The aim of the study was to assess the safety of methionine sulfoxide reductase B2(CaMsrB2) protein as toxicity, allergenecity and identity of inserted gene product that transformed rice. Through bioinfomatical research of CaMsrB2, amino acid sequence of CaMsrB2 did not share overall homology with any known or suspected to be allergen or toxin protein. For the biochemical research, CaMsrB2 protein was expressed and purified. Using purified protein, we made a specific antibody. Purified protein was sequenced by Edman degradation methods and confirmed sequence identify.. The amino acid sequences of purified protein were the same as deduced amino acid sequences exclude N-terminal Histidine. And for the internal sequences of CaMsrB2, we performed MALDI-TOF Mass. The results of MALDI-TOF MS was compared Mascot Database and confirmed the sequence coverage was 56%. These results mean bacterially produced CaMsrB2 was the same with inserted gene product. With these purified and identified CaMsrB2 protein, we performed acute toxicity test. Following the OECD guideline 423, 2,000mg/Kg body weight protein were injected as oral administration. After 2 weeks, there did not shown any death and special symptoms.

Genetically modified (GM) crops have never been cultivated commercially in Korea, it is necessary for a thorough assessment of the risks associated with their environmental release. We quantified the amount determined the frequency of pollen mediated gene flow from disease resistant GM rice to weedy rice (R55). A total of 164,604 seeds were collected from weedy rice, which were planted around GM rice. Resistance of the hybrids was determined by repeated spraying of herbicide and DNA analysis using specific primer to confirm hybrids. Though weedy rice has similar flowering time, the hybrids were found only in non-GM rice and out-crossing ranged from 0.018% at 0.3 m to 0.013% at 0.6 m. All of hybrids were located within 0.6 m distance from the GM rice plot in southerly direction. The meteorological factors including temperature and relative humidity during flowering time were found to be the most important factors for determining rice out-crossing. It should be considered many factors like the local weather condition and flowering time to set up the safety management policy to prevent pollen mediated gene flow between GM and conventional crop.

Rice (Oryza sativa) is the most important staple food of over half the world’s population. This study was conducted to evaluate the possible impact of transgenic rice cultivation on the soil microbial community. Microorganisms were isolated from the rhizosphere of GM and non-GM rice cultivation soils. Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with GM and non-GM rice were similar to each other, and there was no significant difference between GM and non-GM rice. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM rice were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in GM and non-GM rice cultivated soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed similar patterns, but didn’t show significant difference to each other. DNAs were isolated from soils cultivating GM and non-GM rice and analyzed for persistence of inserted gene in the soil by using PCR. The PCR analysis revealed that there were no amplified protox gene in soil DNA. These data suggest that transgenic rice does not have a significant impact on soil microbial communities, although continued research may be necessary.

Korea is a origin of three basic species, P. ussuriensis, P. pyrifolia and P. fauriei. Genetic relationship among Korean pear cultivars compared with their parents were also identified that they are closely related P. pyrifolia, P. ussuriensis and/or hybrid between two species. SSRs or Microsatellites are co-dominant and typically neutral inheritance showing high degree of polymorphism, large number of alleles per locus, abundance in genomes, and suitability for automation. SSR markers were developed in apple and pear where they were used for construction of genetic linkage maps, evaluation of the genetic diversity, cultivar identification, genotype identification, and in the determination of genetic relatedness. Many apple (Malus × domestica Borkh.) SSRs would be useful for genetic mapping in European and Asian pears in previous experiments and cross-species amplification was observed between apple and pear. The objectives of this study were to develop polymorphic SSR markers in ‘Whangkeumbae’ and ‘Minibae’, which were chosen as the representative cultivars of P. pyrifolia and P. ussuriensis in each among Korean pears, from ‘Golden Delicious’ genomic sequences generated by next generation sequencing technology and to evaluate the utility of the SSR markers based on ‘Golden Delicious’ sequences. Of 51 SSR markers, 18 were polymorphic in ‘Whangkeumbae’ and ‘Minibae’. The cross-species transportability of primers designed in ‘Golden Delicious’ sequences makes SSR markers more useful, given the current high level of investment in mapping the genomes of related Rosaceae.

Currently, the type of short insertions and deletions (InDels) polymorphisms are increasingly focused in genomic research. InDels have been known as a source of genetic markers that are widely spread across the genome. Genetic relationship among Korean pear cultivars compared with their parents was also identified that they are closely related P. pyrifolia, P. ussuriensis and/or hybrids between two species. Lack of genetic resources including molecular markers has made it difficult to study pears severely. Recently developed next generation sequencing (NGS) platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The main goal of this study was to develop polymorphic InDel markers in ‘Whangkeumbae’ and ‘Minibae’, which were chosen as the representative cultivars of P. pyrifolia and P. ussuriensis × pyrifolia in each among Korean pears using genomic sequences generated by NGS technology. In this study, more than 18.6 Gbp and 15.8 Gbp sequences were obtained from NGS of ‘Whangkeumbae’ and ‘Minibae’, respectively. ‘Whangkeumbae’ contained 197,210 InDels and 197,272 InDels in ‘Minibae’. In InDels validations between ‘Whangkeumbae’ and ‘Minibae’, the number of polymorphic InDels were 149,338 and non-polymorphic InDels were 122,572. For InDel primer set designing, 11,308 of primers were designed from polymorphic InDels and 10,919 of InDel primers were recommended. The study shows that the utility of NGS technology to design amount of efficient InDels and the developed InDel primers will be used for genetic mapping, breeding by marker assisted selection (MAS) and QTL mapping of Korea native pear as well as further genetic studies.

Single nucleotide polymorphisms (SNPs) are the most frequent type among variations found in genomic regions and are valuable markers for genetic mapping, genetic diversity studies and association mapping in plants. There are three basic species known as Korean native which are Pyrus ussuriensis, P. pyrifolia, and P. fauriei. Genetic relationship among Korean pear cultivars compared with their parents was identified that they are closely related P. pyrifolia, P. ussuriensis and/or hybrids between two species. Lack of genetic resources, including molecular markers to study pears are very severe. Recently developed next generation sequencing (NGS) platforms offer opportunities for high-throughput and inexpensive genome sequencing and rapid marker development. The objective of this study was to develop polymorphic SNP markers in ‘Whangkeumbae’ and ‘Minibae’, which were chosen as the representative cultivars of P. pyrifolia and P. ussuriensis × pyrifolia in each among Korean pears, using genomic sequences generated by NGS technology. In this study, more than 18.6 Gbp and 15.8 Gbp sequences were obtained from NGS of ‘Whangkeumbae’ and ‘Minibae’, respectively. ‘Whangkeumbae’ and ‘Minibae’ contained 2,712,288 and 2,747,224 SNPs, respectively. In SNPs validations between ‘Whangkeumbae’ and ‘Minibae’, the number of polymorphic SNPs were 2,516,438 and non-polymorphic SNPs were 1,179,391. For HRM primer design, 2,125,479 HRM candidate primers were obtained from polymorphic SNPs and 343,731 SNP primers were developed. This study shows that the utility of NGS technology to discover efficiently a large number of SNPs and SNP primers can provide valuable information in the genome study of Pyrus spp.

Genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnology companies. Commercialization of GM crops will be required the assessment of risk associated with the release of GM crops. The purpose of this research is a molecular characterization of introduced T-DNA in transgenic rice T4 ∼ T6 generation lines harboring a pepper MsrB2 gene under the control of stress inducible Rab21 promoter, as a part of biosafety evaluation for drought-tolerant transgenic rice (Agb0103). We identified the structure and sequence of transformation vector of T-DNA and analyzed insertion sites, flanking sequences, and generational stability of inserted T-DNA in transgenic rice lines. The transformation vector was consisted of right border, a drought-tolerant CaMsrB2 gene unit (Rab21 promoter::CaMsrB2::PinII terminator), a selectable marker herbicide resistance unit (CaMV 35S promoter::bar::Nos terminator), and left border in sequential order. Based on the adaptor-ligation PCR and whole genome sequence database, we confirmed that T-DNA was introduced 2 copies (head to head type) at the position of 2,471,957 ∼ 2,472,049 bp of chromosome No. 8. From the generational stability study, T-DNAs were stably inherited through the T4 to T6 generations, and also stable expression of bar gene from T-DNA was confirmed. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene (aadA) was not present in Agb0103 rice genome. These results will be filed to biosafety assessment document of Agb0103

Bulb onion (Allium cepa), which belongs to the family Amaryllidaceae, is one of the oldest vegetative crops known to humans. Despite its high economic value, only a few reports are available on the use of molecular markers in genetic diversity analysis of Allium cepa for its improvement. Molecular genetic markers have been widely used as powerful tools for analyzing the plant genome. In particular, Microsatellites or simple sequence repeats (SSRs) markers are tandem repeats of one to six bp in length and have been proven to be the most powerful polymerase chain reaction (PCR)-based DNA markers in plant diversity analysis. In this study, the genomic DNA was isolated from different Allium cepa lines. The ESTs and gDNA sequences of onion were collected from National Center for Biotechnology information. The SSRs with two to five motifs over a length of 12 bp, were identified using SSRIT (Gramene) software. The PCR products of 100 to 350 bp in length containing SSRs, primers was designed using Primer3 with lengths of 20 to 24 bp and a melting temperature of 60℃. The SSR markers with high polymorphism-information content (PIC) levels was useful for collecting progeny with high genetic homogeneity for onion breeding, and to obtain representative marker sets for genetic tests. The SSR Finder program and the developed SSR markers could be a useful resource for genetic diversity and purity testing in onion.

NABIC(National Agricultural Biotechnology Information Center) established integrated management system of agricultural omics information to achieve and analyze a agricultural bio-information resources in Korea. The amount of bio-information is enormously increasing due to emergence of NGS(Next Generation Sequencing) technology. We are building, maintaining and providing agricultural bio-information databases and information services. Various data type for submission is available such as genome, proteome, transcriptome, metabolome, molecular marker, etc. We issue the submission confirmation which is available for research achievement. Currently, the amount of data submitted on our system is 30Tb. We are also providing various analysis pipelines such as NGS analysis(denovo, rna-seq, reference assembly), Gene annotation, GWAS, marker analysis for breeding ,Microbial community analysis and differential expression profiling analysis using submitted data through web. We have a plan to provide bioinformatics education portal in this year.

Single nucleotide polymorphisms (SNPs) are the most abundant variation in plant genomes. As DNA markers, SNPs are rapidly replacing simple sequence repeats (SSRs) and sequence tagged sites (STSs) markers, because SNPs are more abundant, stable, easy to automation, efficient, and increasingly cost-effective. We developed a 96-plex indica/japonica SNP genotyping set for genetic analysis and molecular breeding in rice using Fluidigm platform. Informative SNPs for indica/japonica populations were selected from 1536 Illumina SNPs and 44K Affymetrix SNP chip data of Rice Diversity and our resequencing data sets. Selected SNPs were evenly distributed across 12 chromosomes and average physical distance between adjacent SNP markers was 4.38Mb. We conducted genetic diversity analysis of 49 Bangladesh germplasm and check varieties to test a 96-plex indica/japonica SNP genotyping set we developed. High-throughput Fluidigm SNP genotyping system will serve a more efficient and valuable tool for genetic diversity analysis, DNA fingerprinting, quantitative trait locus (QTL) mapping and background selection for crosses between indica and japonica in rice. This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008125), Rural Development Administration, Republic of Korea.

The molecuar prossing of upsteam regulation of Pi response genes during Pi starvation remains inadequately understood. R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated and designated OsMYB4P, an R2R3 MYB transcription factor, from rice under low-phosphate conditions. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under low phosphate conditions. To investigate the function of OsMYB4P in Pi-starvation signaling, we developed transgenic rice plants overexpressing OsMYB4P for analysis of Pi signaling and uptake. Shoots and roots of OsMYB4P overexpressing plants grew well in high and low phosphate conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate sufficient and deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P lead to Pi accumulation and acts as a Pi-dependent regulator in controlling the expression of Pi transporters.

We used an efficient system to create rice mutant by Ac/Ds transposon insertion mutagenesis, such as selected homozygous mutant in dwarf phenotypes. We reported here the identification of function of dwarf OsGASD gene(Oryza sativa Gibberellin Acid Sensitive Dwarf). OsGASD gene encodes a 344 amino acid polypeptide and no homology proteins in GeneBank. The osgasd mutnat was sensitive to exogenous GA level. We performed experiment to controlled expression the OsGASD gene, its role in plant development, a quantitative analysis of endogenous GA content and sensitivity to GA. The osgasd mutant includes smaller amount of active GAs than wild-type. osgasd mutant plant of GA biosynthesis pathway causes GA deficiency and dwarf plants, and endogenous GA suppliance can restore the wild type phenotype in this mutant. There sult indicated that OsGASD gene regulated the elongation of shoot, stem and plant height. The increased expression of OsGASD gene dramatically induces expression of the factors associated with GA biosynthesis such as CPS, KO, KAO, GA 20 ox and GA 2 ox, whereas osgasd mutant suppression of the factors associated with GA biosynthesis, loading to dwarf phenotypes. That applied GA3 at the plant development stage to survey the response of OsGASD gene to GA3. These results indicated that OsGASD gene is involved in GA biosynthesis factors, not only in the internodes, but also leaf length at the developing stage.

In this study, we generated and characterized the transgenic rice plant expressing a spider silk protein. Spider silks have great potential as biomaterials with extraordinary properties. We report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22 kDa polypeptide in baculovirus-infected insect cells. To produce transgenic rice plant with high contents of glycine and alanine, the prolamin promoter-driven AvDrag was introduced into rice plant via agrobacterium tumefaciens-mediated gene transformation. The introduction and copy number of the AvDrag gene in transgenic rice plants were determined by PCR and Southern blot analysis. AvDrag expression in transgenic rice seeds was examined by Northern blot and Western blot analysis. Immunofluorescence staining with the AvDrag antiserum revealed that the recombinant AvDrag protein were localized in transgenic rice seed. Furthermore, the amino acid content analysis showed that transgenic rice seeds were greatly increased in glycine and alanine as compared to controls

Onion is one of the most widely consumed vegetables. There are many cultivars, which are grouped according to skin color as yellow, white or red. Onions can also be classified as sweet or non-sweet. Their importance in cooking comes from their typical taste and flavour. The sugars, pyruvic acid accumulation and transcript level of some transcription factors involved in the biosynthesis of high sugars and pyruvic acid was analyzed at different stages of bulb onion (Allium cepa) growing under light and dark condition using High Performance Liquid Chromatography (HPLC) and Quantitative real time PCR. A genetic map and cultivar lines 36101and 36122 were used to identify transcription factors controlling pungency and sugar. We compared 2 different lines for low pungency and high sugars during water and photoperiod stress, which showed significant positive phenotypic and genetic correlations. These results could be presumably used as useful information to obtain onion varieties rich in sugars.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development. BR-induced AtBEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response in Arabidopsis. Here, we identified a poplar (Populus alba x P. glandulosa) BEE3 homolog encoding a putative basic helix-loop-helix (bHLH)-type transcription factor through microarray analysis. Transcripts of PagBEE3 were mainly detected in stems, with the internode having a low level of the transcripts and the node having a relatively higher level. The function of the PagBEE3 gene was investigated through the phenotypic analyses with PagBEE3-overexpressing (ox) transgenic lines. This work mainly focused on a potential role of PagBEE3 in stem growth and development of polar. The PagBEE3-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Microarray analysis revealed that the expression of many genes involved in xylem cell proliferation and development was altered in the PagBEE3-ox plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3-ox plants and PagBEE3 plays a role in the stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Genome sequencing researches for considerable numbers of crops and wild plants are being developed. Cytogenetic researches according to chromosome number and size are essential to confirm and comprehend ploidy level and genome size before genome sequencing project is actually conducted. Cytogenetic researches on six food crop plants were carried out by DAPI staining and fluorescence in situ hybridization (FISH) method. Fagopyrum esculentum Moench showed 2n=2x=16, each chromosome length of 1.42㎛ to 1.77㎛, total chromosome length of 13.31㎛, and karyotypic formula of 2n=8m; Phaseolus angularis W.F. Wight, 2n=2x=22, 2.01㎛ to 3.84㎛, total 28.03㎛, 2n=9m+2sm, Perilla frutescens var. japonica Hara, 2n=2x=40, 1.73㎛ to 2.76㎛, total 44.36㎛, 2n=5m+13sm+2st. Chromosome sizes of the other three species such as, Panicum miliaceum L., 2n=2x=36, total chromosome length of 30.83㎛, Sesamum indicum L., 2n=2x=26, 27.39㎛, lpomoea batatas L., 2n=2x=30, total 33.51㎛ were too small for each chromosome type to be identified and analyzed. The result of FISH analysis using 5S and 45S rDNA probe showed species-specific chromosome locations in the genome. These preliminary analyses were carried out to decide which food crop to prioritize for genome sequencing. This work was supported by the “Cooperative Research Program for Agriculture Science & Technology Development (No.PJ009837), Rural Development Administration, Republic of Korea.

The next-generation sequencing (NGS) technology is being used for more effective genetic mapping and genome analysis. In this study, we performed whole-genome sequencing on the genomic DNA of Milyang23 and Gihobyeo using NGS and developed new cleaved amplified polymorphic sequence (CAPS) markers based on the single nucleotide polymorphisms (SNPs) in coding sequence between these varieties. Approximately, sequences of 60x coverage of the Nipponbare reference genome on average were obtained following Illumina sequencing. Totally, 1,726,798 SNPs between Milyang23 and Gihobyeo were detected. Among them, 149 SNP were selected for CAPS markers and located on genetic map with previously reported 219 PCR-based DNA markers. This map was applied to the detection of quantitative trait loci (QTLs) for stem internode diameters, culm length and panicle length in rice with MGRIL population. Newly 6 QTLs were detected for culm length (CL) and stem diameter (ID) traits including the first internode diameter (I1D), third internode diameter (I3D), and fourth internode diameter (I4D). Among those QTLs, qI1D5 and qCL5 had relatively higher LOD score and explained 8.99% and 4.24% of total variation. This study showed that the NGS allowed the rapid discovery of a large number of SNPs for CAPS marker. Only very small portion of SNPs through re-sequencing were used in this study. Furthermore, the results of QTL analysis described above shows relevance of molecular markers in mapping genes for useful traits.

It is well known that Dharial (Bangladesh origin and weedy rice line) has longer seed longevity than indica and japonica rice varieties. To study the genetic basis of seed longevity of Dharial, we developed 240 BC3F7 backcross recombinant inbred lines derived from the crosses between Dharial (a donor parent) and two korea rice accessions (recurrent parents) including Ilmi and Gopum, respectively. Among these lines, we selected two introgression lines with longer seed longevity and named them Ilmi-NIL and Gopum-NIL. Also, we developed an EMS-induced mutant line from Dharial which has shortened seed longevity, and named it Dharial-EMS. We performed re-sequencing of four rice accessions that are Dharial, Dharial-EMS, Ilmi-NIL, and Gopum-NIL. A total of 706×106 raw reads were generated which provided sequence data over 46x rice genome coverage per each accession. We did genome-wide variation analysis comparing produced re-sequencing data and the re-sequencing data of Ilmi from NABIC database with the Nipponbare reference sequence. By graphical analysis of SNP distribution in rice genome of the five accessions, we could select candidate chromosomal segments introgressed from Dharial in Ilmi-NIL and Gopum-NIL. The introgressed chromosomal segments were in seven regions in Ilmi-NIL and eight regions in Gopum-NIL, and four common introgressed regions between Ilmi-NIL and Gopum-NIL were identified. 2,758 SNPs between Dharial and Dharial-EMS were found in the introgressed regions. Also, we detected 450 genes including at least one SNP among these SNPs. This result will facilitate identification of genes and development of molecular markers for improvement of seed longevity.

The perturbation of the steady state of reactive oxygen species due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol. An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice

Pear (Pyrus spp.) is one of the most important temperate fruit species in the world. The identification of Pyrus spp. based on analysis and comparison of their phenotypes has been causing a number of synonyms and homonyms among Pyrus spp. For accurate identification, genotype analysis with molecular marker such as microsatellite is necessarily to use. However, It is difficult to share and compare of microsatellite profiles obtained among different laboratories because no effort has made to standardize equipment or protocols. A strategy for data comparison by dint of reference to the alleles detected in well-known cultivars will be helpful for co-work with laboratories and developing international databases. Thus the study was performed to compare results between laboratories of microsatellite DNA profiling for reproducibility and standardize allele scoring by defining reference alleles efficiently. In this study, ninety pear species including forty-four comparison Asian pears, six of comparison suspicious Asian pears and reference pears, seven basic pear species, and twenty-seven Korean native pear cultivars to development of a standard set of SSR reference alleles. Twelve primers covering most of pear genome were chosen, which are CH02b10, CH03g07, CH04e03, CH03d12, EMPc117, CH01f07a, EMPc11, CH01d09, GD147, CH01d08, CH05c06, and GD96 located in pear chromosome of 2, 3, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 17 respectively. Genotyping was conducted with Fragment Analyzer. After genotyping, a set of defined standard alleles based on reference pear cultivars can facilitate data comparison among laboratories and will permit the improvement of a common international database.