| Philosamia cynthia ricini,originated in Assam province,Indian.It belongs to the Lepidopteran and is a non-diapause multivoltine insect.Since the introduction in China in the 1940 s,researches have been conducted on the physiological habits,breeding techniques,and other aspects of P.cynthia ricini.There are currently more than 20 P.cynthia ricini germplasm resources are conserved in our laboratory,and a database of phenotypic traits has been established.We have developed molecular markers and performed germplasm identification and DNA barcode development at the genome and transcriptome level using P.cynthia ricini strain B7,which provided important data for the classification and analysis of P.cynthia ricini germplasm resources.The main research results were listed as follows:(1)SSRs were screened at the whole genome level of P.cynthia ricini B7.The MISA program was performed to analyze the full sequence of the P.cynthia ricini reference genome,and a total of 186 790 gSSR loci were retrieved,an average of 1 SSR per 2.4kb;25174 EST-SSR loci were screened based on the assembled transcriptome data,an average of 1 EST-SSR per 2.8kb.P.cynthia ricini genome and transcriptome are dominated by mononucleotide and dinucleotide repeat types.Among the mononucleotide repeat types,both gSSR and EST-SSR have the highest frequency of A/T;Among the dinucleotide repeat types,AT/AT appeared the most frequently.Designed and synthesized 28 pairs of gSSR and40 pairs of EST-SSR primers for PCR amplification detection,of which 25 and 31 pairs of amplified bands of the expected size were obtained,and further automatic capillary nucleic acid electrophoresis detection was used in 20 P.cynthia ricini germplasm resources.We finally found 15 gSSRs and 12 EST-SSRs showed polymorphic bands among 20 samples.Genetic diversity,genetic structure and phylogenetic tree of 20 samples were analyzed by using the software Power Marker,Pop-Gene,and Structure.The results showed that 20 germplasms could be divided into two branches,but the overall diversity was not high,which might be due to the lack of SSR markers and the relative small population size.(2)The mitochondrial genomes of B7 and C2 gx varieties were assembled by using next-generation sequencing technology,and their genomic differences and genetic relationship were compared and analyzed.The results showed that the genome sizes of B7 and C2gx were 15 383 bp and 15 344 bp,and the GC content was 20.20% and 20.05%respectively with obvious AT skewness.The mitochondrial genomes of both B7 and C2 gx contain 37 genes,including 13 protein-coding genes,2 ribosomal RNA genes,22 transfer RNA genes and one non-coding region.Among the 13 protein-coding genes of B7,only the Cox1 uses CGA as the start codon,not typical start codon.Both mitochondrial genomes have some mismatched bases in t RNA genes,most of which are UG mismatched bases in the typical t RNA secondary structure,and there are some UU mismatched bases in the atypical t RNA secondary structure,and there is only a pair of AA mismatch bases.The mitochondrial genome sequence similarity between B7 and C2 gx is as high as 96.83%.Among the 13 protein-coding genes,7 bp insertion/deletion was detected at the beginning of the sequence.Phylogenetic tree analysis shows that B7 and C2 gx are clustered together,which proves the taxonomic status of P.cynthia ricini and P.cynthia cynthia at the molecular level.(3)The conservative mitochondrial protein coding genes Cox1 and Cytb were selected from the B7 mitochondrial genome and amplified in 20 germplasms of P.cynthia ricini.The length of the amplified target fragment of Cox1 is 465 bp,which encode 155 amino acids,and the length of target fragment amplified by Cytb is 915 bp,which encode 305 amino acids.Through the analysis of the base composition,we found that their content of AT was significantly higher than that of GC,and the highest AT content in the third position,and the base composition of gene sequence has obvious bias.A statistical analysis of the frequency of codon usage found that the most frequently used codon for Cox1 fragments is AUU(16times)encoding isoleucine(I).The most frequently used codon for Cytb fragments is UUA(36 times)encoding leucine(L).Analysis of the amino acid composition found that their content of Leucine is the highest,with an average content of 13.55% and 16.72%.Cox1 and Cytb can be used as potential DNA barcodes for interspecific identification of P.cynthia ricini germplasm resources. |
