided into winter rapeseed area and spring rapeseed area.Brassica napus is the main species grown in the winter rapeseed area.Based on the high yield,resistance to lodging and excellent quality,production of Brassica napus occupies the dominant position,and their yields accounted for 86.3%of the total output of rapeseed.Therefore,it plays an important role in rapeseed production in our country.Nonetheless,the Brassica napus were cold sensitive,so there were mainly distributed in the Yangtze River basin.However,in the north of China,the winter Brassica rape is the main type,therefore,improving and breeding germplasm resources of Brassica napus which may have ability to resist cold stress are very important for the development of rapeseed production in north China.In our study,based on the evaluation of cold tolerance in twelve Brassica napus varieties（lines）,we screened out cold-tolerance line and cold-sensitive line.Under freezing stress treatment,we compared and analyzed the differences of morphological characteristics,microstructure,photosynthetic indexes,physiological and biochemical indexes between two different cold-tolerance lines in response to freezing stress,and explored the mechanism of cold-tolerance line in transcript level.The main results were summarized as follows:1.The overwintering survival rate of materials in Tianshui area（34°6′N,altitude1084¹650 m）was 92.1%⁹7.8%.However,the overwintering survival rate decreased rapidly after moving to Lanzhou（36°73′N,altitude 1517 m）and Shangchuan（36°03′N,altitude 2150 m）.The winter Brassica napus lines of“16TS 309-4,16TS 306-3,16TS309-10,15NS 45-4,2016 8（G）and 2016TSG（10）”which were new bred by Gansu Agricultural University could survive over winter,with the average overwintering survival rate of 10.2%¹4.4%,and the overwintering rate significantly higher than“Xinyou 23,Tianyou 14 and Tianyou 2288”.According to the overwintering survival rate,semi-lethal temperature(LT₅₀)and membership function of comprehensive evaluation method,the cold tolerance of the twelve materials was ranked from strong cold-tolerance to cold-sensitive followed by 2016TS（G）10>16TS 309-4>16TS 306-3>15NS 45-4>16TS 309-10>20168（G）>16TS 312-2>16NTS 158>16NPZ 269-1>Tianyou 14>Xinyou 23>Tianyou2288。2.Twelve winter rapeseeds were sowed in early spring and the field performace results shown that the vernalization rate（4.05%）and the average blot height（87.65%）of cold-tolerance line“2016TS（G）10”was the lowest one.The vernalization rate of cold-sensitive lines of“Xinyou 23”and“Tianyou 2288”（87.65%and 85.19%）and the average blots height（80.18 cm and 58.21 cm）,were the highest one among the varieties（lines）.Correlation analysis showed that the vernalization rate was significantly positively correlated with the average plant（bolt）height,the proportion of mature plants and LT₅₀,with R² 0.90⁰.96,and significantly negatively correlated with overwintering survival rate,LT₅₀,comprehensive evaluation value（D）,CAT,POD and soluble protein,with R²-0.96^-0.63.As the results shown that winter rapeseed sowing in early spring（in late march）,the differences of vernalization rate and growth and development of plants could reflect the cold tolerance of winter Brassica napus.3.Revealing the differences of botanical morphology and physiological and biochemical characteristics between cold-tolerance and cold-sensitive line of Brassica napus.Under continuous freezing treatment（-2℃）,the results shown that the palisade tissue cells of cold-sensitive line were decreased,the sponge tissue was loose,the number of chloroplasts was decreased and chloroplast expanded like circular.The relative conductivity（REL）and malondialdehyde（MDA）contents of the leaves were continuously increased.However the contents of soluble protein,soluble sugar and proline were decreased.The palisade tissue of cold-tolerance line“2016TS（G）10”is arranged neatly,the space of spongy tissue cells were increased.The chloroplast expanded,but the number of chloroplast did not decrease significantly,and the starch granules were significantly accumulated in chloroplast.The content of soluble protein,soluble sugar,chlorophyll a,chlorophyll b and the value of chlorophyll a/b were higher than cold-sensitive line.Then,the frozen plants were recovery for 48 h in the room temperature,the net photosynthetic rate（Pn）of cold-tolerance line“2016TS（G）10”was higher than the weak cold resistance line“Tianyou2288”.4.Exploring the mechanism of cold-tolerance Brassica napus line in response to the freezing stress.RNA-seq results revealed that 690,2538 and 2403 differentially expressed genes（DEGs）were respectively identified at frozen 1 h,3 h and 24 h.After comparing the DEGs in three groups,a total of 3905 DEGs were identified,among these genes,2312 were up-regulated and 1593 were down-regulated.The expression of DEGs that enriched in“plant hormone signal transduction”,“carbohydrate metabolism”,“amino acid metabolism”,“lipid metabolism”pathways were induced under freezing stress,the DEGs in biosynthetic pathways such as“ribosome”and“eukaryotic ribosome biosynthesis”was inhibited under freezing stress.It’s worth noting that 3 DEGs（BnaC06g28830D,BnaC04g40760D and BnaC05g37450D）that related to“biosynthetic unsaturated fatty acids”were significantly up-regulated with the extension of freezing time,to adjust the ratio of saturated and unsaturated fatty acids.Although the expression of P5CS（BnaC04g05620D and BnaA05g05760D）that related to“proline metabolism”were inhibited under freezing stress,but the expression of these genes were still high,to increase the content of proline.The DEGs that annotated inβ-amylase（BnaC09g21440D,BnaA07g05790D,BnaC07g34180D,BnaA03g42940D and BnaA01g17940D）were related to the synthesis or degradation of soluble sugars,and the expression were inhibited under freezing stress,resulting in the starch granule significantly accumulated in chloroplasts.In addition,the AP2/ERF,MYB,WRKY and Q-type C2H2 transcription factors were also played an important role in the cold-talerance Brassica napus response to freezing stress.5.The genome-wide identification and analysis the physical and chemical characteristics,chromosome distribution,gene structure,motif distribution,gene replication,phylogenetic relationships and the expression under freezing stress of Q-type C2H2 family menbers in Brassica napus.The results shown that 216 Brassica napus Q-type C2H2 genes were unevenly distributed on 19 chromosomes.Based on the gene structures and highly conserved motifs distribution,the 216 Q-type C2H2 members were divided into 8 clusters on phylogeny.The collinear analysis showed that segment replication played a crucial role in the expansion of the Q-type C2H2 family.In addition,based on the RNA-seq analysis,we analyzed the expression of Q-type C2H2 gene in response to freezing stress.Most of members did not respond,but the expressions of 11 Q-type C2H2 genes,which annotated as the ZAT10,ZAT12,ZAT6 and ZFP2,were continually and significantly increased with the extension of freezing stress time.It is indicated that these genes plays an important role in response to freezing stress.