Integration of transcriptome and whole-genome re-sequencing analyses reveal growth-related candidate genes in Procambarus clarkii.

Growth is a crucial economic trait of all aquaculture species. It is important to explore the molecular regulation on growth, which could help improve the growth rate of species. Mining the growth-related genes is the foundation for revealing its molecular regulation on growth. Presently, the molecular regulation of growth in Procambarus clarkii is not clear, and the study on exploring growth-related genes is limited. In this study, RNA-Seq was used to compare gene expression profiles of the individuals with different growth rates involved in four groups including Big Male (BM), Big Female (BF), Small male (SM), and Small Female (SF) from one P. clarkii family, and the analyses were performed in combination with sex. Meanwhile, whole-genome resequencing data was used to get growth-specific SNP (Single Nucleotide Polymorphism)/InDel (Insertion/Deletion) sites information. Totally, we identified 16,127 genes, of which 9065 were successfully annotated in the GO database. Among these, 1328 DEGs were identified in BM vs. SM, with 357 up-regulated and 971 down-regulated. Additionally, 3507 DEGs were identified in BF vs. SF, with 241 up-regulated and 3266 down-regulated. 96 DEGs were up-regulated and 820 DEGs were down-regulated in Growth-related Group. The expression levels of nine DEGs were validated by RT-qPCR to verify the analysis results of sequencing. 684,040 growth-related SNPs and 182,050 growth-related InDels were obtained after screened. These findings provide candidate growth-related genes and growth-specific SNP/InDel sites for regulation of growth traits in P. clarkii, and new insight into the molecular regulation of P. clarkii growth.

Two Short Repeats in the 5' Untranslated Region of Insulin-like Androgenic Gland Factor in Procambarus clarkii (PcIAG) That Regulate PcIAG Expression.

Insulin-like androgenic gland factor (IAG) plays an important role in sex manipulation in decapods. Understanding the molecular regulation mechanism of IAG in Procambarus clarkii (PcIAG) is important for realizing its sex control. In this study, the promoter and gene structure of PcIAG, mRNA, and miRNA expression profiles after interfering with two siRNAs synthesized according to the two short repeats in the 5' untranslated regions (5'UTR) of PcIAG were analyzed, and miRNAs of exosomes were investigated to explore the role of repeated sequences with tandem two short repeats located in the 5'UTR of PcIAG isolated from the androgenic gland (AG) in the regulation of IAG expression. The results showed that the repeated sequences of 5'UTR only occurred completely in the cDNA from AG, and the function of the two repeats was different in regulating the expression of PcIAG, in which the Wnt signaling pathway may be involved. Furthermore, we found that six miRNAs including miR-133, miR-193, miR-34, miR-1, miR-100, and let-7 might be involved in the regulation of the expression of PcIAG, wherein miR-133 might directly be related with the repeated sequences of 5'UTR.

Exploration of an XX/XY Sex Determination System and Development of PCR-Based Sex-specific Markers in Procambarus clarkii Based on Next-Generation Sequencing Data.

Many economic crustacean species have sex dimorphisms during their growth. Exploring the sex determination system and developing sex-specific molecular marker(s) are very helpful for carrying out sex control breeding, and next-generation sequencing has been used as an efficient way to explore them in recent years. In this study, first, the genetic sex determination system of P. clarkii was explored as an XX/XY system by analyzing the 2b-RAD sequencing data. Furthermore, DNA samples of male and female individuals from a P. clarkii family were pooled separately for whole-genome resequencing. Based on the data of whole-genome resequencing, the 9,163 male- and female-specific bias sites with higher feasibility were obtained based on the assumption of the XX/XY sex determination system, and four sites were selected to design the sex-specific marker primers. One efficient sex-specific marker was identified with a sex discrimination rate of 99.49% (195/196) when applied to five different geographical groups with 196 individuals. The results of this study would provide a foundation for the realization of P. clarkii sex control and could provide some reference for investigating the sex determination system and sex molecular marker(s) of other crustacean species based on next-generation sequencing data.

[Study on dynamic accumulation of index components from Bupleurum chinense in various collecting periods].

OBJECTIVE: To study the dynamic change law of volatile oil, saikosaponin a, d and alcohol-extract from Bupleurum chinense at Songxian region in Henan province, and to explore the optimal harvest period of Bupleurum chinense. METHODS: With the contents of saikosaponin a and d, absorbance of volatile oil and percentage of alcohol-extract as indexes, HPLC-ELSD and ultraviolet spectrophotometry were successively used to analyze them. RESULTS: There are obvious differences among the contents of volatile oil, saikosaponin a, d and alcohol-extract in various collecting periods sample, the absorption of volatile oil in distillation was the highest in October, the content of saikosaponin a was the highest in September, the saikosaponin d in December and the percentage of alcohol-extract in October. CONCLUSION: The optimal harvest period of Bupleurum chinense at Songxian region in Henan is identified, which can provide scientific basis for crude drug production and processing.