Spotted longbarbel catfish, Hemibagrus guttatus (Lacepède, 1803), is a freshwater catfish with high economic value. It is primarily distributed in southern China, Laos, Thai, and Viet Nam (Yang and He 2008; Ha et al. 2017; My et al. 2018). It is carnivorous and feeds on crustaceans, aquatic insects, juvenile fish, and plant debris (Chu et al. 1999). H. guttatus is benthic fish inhabiting lotic environment with gravel substrates (Yang and He 2008). The wild population size of H. guttatus is gradually declining year by year because of overfishing and habitat loss. In 2012, H. guttatus was listed as "Population Decreasing" by IUCN (Zhao 2012). Recently, China has taken further measures to protect the valuable fish resources by designating the wild H. guttatus as a second-level National Key Protected Wildlife species in 2021. Hence, it is imperative to elucidate the genetic diversity and population structure of this species to facilitate its protection and offer guidance for governmental conservation management. However, the availability of molecular markers for H. guttatus is limited, posing challenges to assessing its genetic diversity.
Recently, the genome of H. guttatus has sequenced and assembled (Yang et al. 2024). This genome provides a great amount of information, and we thus developed a series of molecular markers based on this genome. In this study, we reported 57 insertion/deletion (InDel) markers developed by whole-genome resequencing. InDels are widely distributed across the genomes, providing abundant information of genetic variation. With the development of next-generation sequencing (NGS), InDel markers have been extensively used as they are fast, stable, and accurate (Hechanova et al. 2021). While, to our knowledge, no InDel markers have been developed and used for genetic diversity assessment and molecular marker-assisted breeding in spotted longbarbel catfish.
Muscle tissue samples of 32 individuals of H. guttatus were collected form Guangdong Hanyu Ecological Technology Co., LTD, Guangzhou city, Guangdong Province, China (113°30′45″N, 22°55′54″E). The TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) was used to extract the genomic DNA. Afterwards, we employed 1% agarose gel electrophoresis and Qubit 2.0 fluorometer to assess the quality and concentration of DNA. The qualified DNA was then used for library construction and sequenced on an Illumina novaseq 6000 platform. A total of 2186140 InDels were identified using GATK v4.2.6.1 (DePristo et al. 2011), of which 57 InDels were randomly selected for primers design using primer3 web version 4.1.0 (https://primer3.ut.ee/) and evaluated polymorphisms by PCR.
PCR amplification was performed using 25 µl of solution that contained 12.5 µL 2× PCR mix (Sangon, Shanghai, China), 0.25 µΜ of forward and reverse primers, 2 µL genomic DNA, and 10 µl ddH2O. Reaction cycles were set as follows: 95℃ for 5 min; 35 cycles of 95℃ for 30 s, 60℃ for 30 s, 72℃ for 1 min; and a final extension at 72℃ for 7 min. PCR products were sequenced using the ABI 3730 platform. We calculated observed heterozygosity (HO) and expected heterozygosity (HE) using PLINK v1.9 (Purcell et al. 2007), departures from Hardy-Weinberg equilibrium (HWE) was detected using PLINK v1.9 (Purcell et al. 2007). Polymorphism information content (PIC) was calculated using PIC_CALC v0.6 (Botstein et al. 1980).
The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.3333 to 0.5833 and 0.2778 to 0.5000, respectively (Table 1). The range of polymorphism information content (PIC) varied from 0.2692 to 0.3750 (Table 1). Among 57 loci, one locus significantly deviated from the HWE (Table 1). These InDel markers are of great value for genetic and genomic studies of H. guttatus.
Table 1. Characteristics of 57 InDel markers developed for Hemibagrus guttatus