| The Asian yellow pond turtle(Mauremys mutica)is one of the emerging aquaculture species with high edible,medicinal,scientific research and ornamental value in South China.In 2012,there were about 3.53 million individuals cultured as commercial turtles in Guangdong province alone.However,a few of hatchlings in a nest has always been one of the main factors restricting the development of this industry.Since the number of eggs laid by M.mutica is significantly different among individuals,the mothers with high fecundity screened as a breeding parent is one of the effective ways to solve this problem and promote the sustainable development of the industry.In this study,the intrinsic factors affecting the reproductive capacity of Mauremys mutica were discussed from the six aspects as follows.The molecular mechanism of their spawning differences was analyzed from the molecular level,and the SNP loci related to reproductive capacity were screened for Mauremys mutica,which will provide some basic data and effective genetic information for sustainable development of this turtle industry.1.To investigate how maternal size affects offspring size and number,we explored the relationships among the maternal body size and the number and size of cultured Asian yellow pond turtle,Mauremys mutica,hatchlings,using a 4-year dataset.Our results showed that different female not only produced different sizes of offspring,but also produced different numbers of offspring.We did not find maternal body size significantly influenced the mass of offspring according to regression analysis,however,based on which,we detected significant relationships between offspring size and clutch size and maternal age.The mean body mass of offspring increased over the four years,and the clutch size varied significantly,which,in the absence of a correlation between offspring size and maternal body size.However,the number of offspring per females increased with maternal plastron length rather than age.Our results were consistent with optimal offspring size theory in that females did not increase offspring size but rather increased offspring number to increase their fitness,which further our understanding of the evolution of reproductive strategies in cultured reptiles.2.To explore the biotic factors(i.e.,maternal age)as well as abiotic factors(i.e.,weather)effects on the reproductive output of cultured Asian yellow pond turtles,Mauremys mutica,we surveyed and compared the reproductive output of 13-year-old(n = 261)and 25-year-old(n = 265)female turtles on non-raining and raining days,respectively.The results showed that older females produced bigger eggs than those of younger females no matter whether it was raining or not.Females had larger clutch size(the number of eggs per nest)under the non-raining condition compared with that under the raining conditon,Variation of clutch frequency(the number of clutches per day),however,was independent on maternal age and rainwater,and there was also no interaction of maternal age and weather changes on egg size,clutch size and clutch frequency.Our results suggested that maternal age and weather changes had significantly effects on egg size and clutch size,respectively.These results imply that maternal age,together with weather conditions(mainly raining),can affect the reproductive output of M.mutica at different age stages.Our results will provide some useful information for artificial breeding and stock management of M.mutica.3.To disclose how maternal age and size constrain the reproductive capacity(egg size and egg number)of most of oviparous animals including M.mutica,we first identified the full-length cDNAs from estrogen receptor 1(ESR1),bone morphogenetic protein receptor 1B(BMPR1B),and forkhead box L2(FOXL2).The ESR1 open reading frame(ORF)was 1,767 bp encoding 588 amino acids.For BMPR1 B,the ORF was 1,599 bp encoding 532 amino acids,and an ORF of 906 bp encoding 301 amino acids was identified in FOXL2.The effects of maternal age and size on the expression of ESR1,BMPR1 B,and FOXL2 in the ovary,brain,and uterus showed that ESR1 expression in large females was significantly lower than that in small females in the brain,but body size did not affect ESR1 expression in the ovary.The expression of ESR1 was significantly different in the different age groups and size groups,and there was interaction detected between maternal age and body size.Howover,BMPR1 B expression in the ovary,brain,and uterus was independent of maternal age and size.In addition,we found different FOXL2 expression patterns between the brain and uterus,while detected interaction of female age and size in the brain and ovary.Our results imply the complexity and diversity of maternal age and size in regulating the expression of genes related to reproduction.These results provide more information for the maternal effects on the reproduction-related gene expression.4.To investigate whether mutations and copy number variations in mitochondrial genes are related to the reproductive capacity of the Asian yellow pond turtle,Mauremys mutica,there was exploration of the distribution frequency of 129 Single Nucleotide Polymorphism loci of six mitochondrial genes,ND1,ND2,COX2,ND4,Cytb and D-loop,of turtles from a relatively greater and lesser fecundity group by direct sequencing.The validation results for five candidate SNP loci in 83 female turtles indicated that only three SNP loci(C119T,A320 G and A417C)in ND1 were positively correlated with reproductive capacity in M.mutica(P < 0.05).In addition,by constructing linear regression equations of the copy numbers of ND1,ND4,Cytb,D-loop,COX3,and ATP6(Log10 transformed)genes and the mean offspring number of different female turtles during a 4-year period,the copy numbers of ND4 and ATP6(Log10 transformed)genes were positively correlated(P < 0.05)with the fecundity of female turtles.Results from the present study may provide useful genetic markers for breeding M.mutica with greater reproductive capacity.5.To explore the factors affecting the synthesis of sex hormones and the molecular mechanisms by which sex hormones regulating the reproductive capacity of animals,we compared the expression difference in the steroidogenic enzyme genes(P450 cholesterol side chain lyase(P450scc/Cyp11α1),cytochrome P450-17α-hydroxylase(Cyp17a1),3β-hydroxysteroid dehydrogenase(3β-HSD),17β-hydroxysteroid dehydrogenase type 3(17β-HSD3),aromatase(Cyp19a1)and 5α-Reductase(5α-R))and hormone receptor genes(follicle estrogen receptor(FSHR),estrogen receptor type I(ESR1),androgen receptor(AR)and progesterone receptor(PR))"hypothalamic-pituitary-gonadal" axis of between the high and low fecundity turtles,M.mutica.Our results showed that the expression levels of StAR and 17β-HSD3 were significantly higher in the pituitary,ovary and uterus of high fecundity females than that of low fertility females,but there was no significant difference in the hypothalamus between the two groups.The expression levels of Cyp17a1,Cyp19a1 and 5α-R were significantly different in the pituitary,but there was no significant difference in the hypothalamus,ovary and uterus of high and low individuals,however,no significantly different of the Cyp11a1 and 3β-HSD expression levels were detected among the four tissues of high and low fecundity individuals.In the pituitary,the transcription levels of FSHR,ESR1,AR and PR of high fecundity individuals were significantly higher than those in low fecundity individuals,as well as in the hypothalamus for expression levels of ESR1 and PR between the two groups.In addition,there was no significant difference in the expression levels of the above four receptor genes in the ovary and uterus of high and low fecundity individuals.Our research reveals differences in the regulation of hormone synthesis and related genes in prolific and non-prolific females,and will lay a foundation for revealing the mechanisms underlying the differences in reproductive capacity of vertebrates.6.Increasing the number of eggs and obtaining more seedlings is one of the key measures to promote the development of Asian yellow pond turtle industry,but the genetic mechanism behind the difference in spawning has not been known.MicroRNAs play key roles in regulating the reproductive capacity of animals(number of eggs laid or hatchlings).In this study,high-throughput sequencing technology was used to screen miRNAs related to reproductive capacity based on the construction of ovary cDNA library of high and low-fecundity M.mutica.The results showed that 15767494(93.98%)and 15767 494(93.98%)high quality reads were obtained from the high and low fecundity groups,respectively.We screened 131 miRNAs differentially expressed between the high and low fecundity groups,of which 78 were up-regulated and 53 were down-regulated by comparing with the genome of M.mutica.GO enrichment analysis and KEGG pathway analysis of the target genes of differentially expressed miRNA showed that there was significantly different in the enrichment frequency of genes associated with ATP binding and proteolysis between the high and low fecundity groups,while the tricarboxylic acid cycle and the glucagon signaling pathway and the vitamin B6 metabolic pathway may play an important role in determining reproductive capacity.We also verified the 10 miRNAs screened by using qRT-PCR and confirmed the reliability and accuracy of the sequencing results.These results help to further understand the regulation mechanism of miRNAs on oviposition behavior,and will provide some available genetic markers for screening more female turtles with high reproductive capacity. |
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