• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.267-267
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• 2022

Glutinous rice is a key grain quality trait occupying an important part during rice processing in most rice growing areas. Amylose content (AC) of rice determine eating quality which is one of the major traits in rice breeding program. In this study, a gene pyramiding approach was used to introduce two dull genes, responsible for low amylose contents, for glutinous rice breeding using marker assisted selection (MAS). Two dull genes were located on chromosome 6 (wx-mq, AC: 12.7 %) and chromosome 10 (du1, AC: 10.3%), respectively. To test whether these two dull genes have an epistatic interaction, we developed an F₂ population by crossing two nearly isogenic lines(NILs) harboring wx-mq and du1. Gene based marker and KASP marker were used to select NILs(NIL-nor, NIL-wxmq, NIL-du1, and NIL-wxmq/du1) from the F2 population. A two-way ANOVA revealed an epistatic interaction between the two genes in the F2 population. The mean of Amylose contents for NIL-nor, NIL-wxwq, NIL-(du1, and NIL-wxmq/du1 were 17.3%, 12.5%, 9.7%, and 7.2%, respectively. This interaction was confirmed by an analysis of NILs indicating that both genes are involved in the same genetic mechanism controlling amylose contents. This result will be useful for rice breeding related to amylose content.

• Animal Bioscience
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• v.37 no.5
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• pp.929-943
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• 2024

Objective: The present study was executed to explore the molecular mechanism of fibroblast growth factor 10 (FGF10) gene in bovine adipogenesis. Methods: The bovine FGF10 gene was overexpressed through Ad-FGF10 or inhibited through siFGF10 and their negative control (NC) in bovine adipocytes, and the multiplicity of infection, transfection efficiency, interference efficiency were evaluated through quantitative real-time polymerase chain reaction, western blotting and fluorescence microscopy. The lipid droplets, triglycerides (TG) content and the expression levels of adipogenic marker genes were measured during preadipocytes differentiation. The differentially expressed genes were explored through deep RNA sequencing. Results: The highest mRNA level was found in omasum, subcutaneous fat, and intramuscular fat. Moreover, the highest mRNA level was found in adipocytes at day 4 of differentiation. The results of red-oil o staining showed that overexpression (Ad-FGF10) of the FGF10 gene significantly (p<0.05) reduced the lipid droplets and TG content, and their down-regulation (siFGF10) increased the measurement of lipid droplets and TG in differentiated bovine adipocytes. Furthermore, the overexpression of the FGF10 gene down regulated the mRNA levels of adipogenic marker genes such as CCAAT enhancer binding protein alpha (C/EBPα), fatty acid binding protein (FABP4), peroxisome proliferator-activated receptor-γ (PPARγ), lipoprotein lipase (LPL), and Fas cell surface death receptor (FAS), similarly, down-regulation of the FGF10 gene enriched the mRNA levels of C/EBPα, PPARγ, FABP4, and LPL genes (p<0.01). Additionally, the protein levels of PPARγ and FABP4 were reduced (p<0.05) in adipocytes infected with Ad-FGF10 gene and enriched in adipocytes transfected with siFGF10. Moreover, a total of 1,774 differentially expressed genes (DEGs) including 157 up regulated and 1,617 down regulated genes were explored in adipocytes infected with Ad-FGF10 or Ad-NC through deep RNA-sequencing. The top Kyoto encyclopedia of genes and genomes pathways regulated through DEGs were the PPAR signaling pathway, cell cycle, base excision repair, DNA replication, apoptosis, and regulation of lipolysis in adipocytes. Conclusion: Therefore, we can conclude that the FGF10 gene is a negative regulator of bovine adipogenesis and could be used as a candidate gene in marker-assisted selection.

• Journal of Plant Biotechnology
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• v.50
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• pp.56-62
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• 2023

Transgenic lilies have been obtained using Agrobacterium tumefaciens (AGL1) with the plant scale explants, followed by DL-phosphinothricin (PPT) selection. In this study, scales of lily plants cv. "red flame" were transformed with the pCAMBIA3301 vector containing the gus gene as a reporter and the blpR gene as a selectable marker, as well as a gene of interest showing herbicide tolerance, both driven by the CaMV 35S promoter. Using a 20-minute infection time and a 5-day cultivation period, factors that optimized and demonstrated a high transformation efficiency were achieved. With these conditions, approximately 22-27% efficiency was observed for Agrobacterium-mediated transformation in lilies. After transformation with Agrobacterium, scales of lilies were transferred to MS medium without selective agents for 2 weeks. They were then placed on selection MS medium containing 5 mg/L PPT for a month of further selection and then cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets after transferring into hormone-free MS medium. Also, most survived putatively transformed plantlets indicated the presence of the blpR gene by PCR analysis and showed a blue color indicating expression of the gus gene. In conclusion, when 100 scales of lily cv. "red flame" are transformed with Agrobacterium, approximately 22-27 transgenic plantlets can be produced following an optimized protocol. Therefore, this protocol can contribute to the lily breeding program in the future.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.9
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• pp.1244-1249
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• 2014

Goose fatty liver is one of the most delicious and popular foods in the world, but there is no reliable genetic marker for the early selection and breeding of geese with good liver-producing potential. In our study, one hundred and twenty-four 78-day-old Landes geese bred in Shunda Landes goose breeding farm, Jiutai, Jilin, China were selected randomly. The fatty livers were sampled each week after overfeeding during a three week period. Polymerase chain reaction-single strand conformation polymorphism and DNA sequencing were used to identify single nucleotide polymorphisms (SNPs) of fatty acid synthase (FAS), which is an important enzyme involved in the synthesis of fat under both physiological and pathological conditions. Least-squares correlation was established between these SNPs and fatty liver weight, abdominal fat weight, and intestinal fat weight of the overfed Landes geese, respectively. The results showed that fatty liver weight of geese with EF and FF genotypes (amplified by primer P1) was significantly higher than that of the EE genotype (p<0.05), and liver weight of CD and DD genotypes (amplified by primer P2) was significantly higher than that of the CC genotype (p<0.05). Different genotype combinations showed different liver weights, and from highest to lowest were ABDD, DDEF, DDFF, DDEE, ABEF, ABFF, AADD, and CDEF. Further analysis of DNA sequencing showed that there were two SNPs within the 5' promoter region the FAS gene. The geese of EF and FF genotypes carried a change of T to C, and the geese of CD and DD genotypes carried a change of A to G. The changes of the bases could potentially influence the binding of some transcription factors to this region as to regulate FAS gene. To our knowledge, this is the first report of SNPs found within the 5' promoter region of the Landes goose FAS gene, and our data will provide an insight for early selection of geese for liver production.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.45-53
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• 2010

We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of a gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic resistance gene to create a "mazF-cassette". A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.9
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• pp.1221-1225
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• 2005

The peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear hormone receptors. Lots of studies in rodents and humans have shown that PPARs were involved in lipid metabolism and adipocyte differentiation. The main objective of this work was to detect the single nucleotide polymorphisms (SNPs) in whole coding regions of peroxisome proliferator-activated receptor alpha (PPAR-$\alpha$) and gamma (PPAR-$\gamma$) genes with approach of single strand conformation polymorphism (SSCP) in the chicken population of Arber Acres broiler, Hyline layer and three Chinese native breeds (Shiqiza, Beijing You, Bai'r). Two SNPs of C1029T and C297T were found in chicken PPAR-$\alpha$ and PPAR-$\gamma$ genes respectively and each SNP found three genotypes in the experimental populations. The results showed that the distribution frequency of 3 genotypes in Arber Acres broiler, Hyline layer and Chinese native breeds had significant differences on the PPAR-$\alpha$ and PPAR-$\gamma$ gene respectively (p<0.01). Furthermore, in the PPAR-$\alpha$ gene, the results of least square estimation for genotypes and body composition traits showed the BB genotype birds had higher abdominal fat weight (AFW) and percentage of abdominal fat (AFP) than AA genotype birds (p<0.05). From these we conjecture the PPAR-$\alpha$ and PPAR-$\gamma$ genes were suffered intensive selection during the long term commercial breeding and the PPAR-$\alpha$ gene may be a major gene or linked to the major genes that impact chicken fat metabolism and the SNPs could be used in molecular assistant selection (MAS) as a genetic marker for the chicken fatness traits.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.188-188
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• 2017

Rice (Oryza sativa L.) is one of the major crops that is seriously impacted by global soil salinization. Rice is among those crops where most of the high-yielding cultivars are highly sensitive to salinity. The key to a plant survival under NaCl salt stress is by maintaining a high $K^+/Na^+$ ratio in its cells. Selection for salinity tolerance genotypes of rice based on phenotypic performance alone is less reliable and will delay in progress in breeding. Recent advent of molecular markers, microsatellites or simple sequence repeats (SSRs) were used to find out salt tolerant rice genotypes. In the current experiment phenotyping and genotyping studies were correlated to differentiate different rice accessions for salinity tolerance. Eight rice accessions along with check plant Dongjin were screened by physiological studies using Yoshida solution with 50mM NaCl stress condition. The physiology studies identified four tolerant and four susceptible accessions based on their potassium concentration, sodium concentration, $K^+/Na^+$ ratio and biomass. 17 SSR markers were used to evaluate these rice accessions for salt tolerance out of which five molecular markers were able to discriminate tolerant accessions from the susceptible accessions. Banding pattern of the accessions was scored comparing to the banding pattern of Dongjin. The study identifies accessions based on their association of $K^+/Na^+$ ratio with molecular markers which is very reliable. These markers identified can play a significant role in screening large set of rice accessions for salt tolerance; these markers can be utilized to improve salt tolerance of commercial rice varieties with marker-assisted selection (MAS) approach.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.14-14
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• 2017

The discipline of plant breeding is experiencing a renaissance impacting crop improvement as a result of new technologies, however fundamental questions remain for predicting the phenotype and how the environment and genetics shape it. Inexpensive DNA sequencing, genotyping, new statistical methods, high throughput phenotyping and gene-editing are revolutionizing breeding methods and strategies for improving both quantitative and qualitative traits. Genomic selection (GS) models use genome-wide markers to predict performance for both phenotyped and non-phenotyped individuals. Aerial and ground imaging systems generate data on correlated traits such as canopy temperature and normalized difference vegetative index that can be combined with genotypes in multivariate models to further increase prediction accuracy and reduce the cost of advanced trials with limited replication in time and space. Design of a GS training population is crucial to the accuracy of prediction models and can be affected by many factors including population structure and composition. Prediction models can incorporate performance over multiple environments and assess GxE effects to identify a highly predictive subset of environments. We have developed a methodology for analyzing unbalanced datasets using genome-wide marker effects to group environments and identify outlier environments. Environmental covariates can be identified using a crop model and used in a GS model to predict GxE in unobserved environments and to predict performance in climate change scenarios. These new tools and knowledge challenge the plant breeder to ask the right questions and choose the tools that are appropriate for their crop and target traits. Contemporary plant breeding requires teams of people with expertise in genetics, phenotyping and statistics to improve efficiency and increase prediction accuracy in terms of genotypes, experimental design and environment sampling.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.4
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• pp.564-570
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• 2016

The Sal-like 1 gene (Sall1) is essential for kidney development, and mutations in this gene result in abnormalities in the kidneys. Mice lacking Sall1 show agenesis or severe dysgenesis of the kidneys. In a recent study, blastocyst complementation was used to develop mice and pigs with exogenic organs. In the present study, transcription activator-like effector nuclease (TALEN)-mediated homologous recombination was used to produce Sall1-knockout porcine fibroblasts for developing knockout pigs. The vector targeting the Sall1 locus included a 5.5-kb 5' arm, 1.8-kb 3' arm, and a neomycin resistance gene as a positive selection marker. The knockout vector and TALEN were introduced into porcine fibroblasts by electroporation. Antibiotic selection was performed over 11 days by using $300{\mu}g/mL$ G418. DNA of cells from G418-resistant colonies was amplified using polymerase chain reaction (PCR) to confirm the presence of fragments corresponding to the 3' and 5' arms of Sall1. Further, mono- and bi-allelic knockout cells were isolated and analyzed using PCR-restriction fragment length polymorphism. The results of our study indicated that TALEN-mediated homologous recombination induced bi-allelic knockout of the endogenous gene.