Study On DUI Occurring And Gonad Development Of Freshwater Pearl Mussels

Mitochondria have their own genetic material; the general performance of mitochondria is strictly maternally inherited. But recent years, studies have found paternal inheritance in the presence of a wide range of mollusca, termed doubly uniparental inheritance(DUI). Among them, M- type was found only in the gonad of male individuals, while the F- type was found in the gonad and somatic tissues of female individuals as well as in the somatic tissues of male individuals.This study carried out research on the phenomenon of DUI in two kinds of freshwater pearl mussels U. douglasiae and H. cumingii, not only compared on the level of the whole mitochondrial genome sequences, but also analyzed the application of sex identification based on the mt DNA single gene, and observed the gonad development of juvenile freshwater mussels, further researched on the relationship with the occurrence of DUI. The complete F- type and M-type mitochondrial genome of U. douglasiae was firstly determined, verified the DUI phenomenon was universal in freshwater pearl mussels; Combined with the U. douglasiae, H. cumingii and other 4 kinds of freshwater mussel F- type and M- type mt DNA, study on the DUI phenomenon of 6 kinds of freshwater mussel; In addition, based on the specificity of the F- type and M- type COII gene, according to the detection of the differential amplification results, study on sex identification of juvenile H. cumingii; Combined with the histological observation of the juvenile H. cumingii gonad’s development, in order to explore the relationship between the occurrence of DUI and gonad development. Specific contents are as following: 1. Comparative analysis of F- type and M- type mitochondrial genome in U. douglasiaeThe complete F- and M-type mitochondrial genome of U. douglasiae was firstly obtained; they were 15767 bp and 16670 bp, respectively; the Mtype is longer than the F- type 903 bp. Both included 13 protein coding, 22 t RNA, 2 ribosomal r RNA and 25 non coding regions, the length of the non coding region of F-type and M-type is between 1-312 bp and 1-348 bp, respectively, and the location of the largest non coding region is also different. U. douglasiae F- type and M- type mt DNA COI-COIII, ND3-ND5, ND4 L, ATP6, ATP8, t RNAAsp and t RNAHis were coding in H chain, the rest were coding in the L chain. The total length of the 13 protein genes of the M- type mt DNA was longer than the F- type about 600 bp. Among them, there was a special 558 bp fragment at the 3’ end of M- type COII gene. Structural analysis revealed that the F- type and M- type COII genes both had 2 transmembrane structures at the close position of 5’ end, and M- type COII gene has other 4 transmembrane structures at 3’ end, it is inferred that the elongation site of M- type COII gene is a functional gene fragment. 2. Comparison and analysis of DUI in freshwater musselAccording to the complete sequences of U. douglasiae, H. cumingii and other 4 species of freshwater mussel’s F- type and M- type mt DNA, the full-length and COI, COII and ATP8 sequences of six kinds of freshwater mussels were compared. There were no significant difference found between the length of F- type and M- type COI gene; however, great difference of length in two type COII gene sequences was detected, there was a extension of the fragment about 500 bp at the 3 ’end of M- type COII gene; and the F- type ATP8 gene was slightly longer than the M- type. Based on comparison of the gene arrangement of F- type and M- type mt DNA in 6 kinds of freshwater mussels, there were some differences of the t RNA order and the number of t RNA between the ND3-ND5. Among them, the F- type mt DNA gene sequence is ND3â†'Hâ†'A… ND5â†'Qâ†'C, and M- type gene sequence is ND3â†'Aâ†'S…ND5â†'Hâ†'Qâ†'C. In addition, it was found that there was a significant difference in the amino acid sequences of 13 genes of the F- type and M- type mt DNA; The F- type sequence was more conservative and the M- type sequence was mutated easily than the F- type among the species. The amino acid sequences of Ftype and M- type COII and ATP8 had the biggest difference; and COI is the most conservative, with the highest degree of similarity. The NJ phylogenetic trees were constructed using the conserved sequences(COI and CYTB genes), and high mutation rate sequences(COII and ATP8 genes) of the freshwater mussel F-type M- type mt DNA, respectively. F- type and M- type mt DNA were divided into two groups, F- type of all species accumulation in the one branch, and M-type of all species accumulation in the one branch. This is consistent with our expectations of the DUI phenomenon. 3. Sex identification of juvenile H. cumingii based on the COII gene sequencesIn this study, the length differences of the F- type and M- type COII gene, as a molecular marker which were used to detect the presence of Fand M- type mt DNA in the age of 3-10 month juvenile H. cumingii. PCR amplification were used F- type and M- type specific primers for the age of 3 months, 4 months, 5 months old, 5.5 months, 6 months and 6.5 months and 7 months, 7.5 months, 8 months, 9 months and 10 months of 20 individuals H. cumingii’ s DNA extracted from gonad and mantle tissue. The results showed that the specific amplification of F- type and M- type COII gene could be detected at the age of 3 months. The M- type COII gene can be only detected the part of the gonad in of juvenile mussels, and cannot be detected in the mantle tissue; the F- type COII gene could be detected in the gonad and mantle tissue of all juvenile mussels. The same results were also presented in the follow-up of the age of 4-10 months juvenile mussels. The study showed that the DUI phenomenon of the H. cumingii could be identified sex at the age of 3 months. 4. Observation the early gonad development of juvenile H. cumingiiIn this study, the gonad development of juvenile H. cumingii was studied, and the gonad tissues were collected for each one from 10 individuals at the age of 3-10 months H. cumingii to make paraffin sections and stained with HE. Observed the gonad development of the H. cumingii by histological section to explore the relationship between the occurrence of DUI and gonad development. The results showed that it was difficult to observed the gonad tissue at the age of 3- 5 months juvenile H. cumingii, mean while, the digestive tube and a large number of connective tissues are filled with the whole visceral mass; at the age of 5-8 month juvenile H. cumingii, the visceral mass is still filled with a lot of connective tissue, whereas, there was a small number of primordial germ cells, but it is difficult to distinguish between male and female, as the shape is not the rule; at the age of 8 month juvenile H. cumingii’s gonad tissue structure began to mature, male and female could be identified distinct; in addition, the male gonad tissue were matured earlier, compared with the female gonad tissue, which has been able to identify the sperm mother cell very clear at the age of 8 months.This is the first study to obtained the F- type and M- type mt DNA length of U. douglasiae, it would enriched the reference data for the DUI study of freshwater mussels. Meanwhile, the gender identification were carried out based on DUI phenomenon, and compared with the gonad development at the different stage of juvenile H. cumingii which detected using histological section. Thish study would provide basic data for DUI analysis and further used in the practical application.