Pelodiscus sinensis is the most common turtle species raised in East and Southeast Asia. The Japanese strain and the Qingxi black strain are important aquatic breeds of P. sinensis in China with unique characteristics in terms of production performance and appearance, including skin color. In this study, melanin content measure and histological analysis on skin samples of these two strains were carried out to compare their color characteristics. The results showed that Qingxi black turtles clearly have a greater ability to deposit melanin than the Japanese strain especially in the abdomen. Then, de novo transcriptome assembly and differential expression profiling analyses on the ventral skin from the two strains were performed to identify the genes responsible for the differences in skin color using the Illumina RNA-Seq system with three biological replicates. A total of 19,331 annotated unigenes were found by aligning to the reference genome of P. sinensis using TopHat v2.0.12. Differential expression analysis revealed that 670 genes were expressed differently, including 185 upregulated genes and 485 downregulated genes in Qingxi black strain using the DESeq R package (|log₂FoldChange| ≥ 1, padj < 0.05). Sixteen differentially expressed genes (DEGs), which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). GO and KEGG analyses revealed four DEGs (agouti signaling protein, frizzled family receptor 1, phospholipase C, and protein kinase C) were related to melanogenesis pathway. Gene expression levels of the four DEGs as well as three genes from the tyrosinase gene family were measured by qRT-PCR. The results indicated that agouti signaling protein, tyrosinase-related protein, and dopachrome tautomerase could be the main genes responsible for the difference in abdominal skin color between the two turtle strains. This study provided valuable information for further analysis of the melanogenesis mechanisms in different varieties of P. sinensis.

中文翻译：

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转录组分析提供了对黑色素途径在两种不同色素的中华鳖龟品系中的作用的深入了解。


中华鳖是东亚和东南亚最常见的龟种。日本品系和青溪黑品系是我国重要的中华对虾水产品种，在生产性能、外观、皮色等方面具有独特的特点。本研究对这两种菌株的皮肤样本进行了黑色素含量测量和组织学分析，以比较它们的颜色特征。结果显示，青溪黑龟明显比日本品系具有更强的黑色素沉积能力，尤其是在腹部。然后，使用具有三个生物学重复的 Illumina RNA-Seq 系统对两个品系的腹侧皮肤进行从头转录组组装和差异表达谱分析，以确定导致皮肤颜色差异的基因。使用TopHat v2.0.12与中华松的参考基因组比对，共发现19,331个带注释的unigenes。差异表达分析显示，使用DESeq R软件包，青溪黑品系中有670个基因存在差异表达，其中185个上调基因和485个下调基因（|log ₂ FoldChange| ≥ 1，padj < 0.05）。随机选择16个差异表达基因（DEG），通过实时定量PCR（qRT-PCR）进行确认。 GO和KEGG分析显示四个DEG（agouti信号蛋白、卷曲家族受体1、磷脂酶C和蛋白激酶C）与黑素生成途径相关。通过 qRT-PCR 测量了四个 DEG 以及酪氨酸酶基因家族中的三个基因的基因表达水平。 结果表明，agouti信号蛋白、酪氨酸酶相关蛋白和多巴色素互变异构酶可能是造成两个龟品系之间腹部肤色差异的主要基因。该研究为进一步分析不同中华松品种的黑素生成机制提供了有价值的信息。