The Origin Of Northeast Black Bee And Its Adaptive Mechanism To Low Temperature Environment

The Northeast Black bee is an excellent local breed in China.It has the advantages of good cold resistance and strong egg production.However,its origin is still not clear,and the mechanism of cold resistance is lack of systematic research.And the unclear classification status and genetic characteristics have hindered the protection of the Northeast Black bees.In this study,whole genome resequencing technology,determination of free water content and free fat content,and real-time quantitative PCR technology were applied to determine the origin and genetic characteristics of the Northeast Black bee.The main research contents and results are as follows:(1)In this study,whole genome re-sequencing was performed on 100 bee individuals of five bee species including the Northeast Black bee,Yuandong Black bee,Hunchun Black bee,Apis mellifera caucasica and Apis cerana cerana.Several 40 individual re-sequencing data from four bee species,including Apis mellifera carnica,Apis mellifera mellifera,Apis mellifera anatoliaca and Apis mellifera ligustica,were downloaded from the public database.A total of 246.32 G of raw data was obtained by sequencing,and a total of 54.49 G data was obtained by resequencing Northeast Black bee.The average comparison ratio of clean reads and reference genomes was 97.32%,the average sequencing depth was 18.Ox,and 2,114,450 SNPs and 739,093 InDels of Northeast Black bee were identified.(2)By constructing the phylogenetic tree,principal component analysis and population genetic structure analysis,the origin of the Northeast Black bee was explored.The results showed that the Northeast Black bee were closely related to the Yuandong Black bee and the Hunchun Black bee,and its pedigree composition is single.It proves that the Northeast Black bee may originate from the Yuandong Black bee.(3)By analyzing the free water content and free fat content of Northeast Black bees and Apis mellifera ligustica,the cold resistance of the Northeast Black bees was analyzed.The results showed that there was no significant difference in free water content and free fat content between the two bees(P>0.05),which indicated that both of the two bees had adapted to low-temperature environment,and formed a certain mechanism of cold resistance.(4)The selected area of Northeast Black bees were obtained by the comparison of the Northeast Black bee and the Apis mellifera ligustica using Fst&??,and candidate genes were selected by combined with the analysis of GO enrichment and KEGG.Two genes related to the accumulation of small-molecule cold-resistant substances were found out,namely the PH domain leucine-rich repeat-containing protein phosphatase 2 gene associated with glucose accumulation and the adenylate cyclase type 6 gene involved in the metabolism of phospholipids.(5)The real-time quantitative PCR technology was used to analyze the expression of cold-resistance related genes in the Northeast Black bees,Apis mellifera ligustica in Northeast China,and Apis mellifera ligustica in Yangzhou.The results showed that the expression levels of Dachsous,HSP 90 and Hsc70-5 genes were significantly higher in Northeast Black bees than Apis mellifera ligustica in Yangzhou and Apis mellifera ligustica in northeastern China(P<0.05),and there was no significant difference between Apis mellifera ligustica in Yangzhou and Apis mellifera ligustica in Northeast(P>0.05);the expression levels of HSP 60 and Hsc70-4 genes were significantly higher in Northeast Black bees and Apis mellifera ligustica in Northeast China than Apis mellifera ligustica in Yangzhou(P<0.05).The difference between Apis mellifera ligustica in different area was not significant(P>0.05).The results showed that the Northeast Black bees were more resistant to cold than the Apis mellifera ligustica in the northeast,and the Apis mellifera ligustica in the northeast were stronger than the Apis mellifera ligustica in the Yangzhou region,indicating that the environment could influence or even change the cold resistance of the honey bee.This study identified the taxonomic status of the Northeast Black bees,and found that the cold resistance of the Northeast Black bees was its adaptability to the low temperature environment.This study is of great significance to the protection and actual production of the Northeast Black bee genetic resources.