A chromosome-level genome assembly of the oriental river prawn, Macrobrachium nipponense

Abstract Background The oriental river prawn, Macrobrachium nipponense, is an economically important shrimp in China. Male prawns have higher commercial value than females because the former grow faster and reach larger sizes. It is therefore important to reveal sex-differentiation and development mechanisms of the oriental river prawn to enable genetic improvement. Results We sequenced 293.3 Gb of raw Illumina short reads and 405.7 Gb of Pacific Biosciences long reads. The final whole-genome assembly of the Oriental river prawn was ∼4.5 Gb in size, with predictions of 44,086 protein-coding genes. A total of 49 chromosomes were determined, with an anchor ratio of 94.7% and a scaffold N50 of 86.8 Mb. A whole-genome duplication event was deduced to have happened 109.8 million years ago. By integration of genome and transcriptome data, 21 genes were predicted as sex-related candidate genes. Conclusion The first high-quality chromosome-level genome assembly of the oriental river prawn was obtained. These genomic data, along with transcriptome sequences, are essential for understanding sex-differentiation and development mechanisms in the oriental river prawn, as well as providing genetic resources for in-depth studies on developmental and evolutionary biology in arthropods.

: please indicate what do the numbers indicated between brackets mean (confidence intervals, I guess) Reply: Yes, you are right. The numbers within brackets represent confidence intervals. Thais sentence was added to the figure legend of Figure 3 (line 386).
Lines 298-300: please improve the English language used here. Reply: Thanks for your advice. Yes, it is done in lines 304-307 of the manuscript.
The WGD discussion remains unconvincing. The authors mentioned Yuan et al. 2017 (the E. carinicauda genome) stating that this genome was not characterized by a WGD event. Actually, both genome size (5.7Gb) and 2n chromosome number (90) do not indicate significant differences compared with M. rosembergii. On the other hand, the number of genes is more than 2X higher in E. carinicauda. The quality of the E. carinicauda genome assembly was much lower than the genome reported in this study, so do the authors think that clues linked with WGD might have been missed in the study by Yuan et al.? Please also note that other Palaemonidae species have been previously shown to be characterized by very large genomes. For example Palemon serratus, with a c-value > 10. Overall, I believe that the timing of this WGD event cannot be defined with certainty at the present time due to the lack of genome and karyotype information for many species, so I would appreciate if the authors could state more explicitly that many uncertainties about this inference remain. Reply: Thanks for your comments. Yes, you are right; we really think the important clues of WGD event have been missing in the Yuan's study, because the contig N50 of the E. carinicauda genome is only 263bp. These data are indeed fragmental to be used for gene identification. Therefore, too many genes and synteny blocks in the E. carinicauda genome were missed. For the genome size, rich repeat content could be a major reason for the genome expansion. Based on our experience, the Palemon serratus could have much more repeat content. For the WGD analysis, we employed both 4dtv and synteny block analyses that were well-performed and classical methods for WGD identification in various animal and plant genome reports. In the coming future, if there is any newly published genome assembly of a close shrimp species, we will further analyze related WGD event/time. According to your advice, we rewrote related sentences. Please find more details in lines 301-307. industry, because over-reproduction will happen frequently during the farming process, leading 64 to poor survival, low growth rates, and small body size. Therefore, it is critical to understand 65 the mechanism of sexual differentiation and reproductive development in Oriental river prawn 66 in order to obtain genetic improvement.
In our present study, a chromosome-level genome assembly for the Oriental river prawn 68 was constructed by integration of PacBio long reads, Illumina short reads, and Hi-C sequencing 69 data. These genomic data along with transcriptome sequences will be a useful resource for in-70 depth studies on sex-differentiation and the mechanism of reproduction in Oriental river prawn, 71 as well as promoting comparative genomic analyses with other prawn species. [11] and further aligned to the draft genome assembly using Juicer (v1.5, RRID: SCR_017226)

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[12] to generate the Hi-C interaction maps ('merged_nodups.txt' file). Subsequently, scaffold 151 sequences from the draft assembly were ordered and oriented using the 3D-DNA pipeline [13] 152 to integrated into long pseudo-chromosomes. Manual review and refinement were performed 153 for identification and removal of assembly errors with assistance of the Juicebox Assembly   (Table 1). We also predicted 44,086 252 protein-coding genes, of which 39,317 genes have functional assignments with public 253 databases. All distributions of genes, repeat sequences and GC content were shown in Figure   254 2b.   (Table 2). KEGG analysis revealed that these DEGs were involved in "Signal 293 transduction", "Endocrine system", "Neurodegenerative diseases", and "Lipid metabolism".  Here we have identified that the oriental river prawn had undergone a WGD event about 305 109.8 Mya based on large numbers of self-synteny blocks in this species. In a previous study 306 [57], Exopalaemon carinicauda, a Palaemonidae species, was reported as not having genome 307 duplication. According to our analysis of the L. vannamei genome (NCBI accession number 308 QCYY00000000), we identified that this species has not undergone the WGD event either.

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Therefore, this paper is likely the first report of a recent WGD event in the M. nipponense 310 genome.

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Histological observations [58] demonstrated that the testis and androgenic gland of 312 Oriental river prawn in the non-reproductive vs. reproductive season showed significant 313 morphological differences. Therefore, those DEGs in the testis and androgenic gland between 314 the non-reproductive vs. reproductive seasons may participate in the male sexual differentiation 315 and development processes in the Oriental river prawn. We predicted in the present study that 316 a total of 13 DEGs were respectively selected through transcriptomic profiling analysis ( Table   317 2), of which five were co-DEGs. A few plausible sex-related candidate genes were identified, 318 particularly after combining the analysis of genes on Chromosome 25 and differential 319 transcription in testis and androgenic gland between the non-reproductive and reproductive 320 seasons. However, these results require more independent validation.  The data that support the findings of this study have been deposited in CNGB Sequence

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Archive of China National GeneBank DataBase with accession number CNP0001186.

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Genomic data is available via EBI bioproject IDs PRJNA646023 and PRJNA541743.

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Supporting data and materials are also available in the GigaScience GigaDB database [59].