A total of 513 commercial Korean native cattle samples from Gyeungsangbuk, 96, Korea, were included in the study where 10 sires were used to produce this half sib population. Feeding conditions (slaughter age, the fattening period, forage intake, and concentration) were controlled under livestock management of each region (n = 14). They were all steers slaughtered at 941±72 days of age, and the marbling score was measured 24 h after slaughter. First, the carcass was dissected at the last rib and the first lumber vertebra according to the Animal Product Grading System of Korea. Marbling scores were measured or scored in the left carcass cut across the vertebra between the last thoracic vertebra and the first lumbar vertebra. The marbling degree was scored from 1 to 9 with a mean of 5.43 such that the higher score, the more abundant the intramuscular fat (Table 1 ). Genomic DNA was extracted from the longissimus muscle by using the LaboPass Tissue Mini Kit (Cosmo Genetech, Seoul, Korea).
Labopass tissue mini kit
Manufactured by Cosmogenetech
The LaboPass Tissue Mini Kit is a laboratory equipment product designed for the extraction of DNA from small tissue samples. It provides a simple and efficient method for obtaining high-quality DNA.
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6 protocols using labopass tissue mini kit
1
Marbling Evaluation in Korean Native Cattle
2
DNA Extraction and COI Sequencing from Muscle Tissue
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Genomic DNA was extracted from muscle tissue of abdomen using LaboPass Tissue Mini Kit (Cosmo Genetech, Seoul, South Korea), according to the manufacturer’s instructions. COI sequences were obtained using the primer sets, LCO1490-JJ (5’-TAYTCHACYAAYCAYAAAGAYATYGG-3’) and HCO2198-JJ (5’-AWACTTCVGGRTGVCCAAARAATCA-3’) (Astrin and Stüben 2011 (link)). Polymerase chain reaction amplification was performed under the following conditions: initial denaturation at 98 °C for 1 min, followed by 5 cycles of 10 s at 98 °C, 30 s at 43 °C, and 60 s at 72 ° C. This was followed by 30 cycles of 10 s at 98 °C, 60 s at 48 °C, 60 s at 72 °C, and a 5 min extension at 72 °C. The obtained sequences were aligned using Geneious 8.1.9 (Biomatters Ltd., Auckland, New Zealand). The uncorrected p-distance of COI sequences was calculated using MEGA X (Kumar et al. 2018 (link)). The details of the sequences obtained in this study and those downloaded from GenBank are listed in Table 1 .
3
DNA Extraction from SPG Solution
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A tube containing 500 μl of SPG solution was used for DNA extraction. The solution was centrifuged at 13,000 g for 5 min and the resulting pellet was used for DNA extraction. The DNA was extracted using a Labopass Tissue Mini kit (Cosmo Genetech, Co., Ltd., Seoul, South Korea), according to the manufacturer’s instructions. The DNA was eluted in 50 μl of the elution buffer, quantified spectrophotometrically using Eppendorf Biophotomoter® D30 and stored at − 20° until use.
4
Genomic DNA Extraction from Batillaria attramentaria
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A total of 197 individual Batillaria attramentaria were collected from fifteen sites representing the widest distribution around the Korean peninsula in 2006–2007 (Table1 and Figs.1 , 2 ). Although we tried to collect B. attramentaria from the east coast of the Korean peninsula, where the seafloor is mostly sandy bottom with steep gradients and no mud flats, but no live individuals were found. Upon collection, all samples were temporarily stored in 95% ethanol, delivered to our laboratory, and then preserved at −50°C prior to dissection of foot tissues for the extraction of genomic DNA. The genomic DNA was extracted from the head–foot region of each individual with the LaboPass ™ Tissue Mini Kit (Cosmogenetech Inc., Seoul, Korea), following the manufacturer's protocols.
5
Bovine HSPB1 Gene Polymorphism Analysis
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A total of 192 Korean cattle (body weight BW 464.68 45.09 kg; 32.18
months old [SD1.0]) raised in Pyeongchang (Gangwon, Republic of Korea)
were used in this study. All of the steers were maintained under constant
environmental conditions, such as having access to two types of commercial
feeds at six feedlots. Muscle tissues were sampled from slaughtered
individuals. Therefore, there is no certificate of animal ethics. Genomic
DNA was extracted from longissimus dorsi muscle tissue using a
LaboPass™ tissue mini kit (Cosmo Genetech, Seoul, Korea). In
order to discover SNPs, the bovine HSPB1 sequence was obtained from the NCBI
database (AC_000182.1). The primer sequence was designed
using NCBI Premer-BLAST based on the selected polymorphism sites, and the
primer information is shown in Table 1. The sequencing was performed as
outlined in a previous study (Lee et al., 2010), and SNPs were discovered
using the Sequencer v5.2.4 program (Gene Codes Corp., Ann Arbor, MI). In
order to map the functional SNPs on DNA, mRNA, and protein sequences, they
were aligned using the Graphical View Legend on the NCBI database.
months old [SD1.0]) raised in Pyeongchang (Gangwon, Republic of Korea)
were used in this study. All of the steers were maintained under constant
environmental conditions, such as having access to two types of commercial
feeds at six feedlots. Muscle tissues were sampled from slaughtered
individuals. Therefore, there is no certificate of animal ethics. Genomic
DNA was extracted from longissimus dorsi muscle tissue using a
LaboPass™ tissue mini kit (Cosmo Genetech, Seoul, Korea). In
order to discover SNPs, the bovine HSPB1 sequence was obtained from the NCBI
database (AC_000182.1). The primer sequence was designed
using NCBI Premer-BLAST based on the selected polymorphism sites, and the
primer information is shown in Table 1. The sequencing was performed as
outlined in a previous study (Lee et al., 2010), and SNPs were discovered
using the Sequencer v5.2.4 program (Gene Codes Corp., Ann Arbor, MI). In
order to map the functional SNPs on DNA, mRNA, and protein sequences, they
were aligned using the Graphical View Legend on the NCBI database.
6
Molecular Identification of Amoebae
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Amoebal DNA was extracted with the LaboPass Tissue Mini Kit (Cosmogenetech, Daejeon, Korea). PCR was conducted with primers for sequences of amoebal 18S rDNA (forward1, 5′-CCA GCT CCA ATA GCG TAT ATT-3′; forward2, 5′-CCA GCT CCA AGA GTG TAT ATT-3′; reverse, 5′-GTT GAG TCG AAT TAA GCC GC-3′) (Thomas et al. 2006) or for Naegleria 5.8S rDNA with adjacent regions, referred to as internal transcribed spacers (ITS) 1 and ITS2 (forward, 5'-AAC CTG CGT AGG GAT CAT TT-3'; reverse, 5'-TTT CCT CCC CTT ATT AAT AT-3') (De Jonckheere 2011).
The first primers (18S rDNA) were used in order to identify up to genus, while the second primers (5.8S rDNA with adjacent regions ITS1 and 2) were used in order to identify up to species. After electrophoresis, the PCR products were purified using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and submitted to Macrogen Japan (Kyoto, Japan) for direct sequencing. The sequence was analyzed with NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or BioEdit software version 7.2.5 (Hall 1999) . The accession numbers identified in this study were as follows: LC318418 (18S rDNA partial sequence) and LC318419 (ITS1, 5.8S rDNA, and ITS2 region). A phylogenetic tree was constructed using the Neighbor-Joining method (Saitou and Nei 1987) in MEGA software version 7 (Kumar et al. 2016) (link)
The first primers (18S rDNA) were used in order to identify up to genus, while the second primers (5.8S rDNA with adjacent regions ITS1 and 2) were used in order to identify up to species. After electrophoresis, the PCR products were purified using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan) and submitted to Macrogen Japan (Kyoto, Japan) for direct sequencing. The sequence was analyzed with NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or BioEdit software version 7.2.5 (Hall 1999) . The accession numbers identified in this study were as follows: LC318418 (18S rDNA partial sequence) and LC318419 (ITS1, 5.8S rDNA, and ITS2 region). A phylogenetic tree was constructed using the Neighbor-Joining method (Saitou and Nei 1987) in MEGA software version 7 (Kumar et al. 2016) (link)
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