BackgroundInternal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways.FindingsIndividual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed.Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus.ConclusionsThis study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production.

Buffalo DGAT1 (diacylglycerol O-acyltransferase 1) was mainly investigated for the characterization of the gene itself and for the identification of the K232A polymorphism, similar to what has been accomplished in cattle, although no information has been reported so far at the mRNA level. The importance of DGAT1 for lipid metabolism led us to investigate the transcript profiles of lactating buffaloes characterized as high (9.13 ± 0.23) and low (7.94 ± 0.29) for milk fat percentage, and to explore the genetic diversity at the RNA and DNA level. A total of 336 positive clones for the DGAT1 cDNA were analyzed by PCR and chosen for sequencing according to the differences in length. The clone assembling revealed a very complex mRNA pattern with a total of 21 transcripts differently represented in the 2 groups of animals. Apart from the correct transcript (17 exons long), the skipping of exon 12 is the most significant in terms of distribution of clones with 11.6% difference between the 2 groups, whereas a totally different mRNA profile was found in approximately 12% of clones. The sequencing of genomic DNA allowed the identification of 10 polymorphic sites at the intron level, which clarify, at least partially, the genetic events behind the production of complex mRNA. Genetic diversity was found also at the exon level. The single nucleotide polymorphism c.1053C>T represents the first example of polymorphism in a coding region for the DGAT1 in the Italian Mediterranean breed. To establish whether this polymorphism is present in other buffalo breeds, a quick method based on PCR-RFLP was set up for allelic discrimination in the Italian Mediterranean and the Romanian Murrah (200 animals in total). The alleles were equally represented in the overall population, whereas the analysis of the 2 breeds showed different frequencies, likely indicating diverse genetic structure of the 2 breeds. The T allele might be considered as the ancestral condition of the DGAT1 gene, being present in the great part of the sequenced species. These data add knowledge at the transcript and genetic levels for the buffalo DGAT1 and open the opportunity for further investigation of other genes involved in milk fat metabolism for the river buffalo, including the future possibility of selecting alleles with quantitative or qualitative favorable effects (or both).

This is the first study to explore the genetic diversity and population structure of domestic water buffalo (Bubalus bubalis) in Germany and their potential relations to herds in other parts of Europe or worldwide. To this end, animals from different herds in Germany, Bulgaria, Romania, and Hungary were genotyped and compared to genotypes from other populations with worldwide distribution and open to the public. The pilot study analyzed population structure, phylogenetic tree, and inbreeding events in our samples. In buffalos from Germany, a mixed genetic make-up with contributions from Bulgaria (Murrah breed), Romania, and Italy was found. All in all, a high degree of genetic diversity was identified in European buffalos, and a novel genotype was described in Hungarian buffalos by this study. We demonstrate that European buffalos stand out from other buffalo populations worldwide, supporting the idea that buffalos have not completely disappeared from the European continent during the late Pleistocene. The high genetic diversity in European buffalos seems to be an excellent prerequisite for the establishment of local breeds characterized by unique traits and features. This study may also be considered as an initial step on the way to genome characterization for the sustainable development of the buffalo economy in Germany and other parts of Europe in the future.

The overall objective of this study was to evaluate the effects that milking temperament (MT) of water buffaloes has on milking traits, welfare indicators, and reproductive outputs. The study was performed on 60 multiparous buffalo cows (6.7 ± 0.6 lactations) at the beginning of their lactation (100 days in milk, DIM). Each buffalo cow was scored by two independent observers using a temperament scoring system (1: extremely calm, 2: calm, 3: alert, 4: reactive, and 5: aggressive), and then grouped as ‘calm’ (scores 1, 2, and 3; n = 42) or ‘nervous’ (scores 4 and 5; n = 18). Additionally, the milk yield at 100 DIM (MY), milking speed (MS), calving interval (CI), age at first calving (AFC), body condition score (BCS), animal-based welfare parameters, and infrared thermography data (IRT) were evaluated. The MT significantly influenced the MY (p = 0.0082), with calmer cows outperforming their nervous counterparts. The MS was significantly influenced by the MT (p = 0.0015), with calmer animals having a higher milk ejection rate. The MT of the cows had no influence on the CI, AFC, or BCS. The evidence from this study suggests that the responsiveness of buffalo cows during milking affects their milk yield and milking speed, with no associations being found for reproduction efficiency indicators or animal-based welfare indicators.

Genetic parameters for milk yield, fat and protein milk content and "mozzarella" index were estimated in the population of Romanian Buffalo from Sercaia Research and Development Station. A total 609 milk yield and associated characters records, belonging to 87 females, which coming from 11 sire families, for 7 lactations were analyzed. The method used for genetic parameters estimates was REML. There was a large variability in all analyzed traits. Estimated heritability was small to medium for al examined traits. The milk yield is negatively correlated with fat and protein content, and weak positive with "mozzarella" index. Fat and protein milk content are high positive correlated with mozzarella production. In the long term, the female selection for milk yield will be (make) to the detriment of quality. Genetic improvement of the quantity of mozzarella will be make only on account of milk constituents that impart its quality. According to heritability values, in the process of female genetic evaluation appears advisable to use additional information sources in order to increase the accuracy of selection.