Specific DNA Markers Research Articles

Marker assisted pea breeding: eIF4E allele specific markers to pea seed-borne mosaic virus (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.

MoniQA (Monitoring and Quality Assurance)—an EU-funded Network of Excellence (NoE) Contributing Toward a Harmonized Approach to Food Safety Management and Method Validation—Including Food Allergens

Reliable detection and quantification of allergens are essential in order to protect allergic consumers and to comply with labeling regulations. In recent years various allergen-detection methods have been published, and test kits have become commercially available. Due to the nature of the analytes (usually allergenic proteins, specific marker proteins, or specific DNA markers) and their susceptibility to various processing effects, reliability and comparability of results have posed a great challenge. Often processing and matrix effects hamper the extraction efficiency and the quantitative analysis of allergens or markers in food products. Both reference methods and reference materials are urgently needed in the field of allergen testing. The EU-funded Network of Excellence, MoniQA—Monitoring and Quality Assurance in the Total food Supply Chain (www.moniqa.org)—is working toward the harmonization of monitoring and control strategies for food quality and safety assessment and thus focuses on performance criteria for methods used to analyze foods and food products for safety and quality. MoniQA established various analyte-specific working groups: Microbiological Contaminants, Mycotoxins and Phycotoxins, Chemical Contaminants, Food Allergens, Food Additives and Processing Toxicants, Food Authenticity, and Emerging Issues. MoniQA’s Food Allergen Working Group (WG) is compiling information about the most important food allergens, identifying gaps, prioritizing requirements, and developing harmonization guidelines in collaboration with all stakeholder groups, which include industry, food authorities, consumers, and laboratories. The WG works on (1) harmonized validation protocols and certification criteria for allergen testing, (2) status recognition of allergen methods which underwent a validation trial, (3) reference/testing materials, (4) international ring trials for full validation of new reference/testing materials and analytical methods, and (5) the development of a reference method by supporting research toward the improved use of mass spectrometry in food allergen testing. Additionally training for research and industry in the areas of analytical method development, method validation and verification, allergen management, and risk communication and a database on available analytical methods, validation level, and legislation linked with the RASFF—EU’s Rapid Alert System for Food and Feed are provided.

A potential species-specific molecular marker suggests interspecific hybridization between sibling species Littorina arcana and L. saxatilis (Mollusca, Caenogastropoda) in natural populations

Three sister species of rough periwinkles, viz. Littorina saxatilis (Olivi 1792), L. arcana (Hannaford Ellis 1978) and L. compressa (Jeffreys 1865) from the Barents Sea (Russia), the White Sea (Russia) and the Norwegian Sea (Norway) were studied. The identification of two sibling species L. saxatilis and L. arcana is often difficult as both species have extremely similar shell morphology and reproductive systems. Only mature females can be unambiguously distinguished, with a jelly gland present in female L. arcana, but which is replaced by a brood pouch containing developing embryos in L. saxatilis. No clear-cut diagnostic features have been found to discriminate between males or juveniles of the two species. The very first diagnostic DNA marker (DNA fragment A2.8, 271 bp length) for L. arcana and L. saxatilis separation was developed. The marker was derived from apparently species-specific L. arcana DNA fragments obtained via Random Amplified Polymorphic DNA (RAPD) analysis. This fragment was cloned and sequenced, whereupon specific primers were designed and the amplification was surveyed in a large number of morphologically well-identified females of both species. Subsequently, the specific DNA marker was used for the identification of male L. arcana and partners in copulating pairs. In this way, we obtained evidence of possible interspecific hybridization between the sibling species L. arcana and L. saxatilis living in sympatry in natural populations: the presence of A2.8 fragment in 12% of morphologically well identified L. saxatilis females and its absence in 14% of morphologically well identified L. arcana females. The A2.8 fragment never amplified in L. saxatilis from sites without L. arcana. The A2.8 fragment did not amplify in L. compressa, not even in microsympatric populations, and we did not observe interspecific copulations between L. arcana and L. compressa.

Comparison and significance of specific and non-specific cellular immunity in patients with chronic hepatitis B caused by infection with genotypes B or C of hepatitis B virus

The present study was designed to investigate possible relationships between the genotypes of hepatitis B virus (HBV) and the HBV-specific cytotoxic T lymphocyte (CTL) responses. HBV genotypes, HBV specific CTL HBV DNA and other markers of HBV infection were determined in 138 patients with chronic hepatitis B. The results showed that the patients infected with genotype C (n=62) had a significantly lower HBV-specific CTL response than those who were infected with HBV genotype B (P<0.01). HBV DNA titer was higher in patients infected with HBV genotype C than in those infected with HBV genotype B (P<0.01). Both alanine aminotransferase (ALT) and total bilirubin (TBIL) were higher in HBV genotype C infected patients than in those infected with genotype B (P<0.01 and <0.05, respectively). These results suggest that compared with CHB patients infected with HBV genotype B, the higher HBV DNA level and more severe liver damages in the patients infected with genotype C of HBV may be associated with genotype C of the virus.

Identification of genotypes with high essential oil contents in Origanum, Thymus and Sideritis

A large number of medicinal and aromatic plant species naturally grown in the Mediterranean Basin of Turkey contain secondary metabolites such as alkaloids, flavanoids, phenols, polysaccharides, terpenes, and quinones that are used in the food, pharmaceutical, cosmetic, and pesticide industries. Many of these plant species are undergoing domestication and cultivar development. Unfortunately some of these plant species are among the most endangered species which need to be protected to ensure their sustainable use in the region. In the present study several species in Origanum, Thymus and Sideritis were collected from several locations in the Mediterranean Basin of Turkey, and essential oil contents and DNA fingerprinting analyses of each genotype collected from a location and other locations were studied. Preliminary studies indicated that there exist great variations in essential oil and genetic content of plant genotypes of each genus collected within and between the locations. Some of the genotypes within a location showed extremely higher essential oil contents while the other genotypes possess limited contents of essential oil. Using simple sequence repeat (SSR), minisatellite, chloroplast and mitochondrial DNA markers we obtained species specific DNA markers for some species. Upon completion of this study we will be able to define genotypes and locations from Origanum, Thymus and Sideritis with higher essential oil contents and define DNA marker specific to species in each the three genera [1,2]. Since we recorded positions of the each genotype using a global positioning system, we can re-collect those genotypes containing superior essential oil contents and use them in cultivar development studies.

Naegleria fowleri: Light and electron microscopy study of mitosis

DAPI and Feulgen stains were used as specific DNA markers for studying the mitosis process in Naegleria fowleri. Both DAPI and Feulgen stains reacted with DNA in the nuclei of the amoebae. Representative figures of N. fowleri mitotic nuclei with a defined arrangement according to the phase of the cell cycle were observed. A notable characteristic is that the nucleolus is present throughout the stages of mitosis. During metaphase, several deeply stained DNA condensations following an elongated pattern were observed, corresponding almost certainly to tightly grouped chromosomes. Ultrastructural observations demonstrated that the nucleus divides by cryptomitosis, a process in which the nuclear membrane does not disappear during the mitosis. Centrioles were not found, and a spindle of microtubules was observed running the length of the nucleus from pole to pole however, they did not come to a focal point.

CDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.)

Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and Terminal Restriction Fragment Length Polymorphism profiles of 16S rRNA gene

Specific fecal DNA markers were investigated for major pollution sources, cow, human, and pig, and occurrence of the identified markers was analyzed in river waters using Terminal Restriction Fragment Length Polymorphism (T-RFLP) techniques and sequencing of 16S rDNA of Bacteroides-Prevotella. The unique and specific DNA markers for cow and human were identified as a 222 bp and 60 bp peak in HaeIII T-RFLP profiles, respectively, and the pig-specific marker was not identified but the unique T-RFLP profile of pig could be used as a substitution. Human-specific marker was detected in most of the river waters tested (92.1%) and T-RFLP profiles of river waters were shown to be similar to those of human feces. Cluster analysis of T-RFLP data showed that the fecal sources were multiple (human plus cow and human plus dairy cow) in most of the river waters. The phylogenetic analysis for the clones recovered from the fecal and water samples showed that the clones from cow formed a discreet cluster from those of other sources. The other clones from human, pig, and river water formed two groups all together. The results of this study could be used to identify and control the fecal pollution source in the bodies of water in Korea.

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.

Development and utilization of diagnostic DAMD-PCR markers for Capsicum accessions

A polymerase chain reaction (PCR) based approach involving the directed amplification of minisatellite DNA region (DAMD-PCR) was used to identify accession specific DNA markers and study genetic relationships between and within 15 accessions corresponding to 11 species in genus Capsicum. A touch down PCR profile and unique chemical concentration of ingredients resulted in reproducible and reliable DNA amplifications. The number of amplified products varied from 1 to 12 fragments depending on the template DNA and the primers. The DAMD-PCR technique provided a total of 38 accession specific DNA markers (diagnostic DAMD-PCR) which can be utilized in accession identification, preservation and genetic studies of Capsicum germplasm. Based on 1,292 polymorphic and monomorphic DNA markers directed with 22 minisatellite specific primers, accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Capsicum chacoense was found to be the most distinct species.

양배추와 무의 동형 원형질체 융합을 이용한 식물체의 재분화

양배추와 무로부터 원형질체를 분리하였고 PEG 처리를 통하여 융합을 하였다. 고농도의 원형질체 혼합물로부터 많은 미소괴를 관찰할 수 있었다. 미소괴는 정상적인 캘러스로 자랐고 이들 캘러스로부터 총 218개의 신초가 재분화 되었다. 재분화된 개체는 순화 과정을 거처 온실에서 재배하였다. 순화된 208개체를 대상으로 마커 검정을 통하여 융합 여부를 확인한 결과, 모두 무의 NWB-CMS에 특이적인 PCR 산물을 확인할 수 없었다. 그러나 ISSR분석을 통하여 208개체 중 3개체에서 양배추와 무의 세포가 융합됨을 확인하였다. 이를 통하여 원형질체의 융합이 성공적으로 일어났음을 증명할 수 있었다. 세포융합이 일어난 3개체는 모두 양배추와 무의 형질을 보이는 중간적인 형태를 가지고 있었다. 이들은 춘화처리를 거쳐 모두 개화 되었으나 꽃의 색깔이 무의 형질과 같은 흰색이었고 이중 한 개체에서만 역교배를 통하여 3개의 종자를 얻었다. Protoplasts from cabbage and radish were isolated and fused symmetrically by PEG treatment. The PEG treated mixture of high concentrated protoplasts produced lots of micro-calli after <TEX>$2{\sim}3$</TEX> weeks. The microcalli developed to normal calli and shoots were regenerated from the calli. A total of 218 shoots were regenerated, but none of them contained the NWB-CMS specific DNA marker, indicating that the transfer of the radish NWB-CMS character into cabbage did not occur. However, ISSR analysis revealed that the cell fusion between protoplasts from radish and cabbage was occurred (3 out of 208 plantlet). The fused regenerants possessed the characteristics of source plants used for protoplast fusion. After vernalization, three regenerants were flowered with white petal color as seen in radish. Only three seeds were able to obtain from one regenerant by backcrossing with the cabbage pollen.

SsDNA-Binding Protein 2 Is Frequently Hypermethylated and Suppresses Cell Growth in Human Prostate Cancer

Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer-specific DNA marker, especially in high stages of human prostate cancer.

Expression Profiling of PBMC-based Diagnostic Gene Markers Isolated from Vasculitis Patients

Vasculitis (angiitis) is a systemic autoimmune disease that often causes fatal symptoms. We aimed to isolate cDNA markers that would be useful for diagnosing not only vasculitis but also other autoimmune diseases. For this purpose, we used stepwise subtractive hybridization and cDNA microarray analyses to comprehensively isolate the genes whose expressions are augmented in peripheral blood mononuclear cells (PBMCs) pooled from vasculitis patients. Subsequently, we used quantitative real-time polymerase chain reaction (qRT–PCR) to examine the mRNA levels of each candidate gene in individual patients. These analyses indicated that seven genes exhibit remarkably augmented expression in many vasculitis patients. Of these genes, we analyzed G0/G1 switch gene 2 (G0S2) further because G0S2 expression is also enhanced in the PBMCs of patients with systemic lupus erythematodes (SLE). We generated G0S2 transgenic mice that ubiquitously overexpress human G0S2. Although we did not observe any obvious vasculitis-related histopathologic findings in these mice, these mice are unhealthy as they produce only few offspring and showed elevated serum levels of two autoimmunity-related antibodies, anti-nuclear antibody, and anti-double strand DNA antibody. Thus, our large-scale gene profiling study may help finding sensitive and specific DNA markers for diagnosing autoimmune diseases including vasculitis and SLE.

Toward colloid-based biosensors for SNP genotyping and personalised medicine applications

Therapeutic drugs can assist some patients, however, other individuals may exhibit no response. Furthermore, a drug that is normally considered to be safe can have toxic effects in a small proportion of the population, occasionally causing death. Many altered drug responses depend on the genetic constitution of the recipient. The analysis of specific DNA markers within the genome of an individual [single nucleotide polymorphism (SNP) genotyping] could potentially identify subjects at risk in clinical trials and may enable clinicians to individualise medical therapy. The development of technology for personalised medicine relies on the ability to accurately and rapidly identify SNPs in genetic material. Some of the biotechnology tools for SNP identification are colloid-based biosensors, which are proving to possess several advantages over the traditional microarray format. Here, we survey the current technologies used for SNP genotyping and review colloid-based biosensors which are emerging as a higher throughput, potentially more accurate, technology.

Trypanosoma (nannomonas) congolense: separation of chromosomes of PCR product specific to medium-size chromosome

Pulsed field gradient gel electrophoresis was used to separate chromosomes of parasitic protozoan Trypanosome congolense clone IL1180. Total trypanosome DNA was isolated and resolved into 18 chromosomes by pulsed-field gradient gel electrophoresis. The chromosomes fall into four main size categories: the minichromosomes of about 50-150 kb: the medium-size chromosomes 200-750kb; the large chromosomes>1mb and chromosomes that are –specifically trapped in the wells. Four distinct medium –sized chromosomes with sizes 340kb, 360kb, 400kb and 500kb, were identified and designated chromosomes 1-4 respectively. DNA bands from each of the four medium-size chromosomes were excised from the gel, purified and randomly amplified with 23 random oligonucleotide primers (RAPD analysis). The RAPD product, which was unique to each of the four medium –sized chromosomes, were identified and cloned in T-vector plasmid. One RAPD product of approximate size 1.5Kb, which was amplified by oligonucleotide primer, ILO867 was identified as unique to medium –sized chromosome2. This RAPD product referred to as RAPD2/867/1.5 hereinafter only hybridized to medium sized chromosomes2. This cloned fragment can therefore be used as a specific DNA marker for medium –size chromosome 2 of T. congolense. The Kenya Veterinarian Vol. 27 2004: pp. 91-96

Molecular characterization of Kastamonu garlic: An economically important garlic clone in Turkey

Turkey is one of the major garlic producing country in the World and the significant amount of Turkey's production has been made using a garlic variety called Kastamonu garlic. Therefore, the purpose of this study was to assess genetic relationship of Kastamonu garlic with 20 previously characterized garlic clones collected from different regions of the world using AFLP and locus specific DNA markers. One putative Kastamonu garlic genotype was obtained from Taskopru district of Kastamonu province while another putative Kastamonu garlic genotype was collected from a local farmers’ market in Bursa province and called as Kast-Taskopru and Kast-Bursa in this study, respectively. In the UPGMA dendrogram developed by using 120 AFLP markers, Kast-Taskopru was clustered closely over 97% similarity with other non-bolting garlic clones, PI493112, PI493118 and PI383824. This cluster was also supported by bootstrap analysis with 100% bootstrap value. All clones in this cluster also shared same alleles of gene specific DNA markers. However, Kast-Bursa shared 100% polymorphic AFLP markers and gene specific markers with a different garlic clone, PI497951 in another distinct cluster of UPGMA dendrogram and this clustering has also bootstrap value of 100%. These results suggest that Kastamonu garlic is not unique and garlic production in Turkey has been made using several garlic clones, even though most of this production has been sold as Kastamonu garlic due to its high popularity. Therefore, a standard Kastamonu garlic genotype needs to be determined by fingerprinting all available garlic clones cultivated in Kastamonu province and other regions of Turkey.

Suppression Subtractive Hybridization (SSH) and its modifications in microbiological research

Suppression subtractive hybridization (SSH) is an effective approach to identify the genes that vary in expression levels during different biological processes. It is often used in higher eukaryotes to study the molecular regulation in complex pathogenic progress, such as tumorigenesis and other chronic multigene-associated diseases. Because microbes have relatively smaller genomes compared with eukaryotes, aside from the analysis at the mRNA level, SSH as well as its modifications have been further employed to isolate specific chromosomal locus, study genomic diversity related with exceptional bacterial secondary metabolisms or genes with special microbial function. This review introduces the SSH and its associated methods and focus on their applications to detect specific functional genes or DNA markers in microorganisms.

Revealing species‐specific trophic links in soil food webs: molecular identification of scarab predators

Soil food webs are particularly important in terrestrial systems, but studying them is difficult. Here we report on the first study to apply a molecular approach to identify species-specific trophic interactions in below-ground food webs. To identify the invertebrate predator guild of the garden chafer Phyllopertha horticola (Coleoptera, Scarabaeidae) whose root-feeding larvae can be highly abundant in grasslands, a specific DNA marker was developed. It allowed detection of P. horticola egg and white grub meals within the gut content of Poecilus versicolor (Coleoptera, Carabidae) larvae for up to 24 h post-feeding. Soil samples from an alpine grassland revealed a diverse below-ground macro-invertebrate community with earthworms, P. horticola larvae, and centipedes as well as beetle larvae as the most abundant detritivores, herbivores, and predators, respectively. Garden chafer DNA was detected in 18.6%, 4.1%, and 4.4% of field-collected Geophilidae (n = 124), beetle larvae (n = 159), and Lithobiidae (n = 49), respectively. We conclude that most of the investigated predators actively preyed on P. horticola, as secondary predation is unlikely to be detected in below-ground systems. Moreover, scavenging most likely contributes only to a small percentage of the revealed trophic links due to the low availability of carrion. Sampling date did not influence prey detection rates, indicating that both P. horticola eggs and larvae were preyed on. Only 2.7% of the below-ground predators tested positive for earthworms, an alternative, highly abundant prey, suggesting that P. horticola represents an important prey source for centipedes and predatory beetle larvae during summer within the soil food web.

Analysis of melanocortin 1 receptor (MC1R) gene polymorphisms in some cattle breeds: their usefulness and application for breed traceability and authentication ofParmigiano Reggiano cheese

In cattle, the MC1R gene has been the subject of several studies with the aim to elucidate the biology of coat colour. Then, polymorphisms of this gene have been proposed as tools for breed identification and animal products authentication. As a first step to identify breed specific DNA markers that can be used for the traceability of mono-breed dairy cattle products we investigated, using PCR-RFLP and PCR-APLP protocols, the presence and distribution of some alleles at the MC1R locus in 18 cattle breeds for a total of 1360 animals. For each of seven breeds (Italian Holstein, Italian Brown, Italian Simmental, Rendena, Jersey, Reggiana and Modenese) a large number of animals (>70) was genotyped so the obtained results can be considered with more confidence. Allele Ed was identified only in black pied cattle (Italian Holstein and Black Pied Valdostana). Allele E (this nomenclature includes all alleles except Ed, E1 and e) was observed in Italian Brown, Rendena, Jersey, Modenese, Italian Simmental, Grigio Alpina, Piedmontese, Chianina, Romagnola, Marchigiana, Swedish Red and White and Danish Red. Allele E1 was identified in Italian Brown, Rendena, Grigio Alpina, Piedmontese, Swedish Red and White and Danish Red. The recessive allele e, known to cause red coat colour, was fixed in Reggiana and almost fixed in Italian Simmental. This allele was observed also in Italian Holstein, Italian Brown, Rendena, Jersey and Modenese albeit with low frequency. Moreover, this allele was detected in Valdostana, Pezzata Rossa d’Oropa, Piedmontese, Romagnola, Swedish Red and White, Danish Red, Charoleis and Salers. In the case of the Reggiana breed, which is fixed for allele e, the MC1R locus is highly informative with respect to breeds that carry other alleles or in which allele e is at very low frequency. In theory, using the MC1R locus it is possible to identify the presence of milk from some other breeds in Parmigiano Reggiano cheese labelled as exclusively from the Reggiana breed. This possibility was practically tested by setting up protocols to extract and analyse polymorphisms of the MC1R locus in several dairy products, including Parmigiano Reggiano cheese cured for 30 months. The lower detection limit was estimated to be 5% of non expected DNA. This test can represent a first deterrent against fraud and an important tool for the valorisation and authentication of Parmigiano Reggiano cheese obtained from only Reggiana milk.

Assisted Selection by Specific DNA Markers for Genetic Elimination of the Kunitz Trypsin Inhibitor and Lectin in Soybean Seeds

The antinutritional factors found in soybean, lectin (SBL) and Kunitz trypsin inhibitor (SKTI), are usually inactivated by heat treatment. However, residual activity of these factors can be found in several types of soy-derived products. Heat treatment does not completely eliminate these factors, and in addition it may considerably decrease protein solubility. The genetic elimination of these antinutritional factors could be an alternative to the heat treatment. This study aimed to develop soybean lines devoid of SKTI and SBL in their seeds. The population under study was obtained by crossing the normal cv. Monarca with a soybean line lacking SKTI and SBL. Specific DNA primers were designed for the identification of the recessive alleles that condition the absence of SKTI and SBL. F2 seeds presenting the DNA markers that identify the recessive alleles were selected and the corresponding F2 plants were backcrossed with the recurrent parent (‘Monarca’), producing the BC1F1 generation. The F2 generation was analyzed by SDS-PAGE in order to confirm the genotypes of the F2 selected. The segregation tests confirmed that these traits are governed by two genes that segregate independently.