The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring’s eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding.

Genetic diversity is the determinant of the allocation of germplasm resources in the genetic improvement of aquaculture species. In this study, three F₁ families, including a hybrid Scylla family (S. paramamosain ♂ × S. serrata ♀), a paternal family of S. paramamosain, a maternal family of S. serrata, and two wild populations, including a paternal population of S. paramamosain and a maternal population of S. serrata, were used to investigate the genetic diversity and genetic difference. The results indicated that 98 alleles of nine microsatellites loci were observed in five Scylla populations. The highest average value of Ho (observed heterozygosity), He (expected heterozygosity), and PIC (polymorphic information content) of the wild S. paramamosain population were 0.790, 0.799, and 0.771, respectively, suggesting the wild paternal population has high genetic diversity. The comparative analysis of PIC, Fst (fixation index), and HWE (Hardy–Weinberg equilibrium) indicated that the paternal S. paramamosain may be more suitable for artificial breeding than the maternal S. serrata from the perspective of allele frequency. Analysis of molecular variance analysis (AMOVA) showed that the total genetic variation mainly occurred within populations (73.28%), demonstrating that artificial breeding may induce genetic differentiation of the family groups of Scylla. The results of the analysis of Fst value, UPGMA (unweighted pair-group mean analysis) dendrogram, and genetic diversity indicated that the F₁ hybrid offspring had a close genetic distance and high genetic identity with the paternal S. paramamosain populations. It indicated that the F₁ hybrid offspring showed potential paternal genetic affinities and a similar potential for artificial breeding with S. paramamosain. The study will provide valuable information to evaluate the difference in the genetic diversity and population structure between hybrid offspring and distinct parental populations of Scylla.