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SDRT-PCR (SYBR duplex real-time polymerase chain reaction) was designed for an assay that can combine the advantages of real-time PCR and multiplex PCR to identify animal genes more quickly. SDRT-PCR based on melting temperature (tm) discrimination by using SYBR Green fluorescence dye was developed for the analysis of ruminant and poultry contained in foodstuffs. Primers specific to ruminant and poultry were designed for species-specific gene-amplification. Appropriate mixtures of poultry and ruminant meat DNAs were used to develop the assay. Gene products of ruminant and poultry were represented in two melting peaks generated simultaneously at temperatures of 83.2 and 86.5 °C, respectively. Duplexing results obtained with one of the multiplex polymerase mixes correlated extremely well with the singleplex reference. The objective of the study was to design a rapid, specific and accurate duplex real-time PCR assay by using SYBR Green fluorescence dye that is cheaper than double labelled probes to detect a group of mixed meats simultaneously.
BackgroundEpigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence.
The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence. MethodsA retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters.
MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry. ResultsHigher levels of methylation were associated with disease recurrence.
In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively.
ConclusionsHistopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence. Colorectal cancer (CRC), a disease arising from complex and heterogeneous etiological factors and pathogenetic mechanisms, develops in a multi-step manner from normal epithelium, through a pre-malignant lesion (adenoma), into a malignant lesion (carcinoma). Histopathological evaluation of early stage CRC in many cases reveals areas of adenomatous mucosa, but the presence of tissue with histological features ranging from pure tubular to pure villous adenomas accompanied by dysplasia is also frequently detected in invasive colorectal cancer ,. Although individuals with syndromes that strongly predispose to adenomas, e.g. Familial adenomatous polyposis (FAP), invariably develop CRC by the third to fifth decade of life if these lesions are not removed , most adenomas (not FAP) have a low risk of progressing into cancer (about 5%) if not resected.
An adenomatous polyp is a much more frequent finding than CRC and polypectomy has a distinctly protective effect on the subsequent development of CRC. It has been estimated that in the first 10 years after polypectomy, the risk of CRC is reduced to a level similar to that of individuals whose colonoscopy does not reveal the presence of polyps ,.Different molecular mechanisms seem to be related to CRC development. The vast majority of tumors (about 50-80%), present chromosomal instability (CIN) , while a smaller fraction (10-15%) is characterized by microsatellite instability (MSI) ,. In recent years, epigenetic alterations have gained recognition as a key mechanism in carcinogenesis. In particular, hypermethylation of CpG islands present in gene promoter sequences leads to the inactivation of tumor suppressor genes, working in a different way with respect to genetic mutations ,.This aberrant methylation status occurs at the same time as genetic alterations which drive the initiation and progression of colorectal cancer, suggesting that methylation plays an important role in many stages of tumor transformation -. The existence of a methylator phenotype could be related to distinctive biological and/or clinical characteristics.CRCs that show hypermethylation changes in numerous different CpG-rich DNA regions are defined as showing the CpG island methylator phenotype (CIMP).
CIMP-positive cancers have distinct clinical pathological characteristics such as proximal colon location, mucinous and poorly differentiated histology, female preponderance and older age. This phenotype also seems to be associated with MSI and BRAF mutations ,. Conversely, hypomethylation of specific sequences may decrease the fidelity of chromosomal segregation , suggesting that it may be involved in the chromosomal instability phenotype. DNA methylation changes probably lead adenomatous precursor lesions to progress into malignant tumors. In fact, sessile serrated adenomas, considered important precursors of cancer, are often CIMP-positive.Taking the above considerations into account, a better understanding of the epigenetic mechanisms associated with adenoma-carcinoma transition could represent an important tool for CRC prevention.
In accordance with international guidelines, pre-neoplastic lesions of the colon and rectum are classified according to pathological parameters (size, histology, number of polyps and dysplasia) as having high or low risk of recurrence. In high risk patients a new colonoscopy is performed after 3 years, while in low risk subjects the time interval is extended to 5 years. However, this type of subdivision is unable to predict the real risk of developing a new lesion. In fact, it has been seen that patients who are classified as high risk may not experience any further problems, while those who are classed as low risk may relapse after a short time.Little data is available on the relationship between risk of recurrence of pre-neoplastic lesions and molecular alterations of colorectal lesions, whereas a great deal is known about the mechanisms of CRC transformation. Although a number of gene promoter methylation profiles have been shown to characterize specific stages of tumor progression, no data are available on epigenetic alterations or risk of disease evolution/recurrence.
The identification of these specific epigenetic profiles could help us to better understand the mechanisms of adenoma recurrence and, possibly, adenoma-carcinoma transition, resulting in a more accurate classification of the risk of recurrence of pre-neoplastic and permitting a personalized program of cancer prevention.The aim of this study was to evaluate whether altered methylation profiles in pre-neoplastic lesions sampled by colonoscopy is capable of identifying patients at high risk of recurrence with greater accuracy than conventional clinical pathological parameters.
BackgroundConservation of genetic diversity is an essential prerequisite for developing new cultivars with desirable agronomic traits. Although a large number of germplasm collections have been established worldwide, many of them face major difficulties due to large size and a lack of adequate information about population structure and genetic diversity.
Core collection with a minimum number of accessions and maximum genetic diversity of pepper species and its wild relatives will facilitate easy access to genetic material as well as the use of hidden genetic diversity in Capsicum. ResultsTo explore genetic diversity and population structure, we investigated patterns of molecular diversity using a transcriptome-based 48 single nucleotide polymorphisms (SNPs) in a large germplasm collection comprising 3,821 accessions. Among the 11 species examined, Capsicum annuum showed the highest genetic diversity (H E = 0.44, I = 0.69), whereas the wild species C. Galapagoense showed the lowest genetic diversity (H E = 0.06, I = 0.07). The Capsicum germplasm collection was divided into 10 clusters (cluster 1 to 10) based on population structure analysis, and five groups (group A to E) based on phylogenetic analysis. Capsicum accessions from the five distinct groups in an unrooted phylogenetic tree showed taxonomic distinctness and reflected their geographic origins.
Most of the accessions from European countries are distributed in the A and B groups, whereas the accessions from Asian countries are mainly distributed in C and D groups. Five different sampling strategies with diverse genetic clustering methods were used to select the optimal method for constructing the core collection. Using a number of allelic variations based on 48 SNP markers and 32 different phenotypic/morphological traits, a core collection ‘CC240’ with a total of 240 accessions (5.2%) was selected from within the entire Capsicum germplasm. Compared to the other core collections, CC240 displayed higher genetic diversity (I = 0.95) and genetic evenness (J’ = 0.80), and represented a wider range of phenotypic variation (MD = 9.45%, CR = 98.40%). ConclusionsA total of 240 accessions were selected from 3,821 Capsicum accessions based on transcriptome-based 48 SNP markers with genome-wide distribution and 32 traits using a systematic approach. This core collection will be a primary resource for pepper breeders and researchers for further genetic association and functional analyses.
Pepper ( Capsicum spp.) is one of the major vegetable and spice crops grown worldwide, and is rich in bioactive compounds, such as capsaicinoids and carotenoids, which contribute to the improvement of human health ,. Because of its economic and nutritional importance, breeders have improved agronomic traits of pepper, such as pungency, fruit shape, abiotic stress tolerance, and disease resistance. Meanwhile, genetic diversity of breeding lines has become smaller and some useful genes in the landraces are lost due to the breeding activities ,. Therefore, conservation and sustainable utilization of genetic resources are keys to continuous improvement of peppers.During the last several decades, there has been remarkable progress in germplasm collection and conservation of various plants. Although a large number of germplasms have been collected, their management has become more and more complicated due to their huge sizes. Furthermore, little is known about the genetic diversity and structure of such collections at the interspecific and intraspecific levels.
To make efficient use of large germplasm collections, the concept of core collections has been proposed. A core collection is a subset of a germplasm collection of a species that represents the genetic diversity of the entire collection. A good core collection is one that has no redundant accessions, is small enough to be easily managed, and represents the total genetic diversity.Various types of data including passport data, geographic origin , , agronomic traits –, and molecular markers can be used for selecting a core set. Although the major reason for establishing a core set is to reduce the number of representative accessions up to 10% while maintaining the diversity of the entire collection, there are a number of possible methods for selection of a core set depending on the research goals. In the early 2000s, most researchers performed random sampling using various assignment methods ,. Later, the M (maximization) strategy was proposed as a more effective method to select a core set representing the maximum genetic diversity without redundancy ,.Several research institutions have collected and conserved thousands of Capsicum accessions, ranging from 1,000 in the Centre for Genetic Resources (CGN), the Netherlands to almost 8,000 in the Asian Vegetable Research and Development Center (AVRDC), Taiwan.
Researchers and institutions have attempted to construct core collections of Capscicum spp. For various purposes.
, Nicolai et al. , and Zewdie et al. established core collections to reveal phenotypic and genetic variation.
Thies and Fery , and Quenouille et al. constructed a core collection for disease resistance against northern root-knot nematode and Potato virus Y (PVY), respectively.
Hanson et al. developed a core collection to analyze antioxidant activities. However, most studies involved a relatively small number of accessions, using fewer than 1,000 accessions with limited numbers of morphological traits and molecular markers ,. The limited number of morphological traits and markers allow us to survey only a small portion of the genetic diversity of the entire germplasm, and the resulting data cannot be used for genome-wide variation studies.In this study, we performed population structure analysis in a large Capsicum germplasm collection consisting of 3,821 accessions by applying 48 genome-wide SNPs, and selected a core set using the SNP data together with data for 32 morphological traits. This allowed us to 1) examine the level of genetic diversity and the population structure within the worldwide Capsicum germplasm collection; 2) optimize selection methods by comparing different core sets, which were selected using a stepwise selection strategy based on various combinations of data and clustering methods; and 3) ultimately construct a Capsicum core collection that represents the entire germplasm collection without redundancy. Finally, we validated the core collection by evaluating the diversity of a range of traits and genotyping additional molecular markers. This core collection will be a valuable data set for both pepper breeding and genome-wide association studies.
Plant materialsA total of 4,652 Capsicum accessions used in this study originated from 97 countries and included 11 species: C. Cardenasii, C. Chacoense, C. Frutescens, C. Galapagoense, C. Praetermissum, C.
Pubescens, and C. The geographic origin and passport data of the germplasm accessions were obtained from the Rural Development Administration (RDA, Jeonju, Korea) and Seoul National University (SNU, Seoul, Korea). Among the germplasm accessions, 3,599 were obtained from the RDA, and 1,053 were obtained from SNU. Most of the accessions were C. Annuum, accounting for 4,163 accessions.
Four other domesticated species, C. Frutescens, and C. Pubescens accounted for 163, 122, 152, and 11 accessions, respectively. Among the wild Capsicum species, C.
Cardenasii, C. Chacoense, C. Galapagoense, C. Praetermissum and C. Tovarii accounted for 1, 28, 4, 2, 5, and 1 accessions, respectively. DNA extraction and SNP genotypingTwo young leaves from each accession were used for DNA extraction. DNA was extracted using the cetyl trimethylammonium bromide (CTAB) method as described previously.
The concentration and purity of DNA samples were determined with a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). DNA samples showing absorbance ratios above 1.8 at 260/280 nm were used for marker analysis.A set of 48 SNP markers evenly distributed in 12 pepper chromosomes were used in this study (Additional file: Table S1).
Rotor Gene Software
In a preliminary study a total of 282 accessions were randomly selected from entire germplasm collection for genetic diversity study with 412 SNP markers developed by Kang et al. Based on this analysis, highly polymorphic SNP markers (PIC 0.45) were selected. Genotyping was performed using the BioMark™ HD system (Fluidigm, San Francisco, CA, USA), EP1™ system (Fluidigm, San Francisco, CA, USA), and 48 × 48 Dynamic Array IFCs (Fluidigm, San Francisco, CA, USA) according to the manufacturer’s protocol. Specific target amplification (STA) was performed prior to SNP genotyping analysis. PCR was performed in a 5-μL reaction containing 60 ng of the DNA sample according to the manufacturer’s protocol.
Thermal cycling conditions were 15 min at 95 °C, followed by 14 cycles of a 2-step amplification profile of 15 s at 95 °C and 2 min at 60 °C. For genotyping, SNPtype assays were performed using STA products following manufacturer’s protocol. Thermal cycling was carried out at 95 °C for 15 s, 64 °C for 45 s and 72 °C for 15 s with a touchdown of −1 °C per cycle from 64 to 61 °C, followed by 34 cycles of 95 °C for 15 s, 60 °C for 45 s and 72 °C for 15 s. For the species verification and/or identification of pepper accessions with missing species information, SNP markers C2At5g04590, C2At1g50020, and C2At2g19560 were used based on high resolution melting (HRM) analysis.
Genotyping analysis was performed using a Rotor Gene 6000 (Qiagen, Valencia, CA, USA). Population structure analysisTo analyze the population structure of the entire germplasm collection used in this study, we used a model based genetic clustering algorithm as implemented in the STRUCTURE program ver.
The number of sub-populations (ΔK) was determined using the ad-hoc statistical method, based on the rate change in the log probability of data between successive K values.
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