Pacific abalone (Haliotis discus hannai) is a commercially important high valued molluscan species. Its wild population has decreased in recent years. Pacific abalone is widely cultured in Korea. Traditional breeding programs have been implemented for hatchery production of abalone seeds. To obtain more genetic information for the molecular breeding program, a high-density linkage map and quantitative trait locus (QTL) for three growth-related traits was constructed for Pacific abalone. F1 cross population with two parents were sampled to construct the linkage map using genotyping by sequencing (GBS). A total of 664,630,534 clean reads and 56,686 SNPs were generated. In sum, 3,345 segregating SNPs were used to construct a consensus linkage map. The map spanned 1,747.023 cM with 18 linkage groups and an average interval of 0.55 cM. QTL analysis revealed two significant QTL in LG10 on the consensus linkage map in each growth-related trait. Both the QTLs are located in the telomere region of the chromosome. Moreover, four potential candidate genes for growth-related traits were identified in the QTL region. Expression analysis revealed that identified genes are involved in growth regulation of abalone. The newly constructed genetic linkage map, growth-related QTLs and potential candidate genes identified in the present study can be used as valuable genetic resources and will be useful for marker-assisted selection (MAS) of Pacific abalone in molecular breeding program.

1. Mapping Family

An F1 family of Pacific abalone was produced in May 2019 for genetic mapping. Female and male parents were obtained from two geographically distant zones in Korea: a smaller size female (95.64 g; 94.97 mm) from Wando (34°19'12.8"N 126°39'02.6"E) in South coast of Korea and a larger size male (115.92 g; 105.21 mm) from Boryeong (36°18'56.3"N 126°26'17.8"E) in West coast of Korea. The parents were maintained in a commercial abalone hatchery, Tou-Jeong Soosan abalone hatchery, Dolsan-eup, Yeosu, Korea. Abalones were reared in recirculating sea water tanks. After reaching at full maturity, induced spawning was performed by heat-induction and UV-irradiation. After fertilization and production of abalone larvae, 15,000 progenies were reared under commercial condition at the larval rearing unit with recirculating sea water.

2. Sample Collection and Trait Measurement

Twelve months after hatching in May 2020, 96 F1 offspring (48 largest and 48 smallest) were randomly selected and sampled. For linkage analysis, three quantitative growth traits of abalone, i.e., total weight (TW), shell length (SL) and shell width (SW), were measured. A portion of nondestructive cephalic tentacles were seized from each juvenile abalone for extraction of genomic DNA (gDNA), washed with PBS (phosphate buffered saline), immediately flash frozen in liquid nitrogen and stored at –80 °C until gDNA extraction.

3. Genotyping-by-Sequencing (GBS) Library Preparation, Sequencing and SNP Genotyping
   1. Extraction of genomic DNA

Genomic DNA of each F1 progeny and parent abalone were extracted from cephalic tentacle using the CTAB (Cetyl trimethylammonium bromide) method. Briefly, samples were cryogenically grinded to fine power in a bead beater. After adding 200 µL of CTAB extraction buffer (0.1 m Tris‐HCl pH 8.0, 20 mm EDTA pH 8.0, 1.4 M NaCl, 2% CTAB w/v) containing 73 uL 2-mercaptoethanol to each 100 mg homogenized tissue, the mixture was vortexed thoroughly and the homogenate was then incubated at 65°C in a water bath for 1h. Following incubation, it was centrifuged at 14,000 g for 5 min. The supernatant was then transferred to a new tube. After adding 5 µL of RNase A, the mixture was incubated at 37°C for 20 minutes. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and vortexed for 5 seconds, incubated at room temperature for 5 min and then centrifuged at 13,000 g for 5 min to separate the aqueous phases. The clear aqueous upper phase was then transferred to a new tube. After adding 200 µL of chloroform/isoamyl alcohol (24:1), previous steps were repeated and the upper clear aqueous phase was transferred to a new tube. Later, DNA samples were precipitated by adding 2.5 volume of ice-cold 100% ethanol and incubated at -70°C for 30 minutes. After the incubation, samples were centrifuged at 13,000 g for 30 min at - 4°C. The supernatant was then decanted without disturbing the pellet. The pellet was subsequently washed with 500 µL ice-cold 70% ethanol. Ethanol was decanted and residual ethanol was removed by air-drying. Finally, DNA was dissolved in 20 µL TE buffer (10 mM Tris, pH 8, 1 mM EDTA). The integrity of each DNA sample was evaluated by 1.2% agarose gel electrophoresis and the concentration of DNA was determined with a Nano Photometer spectrophotometer (IMPLEN, USA). The ration of absorbance at 260/280 nm was used to assess the purity of DNA.

1. 2. Construction of GBS library and sequencing

Construction of GBS library and Illumina HiSeq X sequencing were performed at SEEDERS, Korea using genomic DNA of each female and male parents and 90 progenies. A GBS library was constructed by double digestion with two restriction enzymes: PstI and MspI. Chronological steps of GBS library construction included adaptor annealing, DNA digestion with PstI and MspI, adaptor ligation, sample pooling, DNA purification and multiplexed PCR. The quality of the constructed library was analyzed through a quality control check and agarose gel electrophoresis. Pooled GBS library was then sequenced using a HiSeq X (Illumina, Inc., USA) by the paired-end read method.

1. 3. Sequence data analysis

Raw sequences were demultiplexed into 96 samples according to barcode sequences. Barcode and adapter sequences were removed using Cutadapt (version 1.8.3) software. Low quality sequences were trimmed using DynamicTrim and LengthSort programs of SolexaQA (v.1.13) package. For DynamicTrim, a phred score ≥ 20 was used as the criteria. For LengthSort, a read length of ≥ 25 pb was applied. DynamicTrim removed low-quality bases at either ends of short reads based on the phred score to carry out a purification process for high-quality clean reads. LengthSort removed the reads, from which excess bases were then cut with DynamicTrim.

1. 4. Raw SNP detection and generation of SNP matrix

Cleaned sequences were aligned to the reference genome sequence of Pacific abalone consisting of 80,032 scaffolds using Burrows-Wheeler Aligner (BWA) program version 0.6.1-r104. A BAM format file was generated to detect raw SNP (In/Del) using default values and the following options: a seed length (-l) of 30, a maximum difference in the seed (-k) of 1, number of threads (-t) of 16, a mismatch penalty (-M) of six, a gap open penalty (-O) of 15 and a gap extension penalty (-E) of eight. Raw SNPs (In/Del) were detected and consensus sequences were extracted using SAMTools version 0.1.16. Prior to raw SNP detection, SNP validation was carried out using an in-house script of SEEDERS. To compare SNPs among analytic targets, an integrated SNP matrix was produced for samples. Based on respective coordinates, the SNP type was classified as homozygous (SNP read rate ≥ 90%), heterozygous (40% ≤ SNP read rate ≤ 60%) and ambiguous (20% ≤ SNP read rate ≤ 40% or 60% < SNP read rate < 90%).

4. SNP Filtering, Genotyping and Construction of Genetic Linkage Map

Genetic linkage maps were constructed using the JoinMap 4.1 program with population type cross-pollination (CP) under the condition of logarithm of odds (LOD) 6.0 or higher with a maximum distance of 30 cM. To construct an integrated map of male and female parents, all segregating markers that showed polymorphism in at least one parent were used in the JoinMap configuration for CP mode (lmxll, nnxnp, hkxhk). Ratios of marker segregation were calculated using Chi-square test. Markers that satisfied the expected Mendelian segregation ratio were included for mapping. Filtered markers contained different segregation types, including < nn x np >, < lm x ll > and < hk x hk >. Markers showing significantly distorted segregation (p < 0.05) were excluded from map construction. Markers were grouped with a minimum logarithm of odds (LOD) score of 6.0 and a recombination frequency of 0.45. A regression mapping algorithm was used to build the linkage map. Map distances were calculated in centiMorgans (cM) according to the Kosambi mapping function.