Restriction Site Research Articles

Simultaneous Short- and Long-Patch Base Excision Repair (BER) Assay in Live Mammalian Cells.

The base excision repair (BER) pathway repairs small, non-bulky DNA lesions, including oxidized, alkylated, and deaminated bases, and is responsible for the removal of at least 20,000 DNA lesions per cell per day. BER is initiated by DNA damage-specific DNA glycosylases that excise the damaged base and generates an abasic (AP) site or single-strand breaks, which are subsequently repaired in mammalian cells either by single-nucleotide (SN) or multiple-nucleotide incorporation via the SN-BER or long-patch BER (LP-BER) pathway, respectively. This chapter describes a plaque-based host cell reactivation (PL-HCR) assay system for measuring BER mechanisms in live mammalian cells using a plasmid-based assay. After transfection of a phagemid (M13mp18) containing a single modified base (representative BER DNA substrates) within a restriction site into human cells, restriction digestions detect the presence or absence (complete repair) of the adduct by the transformation of the digestion products into E. coli and counting the transformants as plaques. To monitor the patch size, different plasmids are constructed containing C:A mismatches within different restriction sites (inhibiting digestion) at various distances on both sides (5' or 3') of the modified base-containing restriction sites. Using this assay, the percentage of repair events that occur via 5' and 3' patch formation can be quantified.

BIOLOGICAL SAMPLES CONTAMINATION CONTROL OF THE SUS SCROFA USING HAPLOID DNA MARKERS

This paper proposes an effective method for controlling the contamination of biological samples of Sus scrofa with alien material in the preanalytical phase of a PCR study. Because PCR is highly sensitive, even a small amount of DNA containing alien biological substances can lead to false results. In the case of analysis of contaminated biological samples using diploid DNA markers, a mixture of two different homozygotes will be defined as a heterozygote. Unlike diploid DNA markers, a mixture of two different haplotypes is uniquely determined. To perform the study in the slaughter shop of the Globinsky Meat Processing Plant, after slaughter, one ear was cut off from the carcasses of pigs with an animal identification number tag. DNA was isolated from the epithelial tissue of the auricle. Five SNPs of the mitochondrial genome were used as a haploid marker. The study was carried out using a multisite PCR-RFLP method, the peculiarity of which was the analysis of the D-loop fragment between positions 15531 and 15959 of the porcine mitochondrial genome (GenBank: AJ002189.1). This sequence contains one monomorphic site (15558W) and five polymorphic Tas I restriction enzyme sites (15616T > C, 15714T > C, 15758T > C, and 15916A > T). The presence or absence of the Tas I site in the above positions determines mitochondrial haplotypes, denoted by Latin letters from A to P. PCR-RFLP analysis of DNA samples revealed DNA fragments on the electrophoregram, indicating a mixture of two or more haplotypes. It was possible to establish the presence of contamination due to the use of a multisite PCR-RFLP method, which implies a strictly discrete set of restrictive fragments for the mitochondrial DNA of an individual animal. The total size of the restriction fragments should be 428 bp and the presence of additional DNA bands indicate the association of two or more haplotypes. Thus, it was demonstrated that the use of haploid DNA markers makes it possible to determine the contamination of samples with alien material. This method can be used in the study of porcine nuclear DNA as a laboratory quality assurance of the preanalytical phase, which will reduce laboratory costs, improve the organization of work and avoid dramatic errors when performing genetic examinations. Keywords: pigs, epithelium, mitochondrial genome, haploid DNA markers, PCR, contamination.

The Creation of a Gene Library of Valuable Plant Species in Armenia

Using DNA banks or gene libraries is the most efficient method of protecting and managing valuable, endangered, and rare gene pools. Genomic libraries are collections of bacteria colonies with restriction sites on their genomic DNA that contain desired genes from a specific organism. The “Scientific Center of AgroBiotechnology” branch of ANAU has created gene libraries of 34 plant species, which are preserved in the National Genebank of Crops and their Wild Relatives, which forms a basis for genomic selection. This allows genetic identification, barcoding, DNA formula decoding, and, most importantly, is the basis for genome identification. Based on the results, plasmids can incorporate restriction segments of genomic DNA and be efficiently transformed. The high level of correlation between the number of necessary clones and the efficiency of transformation of all studied plant species proves the completeness of the gene pool.

ORE-Seq: Genome-Wide Absolute Occupancy Measurement by Restriction Enzyme Accessibilities.

Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1-accessibility) in absolute terms as the percentage of cut or uncut molecules, respectively, out of all molecules. The protocol presented here takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared, and converted into libraries for high-throughput sequencing. Bioinformatic analysis counts uncut DNA fragments as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straightforward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments. Our protocol includes two orthogonal approaches to correct for this bias, the "corrected cut-uncut" and the "cut-all cut" method, so that accurate measurements of absolute accessibility or occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.

Computational Protocol for DNA Methylation Profiling in Plants Using Restriction Enzyme-Based Genome Reduction.

Epigenetics can be described as heritable phenotype changes that do not involve alterations in the underlying DNA sequence. Having widespread implications in fundamental biological phenomena, there is an increased interest in characterizing epigenetic modifications and studying their functional implications. DNA methylation, particularly 5-methylcytosine (5mC), stands out as the most studied epigenetic mark and several methodologies have been created to investigate it. With the development of next-generation sequencing technologies, several approaches to DNA methylation profiling were conceived, with differences in resolution and genomic scope. Besides the gold standard whole-genome bisulfite sequencing, which is costly for population-scale studies, genomic reduced representation methods emerged as viable alternatives to investigate methylation loci. Whole-genome bisulfite sequencing provides single-base methylation resolution but is costly for population-scale studies. Genomic reduction methods emerged as viable alternatives to investigate a fraction of methylated loci. One of such approaches uses double digestion with the restriction enzymes PstI and one of the isoschizomers, MspI and HpaII, with differential sensitivity to 5mC at the restriction site. Statistical comparison of sequencing reads counts obtained from the two libraries for each sample (PstI-MspI and PstI-HpaII) is used to infer the methylation status of thousands of cytosines. Here, we describe a general overview of the technique and a computational protocol to process the generated data to provide a medium-scale inventory of methylated sites in plant genomes. The software is available at https://github.com/wendelljpereira/DArTseqMet .

Assembling Multiple Fragments: The Gibson Assembly.

The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.

HDR-based CRISPR/Cas9-mediated Knockout of PD-L1 in C57BL/6 Mice.

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.

Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome.

Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

Modification of a previously patented method to unequivocally score A2-like and A1-like bovine β-casein variants

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 β-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the β-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like β-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of β-casein. The advantages of this method are that it:• enables to unequivocally score A2-like as well as A1-like β-casein variants,• can be performed at low cost in simply equipped molecular biology laboratories, and• can be scaled up to analyze hundreds of samples per day.For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

Immunoinformatics design of multi-epitope vaccine using surface cell antigen OmpB and heat shock protein GroEL against rickettsioses

Rickettsioses, caused by intracellular bacteria of the rickettsia genus, pose a global health threat, particularly affecting underserved communities. Delays in diagnosis and treatment due to limited healthcare access result in severe cases and fatalities. To address this, a multi-epitope vaccine targeting OmpB and GroEL proteins in epidemic and endemic typhus rickettsia was developed using immunoinformatics techniques. T-cell and B-cell epitopes were predicted from OmpB and GroEL proteins, resulting in 5 CTL, 4 HTL, and 4 B-cell epitopes. These epitopes were linked appropriately and combined with suitable adjuvants to construct the vaccine. The vaccine's physicochemical properties and tertiary structure were validated within acceptable ranges. Molecular docking analysis demonstrated favorable binding with TLR4 receptors and MHC molecules, further confirmed by molecular dynamics simulation. Immune simulation analysis predicted T-cell, IFN, and IL-2 responses upon vaccine administration. The vaccine sequence was then optimized and cloned into a plasmid vector pET-23a(+) at the Hind and AlwNI restriction sites. In this in silico experiment, a multi-epitope vaccine derived from OmpB and GroEL proteins is presented, the first validation steps of which have been completed. Subject to successful in vivo studies, this vaccine could prove to be a promising therapeutic strategy against rickettsiosis and address a critical public health challenge.

Efficacy of genetic transformation of E.coli field and reference (ATCC 8739) strains at different concentrations of CaCl2 for creation of Gene libraries

The importance of genetic transformation in various fields of biological research, and especially in the process of gene libraries formation, is very important. It has been proven by numerous studies that during the phase of obtaining competent cells, Ca2+ ions neutralize the electrostatic repulsion of membrane proteins and DNA of the bacterial cell, thereby stimulating the introduction of foreign DNA into the membrane pores. Our research aimed to elucidate the maximum efficiency of transformation in field and ATCC 8739 reference strains of E.coli at 0.05; 0.1 and 0.15 M concentrations of CaCL2. Microbiological, molecular-biological modern methods were used in the experimental studies. From the research results, it became evident that the efficiency of genetic transformation increases with the addition of concentration of CaCL2 and reaches the maximum at 0.1 M. Among the studied strains of E.coli, at 0.05 and 0.1 M concentrations of CaCL2, the highest efficiency was recorded in the field strain. At a 0.15 M concentration of CaCL2, the transformation efficiency in the field and reference strains is highly variable due to the size of the genomic DNA of the internalizing bacteria and the number of restriction sites.

Spotted-wing drosophila, Drosophila suzukii (Diptera: Drosophilidae), exhibits large-scale spatial genetic structure across Canada but not fruit host–associated genetic structure

Abstract Spotted-wing drosophila, Drosophila suzukii, is a global pest of soft fruits that is capable of reproducing on a wide range of cultivated and wild plant species. In Canada, D. suzukii was first reported in British Columbia in 2009 and is now widespread across the country. Understanding the genetic structure of D. suzukii populations could be important for pest management if there are phenotypic differences between genetically distinct populations. For example, insect pest populations could respond differently to directional selection imposed by insecticides, differ in their host plant preferences, and vary in their susceptibility to biological control agents. Here, we used double-digest restriction site–associated DNA sequencing to examine large- and fine-scale patterns of the genetic structure of D. suzukii reared from fruit hosts in Canada. We found that this species has a large-scale spatial genetic structure; the flies collected formed two distinct genetic clusters, one of which was distinct to western Canada and the other to eastern Canada. At the local scale, D. suzukii populations showed no evidence of host-associated structuring in British Columbia, suggesting that pest management tactics may be best applied at the landscape level. Our results highlight the need to investigate phenotypic differences between western and eastern D. suzukii populations in Canada.

Association of Melatonin receptor 1A (MTNR1A) gene polymorphism with seasonal suppression of fertility in buffaloes

The polymorphism in Melatonin receptor 1A (MTNR1A) gene and its association with reproductive cyclicity (cyclic, n=51; non-cyclic, n=50) during summer season and age at first calving (&lt;3.5 years, n=38; &gt;3.5 years, n=60) was investigated in buffaloes subjected to blood sampling. Polymerase chain reaction (PCR) of genomic DNA was carried out using three primers viz. 824 bp (primer pair 1), 856 bp (primer pair 2) and 268 bp (primer pair 3) corresponding to exon-II of MTNR1A gene. Restriction fragment length polymorphism was performed using PCR products of primer pair 1 and 2. The digestion of PCR product of primer pair 1 with restriction enzymes viz. MnII, RsaI or Eco3II generated total restriction sites respectively as 14, 4 and 5. Based upon differential restriction sites, six genotypes revealed in buffaloes using MnII were MM-1 to MM-6, four genotypes using RsaI were RR-1 to RR-4, and four genotypes using Eco3II were EE-1 to EE-4. The digestion of PCR product of primer pair 2 with MnII, RsaI or Eco3II generated restriction sites respectively as 13, 5 and 6. Based upon differential restriction sites, six genotypes revealed using MnII were mm-1 to mm-6, five genotypes using RsaI were rr-1to rr-5, and three genotypes using Eco3II were ee-1 to ee-3. For primer pair 3, single stranded conformational polymorphism analysis exhibited 5 genotypes viz. AA, BB, AB, CC and DD. The results revealed that the buffaloes during summer had polymorphic genotypes for MTNR1A gene as buffaloes showing reproductive cyclicity had MM-3, MM-4, RR-2, EE-3, mm-2, mm-4, rr-3, ee-3, CC and DD genotypes, whereas their non-cyclic counterparts had MM-2, MM-6, EE-4, mm-3, mm-6, AA, BB and AB genotypes. However, no such differences were recorded with respect to age at first calving. In conclusion, polymorphic genotypes of MTNR1A gene may be considered as genetic markers to identify buffaloes with better reproductive potential during summer season.

Virtual Clinical Trials: An Overview

The production of successful and promising innovation in pharmaceutical and clinical care delivery is subjected to factual and efficient clinical trials. The most crucial step for a successful trial is dependent on subject participation. The extent of subject participation has a high impact on the conclusions of a trial. The biggest challenges with the conventional trials are patient enrollment and retention, inefficiency, costly and time-consuming methods. A new concept of digitalizing the clinical research process, ‘Virtual Clinical Trials’ (VCTs), has captured the attention of researchers with its phenomenal time and cost-saving attributes along with the advantage of data collection from multiple sources, ease of access to restricted study sites breaking the location barriers, increased patient retention and automated data collection. The concept of virtual trials is not fully explored yet and seems to face various challenges but is expected to revolutionize the methods of clinical research with its potential benefits.

Simple and Rapid Non-ribosomal Peptide Synthetase Gene Assembly Using the SEAM-OGAB Method.

Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.

Cryptic persistence and loss of local endemism in Lake Constance charr subject to anthropogenic disturbance.

In the welcome circumstance that species believed extinct are rediscovered, it is often the case that biological knowledge acquired before the presumed extinction is limited. Efforts to address these knowledge gaps, in particular to assess the taxonomic integrity and conservation status of such species, can be hampered by a lack of genetic data and scarcity of samples in museum collections. Here, we present a proof-of-concept case study based on a multidisciplinary data evaluation approach to tackle such problems. The approach was developed after the rediscovery, 40 years after its presumed extinction, of the enigmatic Lake Constance deep-water charr Salvelinus profundus. Targeted surveys led to the capture of further species and additional sympatric normal charr, Salvelinus cf. umbla. Since the lake had been subject to massive stocking in the past, an evaluation of the genetic integrity of both extant forms was called for in order to assess possible introgression. A two-step genomic approach was developed based on restriction site associated DNA (RAD). Diagnostic population genomic (single nucleotide polymorphism [SNP]) data were harvested from contemporary samples and used for RNA bait design to perform target capture in DNA libraries of archival scale material, enabling a comparison between extant and historic samples. Furthermore, life history traits and morphological data for both extant forms were gathered and compared with historical data from the past 60-120 years. While extant deep-water charr matched historical deep-water specimens in body shape, gill raker count, and growth rates, significant differences were discovered between historical and extant normal charr. These resulted were supported by genomic analyses of contemporary samples, revealing the two extant forms to be highly divergent. The results of population assignment tests suggest that the endemic deep-water charr persisted in Lake Constance during the eutrophic phase, but not one of the historical genomic samples could be assigned to the extant normal charr taxon. Stocking with non-endemic charr seems to be the most likely reason for these changes. This proof-of-concept study presents a multidisciplinary data evaluation approach that simultaneously tests population genomic integrity and addresses some of the conservation issues arising from rediscovery of a species characterized by limited data availability.

Molecular Characterization of Aspergillus flavus Strains Isolated from Animal Feeds.

Aflatoxin (AF)-producing fungi such as Aspergillus flavus commonly contaminate animal feeds, causing high economic losses. A. flavus is the most prevalent and produces AFB1, a potent mutagen, and carcinogen threatening human and animal health. Aspergillaceae is a large group of closely related fungi sharing number of morphological and genetic similarities that complicate the diagnosis of highly pathogenic strains. We used here morphological and molecular assays to characterize fungal isolates from animal feeds in Southwestern Algeria. These tools helped to identify 20 out of 30 Aspergillus strains, and 15 of them belonged to the Aspergillus section Flavi. Further analyses detected four out of 15 as belonging to Aspergillus flavus-parasiticus group. PCR targeting the AF genes' aflR-aflS(J) intergenic region amplified a single 674 bp amplicon in all four isolates. The amplicons were digested with a BglII endonuclease, and three specific fragments were observed for A. flavus but A. parasitucus lacked two typical fragments. Sequencing data of four amplicons confirmed the presence of the two BglII restriction sites yielding the three fragments, confirming that all four strains were A. flavus. In addition, this analysis illustrated the genetic variability within the A. flavus strains.

Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

Long and Covalently Conjugated Branched DNA Structures for in Situ Gelation in Vivo

Crystal structure of the feruloyl esterase from Lentilactobacillus buchneri reveals a novel homodimeric state.

Ferulic acid is a common constituent of the plant cell-wall matrix where it decorates and can crosslink mainly arabinoxylans to provide structural reinforcement. Microbial feruloyl esterases (FAEs) specialize in catalyzing hydrolysis of the ester bonds between phenolic acids and sugar residues in plant cell-wall polysaccharides such as arabinoxylan to release cinnamoyl compounds. Feruloyl esterases from lactic acid bacteria (LAB) have been highlighted as interesting enzymes for their potential applications in the food and pharmaceutical industries; however, there are few studies on the activity and structure of FAEs of LAB origin. Here, we report the crystal structure and biochemical characterization of a feruloyl esterase (LbFAE) from Lentilactobacillus buchneri, a LAB strain that has been used as a silage additive. The LbFAE structure was determined in the absence and presence of product (FA) and reveals a new type of homodimer association not previously observed for fungal or bacterial FAEs. The two subunits associate to restrict access to the active site such that only single FA chains attached to arabinoxylan can be accommodated, an arrangement that excludes access to FA cross-links between arabinoxylan chains. This narrow specificity is further corroborated by the observation that no FA dimers are produced, only FA, when feruloylated arabinoxylan is used as substrate. Docking of arabinofuranosyl-ferulate in the LbFAE structure highlights the restricted active site and lends further support to our hypothesis that LbFAE is specific for single FA side chains in arabinoxylan.

Association and expression study of SEPW1 and JAML as preliminary candidate genes related to lamb odor and flavor

The current study aimed to identify the SEPW1 and JAML genes in lamb as candidate genes related to lamb odor and flavor. The polymorphism study showed that the SEPW1 gene was polymorphic at the BanI restriction site with three genotypes (AA, AG, and GG), whereas the JAML gene was monomorphic at HhaI with genotype (GG). The association of SEPW1 between genotype and lamb odor and flavor (BCFAs and skatole) was analyzed using GLM (General Linear Model). MNA (4-methylnonanoic) was significantly associated (p < 0.05) with lamb odor and flavor. AA genotype has a lower level of MNA than AG and GG, while MOA (4-methyloctanoic), EOA(4-ethyloctanoic), MI (3-methylindole) and MP (3-methylphenol) was not significantly associated with lamb odor and flavor (p > 0.05). Furthermore, to analyze the mRNA expression of SEPW1 in liver tissues, the lambs were divided into three groups based on the genotypes AA, AG, and GG, however, mRNA expression was not differentially expressed between AA, AG, and GG (p > 0.05). These results will enhance the understanding of the functions of SEPW1 gene relation to odor and flavor traits and will shed light on the polymorphism of SEPW1 gene in lamb as a candidate gene for reducing MNA in lamb.