Mutations in the bovine prolactin receptor (PRLR) gene: allele and haplotype frequencies in the Reggiana cattle breed

AbstractProlactin receptor (PRLR) is a member of the cytokine receptor superfamily. PRLR exerts its functions binding three types of ligands (prolactin, placental lactogen and growth hormone) invol..

Identification of mutations in the bovine KIT gene, a candidate for the Spotted locus in cattle

AbstractIn mammals, abnormal migration of melanoblasts from the neural crest during embryonic development may be the reason of the pielbaldism phenotype that is a mixture of pigmented and unpigmented areas in the coat. Several cattle breeds, like for example Holstein, show the piebald spotted coat colour phenotype, that, according to crossbreeding studies, is due to a recessive allele (s), member of the allele series of the Spotted (S) locus. Dominant alleles at this locus act as suppressors of the spotted pattern and produce uniformly pigmented animals while others determine the colour-sided pattern known, for example, in the Hereford breed. The bovine v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (KIT) gene was localized in the region of chromosome 6 where the Spotted locus was mapped. KIT plays a major role during the embryonic development in directing the migration of the melanoblasts from the neural crest. Mutations in this gene cause different coat colour patterns in mouse and human. In pigs..

Employment of an auto-regressive model for knock detection supported by 1D and 3D analyses

In this work, experimental data, carried out on a twin-cylinder turbocharged engine at full load operations and referred to a spark advance of borderline knock, are used to characterize the effects of cyclic dispersion on knock phenomena. 200 consecutive incylinder pressure signals are processed through a refined Auto-Regressive Moving Average (ARMA) mathematical technique, adopted to define the percentage of knocking cycles, through a prefixed threshold level. The heuristic method used for the threshold selection is then verified by 1D and 3D analyses. In particular, a 1D model, properly accounting for cycle-by-cycle variations, and coupled to a reduced kinetic sub-model, is used to reproduce the measured cycles, in terms of statistical distribution of a theoretical knock index. In addition, few individual cycles, representative of the whole dataset, are selected in a single operating condition in order to perform a more detailed knock analysis by means of a 3D CFD approach, coupled to a tabulated chemistry technique for auto-ignition modeling. Outcomes of 1D and 3D models are compared to the ARMA results and a substantial coherence of the numerical and experimental results is demonstrated. The integrated 1D and 3D analyses can hence help in supporting the choice of the experimental threshold level for knock identification, following a more standardized theoretical approach

Analysis of PRKAG3 gene polymorphisms in Italian autochthonous pig breeds.

The PRKAG3 gene encodes for the regulatory gamma 3 subunit of adenosine monophosfate-activated protein kinase (AMPK) protein that acts as a regulator of carbohydrate and fat metabolism. Several single nucleotide polymorphisms (SNPs) have been identified in the porcine PRKAG3 gene. Two of them (c.595A>G or p.I199V and c.599G>A or p.R200Q) have been associated with variability of meat quality and carcass traits. In this study, we investigated the frequency of the c.595A>G and c.599G>A SNPs in 372 animals of five Italian autochthonous pig breeds (Apulo-Calabrese, Casertana, Cinta Senese, Mora Romagnola and Nero Siciliano). Genomic DNA were extracted from hair roots or blood and SNPs genotyping was performed by PCR-RFLP protocols. The c.599A (p.200Q) allele was carried by only 3 Nero Siciliano pigs. All five breeds were polymorphic at the c.595A>G site. The c.595A (p.I199) allele was the less frequent in the analysed breeds with minor allele frequency ranging from 0.144 (Nero Siciliano) to 0.464 (Casertana). Based on identified allele frequencies, the c.595A>G SNP can be useful in association studies with meat, carcass and ham quality traits in the Italian local pig breeds

Nero Siciliano pig: analysis of coat colour affecting genes and perspectives for breed traceability

Nero Siciliano is an autochthonous pig breed reared in the internal areas of Sicily region mainly in the Nebrodi mountains. The animals are usually completely black with a dorsal stripe but a few present white portions mainly in the face or in the fore legs. According to the increased requests of the consumers for local and typical products, meat and cured products of Nero Siciliano pigs are sold at a higher price compared to other pig products. Thus there is the need to guarantee both consumers and the whole Nero Siciliano production chain from possible frauds. The identification and/or use of DNA markers that may be breed specific could make it possible to establish breed traceability and authenticity systems for the products obtained with this local pig breed. Mutations in coat colour genes have been already described and utilized for porcine breed traceability. In this trial we analysed mutations identified in two coat colour affecting genes, the melanocortin 1 receptor (MC1R) and the v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene (KIT), with the aim to characterize the Nero Siciliano pig at these loci and provide useful information to establish authenticity systems for the meat products. Fragment analysis of PCR products and PCRRFLP methods were used to identify the polymorphic sites that can distinguish known alleles at these two loci in 104 Nero Siciliano pigs. Four alleles were identified at the MC1R locus: the two dominant black alleles (ED2, frequency of 0.673; ED1, 0.187), allele EP (0.106) and the recessive e allele (0.034). The results showed that different alleles were observed at this locus, polymorphisms at the MC1R gene cannot be used for product traceability and authentication of this breed. As regards the KIT locus, all the animals were negative for the splice site mutation of exon/intron 17. Thus, meat of Nero Siciliano pigs can be distinguished from meat of white pigs that are positive for this polymorphic site. Moreover, at this locus only 4 pigs showed the 3'-5' duplication breakpoint suggesting that they carried the Ip allele. Studies are in progress to evaluate the effect of this allele on coat colour phenotypes in Nero Siciliano pig

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

l legame tra un prodotto di origine animale e la razza da cui questo \ue8 originato rappresenta un aspetto importante per la valorizzazione di alcune produzioni. Il maggior prezzo che questi prodotti spuntano sul mercato fa emergere l\u2019esigenza di poter autenticare o tracciare i prodotti mono-razza per smascherare e scoraggiare possibili frodi. A questo scopo sono stati proposti sistemi di analisi del DNA, alcuni dei quali utilizzano marcatori in geni che determinano il colore del mantello, che \ue8 uno dei principali caratteri che differenziano tra di loro le razze. Diverse mutazioni nel gene melanocortin 1 receptor (MC1R) sono gi\ue0 state associate a particolari effetti sul colore del mantello nella specie bovina. In questa ricerca abbiamo studiato la presenza dei principali alleli al locus MC1R, per valutare la possibilit\ue0 di utilizzare questo gene per l\u2019autenticazione e la tracciabilit\ue0 di razza dei prodotti lattiero-caseari. Le mutazioni che permettono di distinguere questi alleli sono state analizzate utilizzando protocolli di PCR-RFLP e PCR-APLP su un totale di 1360 animali appartenenti a 18 razze bovine. Per ognuna delle seguenti razze, Frisona Italiana, Bruna Italiana, Pezzata Rossa Italiana, Jersey, Rendena, Reggiana e Modenese, \ue8 stato possibile analizzare pi\uf9 di 70 animali. L\u2019allele Ed \ue8 stato identificato nella razza Frisona Italiana con una frequenza dello 0,886. L\u2019allele E (nomenclatura che include tutti gli alleli tranne che e, Ed e E1) \ue8 stato identificato con alta frequenza nella Bruna Italiana (0,591), Rendena (0,738), Jersey (0,955) e Modenese (0,961) e con bassa frequenza nella Pezzata Rossa Italiana (0,029). Inoltre, questo allele \ue8 stato osservato nella Rossa Svedese, Rossa Danese, Grigio Alpina, Piemontese, Romagnola, Marchigiana e Chianina. In alcune di queste razze (Bruna Italiana, Rendena, Grigio Alpina, Piemontese, Rossa Svedese e Rossa Danese) \ue8 stato identificato anche l\u2019allele E1. L\u2019allele e \ue8 risultato fissato nella razza Reggiana e quasi fissato nella razza Pezzata Rossa Italiana. Inoltre, con bassa frequenza, \ue8 stato identificato in tutte le altre razze analizzate, tranne che nella Marchigiana. Le differenze osservate tra razze esaminate indicano che, almeno in alcuni casi, \ue8 possibile utilizzare i polimorfismi del gene MC1R per escludere o confermare l\u2019impiego di latte di una determinata razza nella produzione di un prodotto lattiero-caseario. Il caso pi\uf9 interessante \ue8 quello del formaggio Parmigiano Reggiano prodotto con l\u2019uso esclusivo di latte di bovine di razza Reggiana. Infatti, essendo presente in questa razza soltanto l\u2019allele e il rilievo analitico di qualsiasi altro allele nel DNA estratto dal formaggio rivela l\u2019uso di latte proveniente da altre razze. La messa a punto di un metodo PCR-RFLP per l\u2019analisi del DNA estratto da prodotti lattiero caseari, incluso il Parmigiano Reggiano di oltre 24 mesi di stagionatura, rappresenta uno strumento importante per la difesa di questo prodotto mono-razza da eventuali frodi. I risultati ottenuti su 10 forme di formaggio prodotto esclusivamente con latte di bovine di razza Reggiana e su 15 forme di Parmigiano Reggiano commerciale ottenuto senza restrizione della razza di origine del latte hanno mostrato la validit\ue0 del metodo del quale \ue8 stata valutata anche la sensibilit\ue0n 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\u2019Oropa, 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

Investigation of SNPs in the ATP1A2, CA3 and DECR1 genes mapped to porcine chromosome 4: analysis in groups of pigs divergent for meat production and quality traits

STUDIO DI TRE GENI (ATP1A2, CA3E DECR1) LOCALIZZATI SUL CROMOSOMA SUINO 4: ANALISIDELLE FREQUENZE ALLELICHE DI SNP IN SUINI ESTREMI PER ALCUNI CARATTERI PRODUTTIVITre geni (ATPase, Na+/K+transporting, \u3b12(+) polypeptide, ATP1A2; carbonic anhydrase III, CA3; 2,4-die-noyl CoA reductase 1, mitochondrial, DECR1), isolati da una libreria a cDNA ottenuta da muscolo scheletri-co di suino, sono stati scelti per questo studio sulla base del ruolo fisiologico della proteina codificata in pro-cessi cellulari e metabolici collegabili in modo diretto o indiretto con alcuni caratteri produttivi. La scelta dei tre geni \ue8 stata anche basata sulla loro localizzazione sul cromosoma 4 dove diversi QTL per caratteri pro-duttivi sono gi\ue0 stati identificati.Nella regione 3\u2019 non tradotta del gene CA3\ue8 stato identificato, mediante analisi SSCP, un polimorfismo bial-lelico. Il sequenziamento dei due alleli ha permesso di identificare una nuova mutazione puntiforme, che \ue8stata successivamente analizzata mediante PCR-RFLP. Per questo gene \ue8 stato effettuato il mappaggiogenetico sul cromosoma 4 mediante l\u2019analisi della mutazione nei campioni delle famiglie di riferimento delprogetto europeo di mappaggio del genoma suino (PiGMaP). Utilizzando il polimorfismo PCR-RFLP del geneATP1A2, gi\ue0 decritto in un precedente lavoro come SSCP, sono state tipizzate diverse famiglie di riferimen-to PiGMaP ed \ue8 stato confermato il mappaggio genetico di ATP1A2. Utilizzando queste informazioni e quel-le gi\ue0 disponibili per DECR1, per la prima volta, \ue8 stata ottenuta una mappa di linkage del cromosoma 4che comprende tutti e tre i geni analizzati. Il mappaggio genetico dei tre geni \ue8 stato anche confermatomediante tipizzazione di un pannello di ibridi di cellule irradiate (IMpRH 7000 rad).Le frequenze alleliche delle mutazioni identificate nei tre loci sono state studiate in 11 razze suine (LargeWhite Italiana, Landrace Italiana, Duroc Italiana, Landrace Belga, Hampshire, Pi\ue9train, Meishan, CintaSenese, Casertana, Calabrese and Nero Siciliano) per un totale di 272 animali. Inoltre, come approccio ini-ziale per poi scegliere i geni da analizzare in futuri studi di associazione, abbiamo confrontato le frequenzealleliche di mutazioni puntiformi per questi tre loci in gruppi di suini di razza Large White Italiana e DurocItaliana, analizzando animali con valori degli indici genetici estremi e divergenti per alcuni caratteri produt-tivi (accrescimento, spessore lardo dorsale, tagli magri e grasso intermuscolare visibile). Per il gene CA3 \ue8stata osservata una differenza nella distribuzione delle frequenze alleliche (P< 0,05) per i due caratteri taglimagri (nella razza Large White Italiana) e grasso intermusculare visibile (nella razza Duroc Italiana). Per ilgene DECR1, differenze significative sono state osservate per il carattere grasso intermuscolare visibile. Ilgene ATP1A2, che mappa vicino al locus FAT1, non ha presentato differenze statisticamente significativenelle frequenze tra i gruppi estremi per i caratteri oggetto di studio. Analizzando il livello di linkage disequi-librium(LD) tra i tre loci, \ue8 stato evidenziato un elevato livello di LD (D\u2019= 0,967; P< 0,0001) tra i geni CA3e DECR1, solo nella popolazione Duroc Italiana. Questi primi risultati pongono le basi per ulteriori studi perverificare se i geni CA3e DECR1sono associati con i caratteri oggetto di selezione nel suino pesante.Three genes (ATPase, Na+/K+ transporting, \u3b1 2(+) polypeptide, ATP1A2; carbonic anhydrase III, CA3; 2,4-dienoyl CoA reductase 1, mitochondrial, DECR1), isolated from a porcine skeletal muscle cDNA library and mapped on porcine chromosome 4 (SSC4), were investigated. A new single nucleotide polymorphism (SNP) was identified in the 3\u2019-untranslated region of the CA3 gene and used to genetically map this locus on SSC4 together with the ATP1A2 and DECR1 loci for which SNPs were already reported. Allele frequencies of the three loci were reported for 11 pig breeds (Italian Large White, Italian Landrace, Italian Duroc, Belgian Landrace, Hampshire, Pi\uf9train, Meishan, Cinta Senese, Casertana, Calabrese and Nero Siciliano). Radiation hybrid mapping of these genes confirmed the linkage mapping results as well as mapping information reported by other authors. Then, the SNPs identified in the ATP1A2, CA3 and DECR1 genes were genotyped in Italian Large White and Italian Duroc animal groups with extreme and divergent estimated breeding value for several production traits. For CA3 significant differences in allele frequencies (P< 0.05) were observed between the extreme groups of pigs for the lean cuts (Italian Large White) and visible intermuscular fat (Italian Duroc) traits. For DECR1, a significant difference in allele frequencies was observed only for the visible intermuscular fat trait. ATP1A2, which maps close to the FAT1 locus, did not show any significant difference. A very high linkage disequilibrium (D\u2019= 0.967; P< 0.0001) was identified between CA3 and DECR1 in the Italian Duroc population. Further investigations are needed to evaluate the effect of CA3 and DECR1 on the considered trait

investigation of the dgat1 k232a and vntr mutations in dairy and dual purpose cattle breeds

AbstractSeveral studies have reported that the centromeric end of bovine chromosome 14 harbours QTL for milk production and composition traits. The acyl-Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) gene was indicated to be the quantitative trait gene affecting these traits with a major effect on milk fat content. A two bp mutation in exon 8 causing a nonconservative lysine to alanine amino acid substitution at codon 232 (K232A) showed a confirmed effect across breeds with allele K resulting associated with an increase on fat yield, fat percentage and protein percentage while allele A was associated with higher milk yield. Another mutation in the 5' regulatory region of this gene, a variable number of tandem repeat (VNTR) of 18 bp, was suggested to affect fat percentage. The objective of the present work was to investigate the occurrence of the DGAT1 K232A and VNTR polymorphisms in several Italian dairy and dual purpose cattle breeds as a first step to evaluate their effects on milk production trait..

How do roots respond to osmotic stress? A transcriptomic approach to address this question in a non-model crop

Drought is a complex phenomenon that is relevant for many crops. Performing high-throughput transcriptomics in non-model crops is challenging. The non-model crop where our workflow has been tested on is banana (Musa spp.), which ranks among the top ten staple foods (total production over 145 million tons in 2013 (FAOstat)[1]). Bananas need vast amounts of water and even mild-drought conditions are responsible for considerable yield losses[2]. To characterize drought in the roots of different banana genotypes, we designed a lab model based on osmotic stress (5% PEG treatment for 3 days) and performed mRNA-seq analysis[3]. Using Illumina technology, 18 cDNA libraries were sequenced producing around 568 million high quality reads, of which 70-84% were mapped to the diploid reference genome[4]. We show that the applied stress leads to a drop in energy levels inducing a metabolic shift towards (i) higher oxidative respiration, (ii) alternative respiration and (iii) fermentation. We also analyzed the expression patterns of paralogous genes belonging to the same gene families and detected possible cases of sub-functionalization