Shavindra Bajaj, Yidong Ran, Jonathan Phillips, Gunaseelan Kulrajathevan, Sunil Pal, Dan Cohen, Kieran Elborough and Sathish Puthigae

Plant Cell Reports , accepted for publication

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium strain EHA101 carrying the plasmid pCAMBIA 1305.1 in presence of acetosyringone for 3-4 days. The calli were grown under 50 and 80 mg/l hygromycin selection, respectively for 2 cycles of 2-weeks each, followed by transfer to regeneration medium with 25 mg/l hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After the development of transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past two years, we have produced more than one thousand plants with constructs encoding different genes of interest from perennial ryegrass.

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Emerging technologies for identifying superior dairy cows in New Zealand

Authors: Garrick DJ, Snell RG.

New Zealand Veterinary Journal 53: 391-400, December 2005

The performance of animals is determined by the interaction of their genes with environmental circumstances. Accordingly, animals exhibiting superior performance are not necessarily the animals with the best genes nor are they the best choice of parents. Statistical analyses of production records for repeated traits, e.g. lactation yields and reproductive performance, show that part of the variation in performance among animals in the same herd and year is due to genetic differences, and the remainder is due to so-called residual or environmental factors that are not passed on to offspring. These within-herd environmental factors can be partitioned into a component that affects performance throughout an animal's lifetime, and a part that is unique to each observation. The process of animal evaluation from pedigree and performance records partitions the superiority of each cow into these three components. Reliable assessment of the genetic merit of bulls has required progeny testing, and for cows has required observation of their own individual performance. Selection on the genetic or breeding value component has systematically improved animal performance over recent decades, but has been limited by the age at which assessments of genetic merit are available. Emerging molecular technologies can read DNA sequences or measure RNA expression and have allowed the identification of a number of chromosome regions, and a few specific genes in those regions, that influence economic performance. This information allows better characterisation of the relationships between animals and more accurate predictions of genetic merit in bulls without progeny information and in cows that have yet to produce their own performance record. At some stage, enough genes responsible for variation in performance will be identified to allow faster genetic progress through selection of animals at young ages and therefore more rapid turnover of the generations. Mechanisms that modify gene expression have been identified and these may ultimately allow animals to be selected at an early age for lifetime productivity, accounting for processes that modify gene expression and lead to differences in performance that are not reflected by DNA sequence information. This review describes the status of these emerging technologies and their likely role in the improvement of dairy cattle.

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Lactation traits of importance in dairy cows and applications for emerging biotechnologies

Author: Davis SR

New Zealand Veterinary Journal 53: 401-406, December 2005

New molecular and quantitative genetic technologies are the latest in a long list of technologies that have been introduced to dairy industries over many decades to improve the performance of cows. The catalysts for future advances will be sequencing of the bovine genome and development of high throughput technologies to identify and exploit relevant variation in DNA sequences. The new technologies will allow the selection of animals based on specific genotypes that suit specific applications such as once-daily milking or extended lactation.

These technologies will also allow exploitation of between-cow variation in milk composition, which is currently hidden by bulking of milk on and between farms. Thus, there are opportunities to produce milk from herds of cows selected for specific milk composition that might be e.g. more suitable for cheese-making or had milkfat with specific properties to enhance human health. Identification of genes and gene polymorphisms associated with improved resistance to specific diseases in cows and other farm species represents a real opportunity to improve animal health over the coming decade.

New levels of genetic regulation have been identified eg. microRNA and epigenetics, the impact of which on cow and human performance are only just beginning to be understood.

There is a potential role for veterinarians to provide or be the interface for provision of genetic advice to farmers in much the same way that nutritional advice is currently given.

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Dissecting the regulation of fructan metabolism in perennial ryegrass (Lolium perenne) with quantitative trait locus mapping

Authors: L. B. Turner, A. J. Cairns, I. P. Armstead, J. Ashton, K. Skøt, D. Whittaker, and M. O. Humphreys

New Phytologist 2005; doi: 10.1111/j.1469-8137.2005.01575.x

Quantitative trait locus (QTL) mapping, which can be a useful tool for dissecting complex traits, has been used here to study the regulation of fructan metabolism in temperate forage grasses.

An F2 mapping family, derived from a high water-soluble carbohydrate (WSC) × low WSC cross, was used to map fructans and the other components of WSC (sucrose, glucose and fructose) in leaves and tiller bases of perennial ryegrass (Lolium perenne) in spring and autumn. To characterize regions of the genome that control basic carbohydrate metabolism, a strategy to minimize the impact of genotype (G) × environment (E), and E-effects on the characterization of G-effects, was adopted.

Most traits were highly variable within the family. There was also considerable year-to-year environmental variation. However, significant genetic effects were detected, and several traits had high broad-sense heritability. QTL were identified on chromosomes 1, 2, 5 and 6. Leaf and tiller base QTL did not coincide. Individual QTL explained between 8 and 59% of the total phenotypic variation in the traits.

Fructan turnover, metabolism and their genetic control, and the effect of environment, are discussed in the context of the results.

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Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora

Authors: Manuel Ferrer, Olga V. Golyshina,, Tatyana N. Chernikova, Amit N. Khachane, Dolores Reyes-Duarte, Vitor A. P. Martins Dos Santos, Carsten Strompl, Kieran Elborough, Graeme Jarvis, Alexander Neef, Michail M. Yakimov, Kenneth N. Timmis, and Peter N. Golyshin

Environmental Microbiology 7: 1996-2010, December 2005; doi:10.1111/j.1462-2920.2005.00920.x

A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo--1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.

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Conversion of a Carboxylesterase into a Triacylglycerol Lipase by a Random Mutation

Authors: Dolores Reyes-Duarte, Julio Polaina, Nieves López-Cortés, Miguel Alcalde, Francisco J.Plou, Kieran Elborough, Antonio Ballesteros, Kenneth N.T immis, Peter N.Golyshin, and Manuel Ferrer

Angewandte Chemie (International Edition) 44: 1-5, October 2005; doi: 10.1002/anie.200502461

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A mutation in bovine keratin 5 causing epidermolysis bullosa simplex, transmitted by a mosaic sire

Authors: Ford CA, Stanfield AM, Spelman RJ, Smits B, Ankersmidt-Udy AE, Cottier K, Holloway H, Walden A, Al-Wahb M, Bohm E, Snell RG, Sutherland GT

The Journal of Investigative Dermatology 124(6): 1170-6, June 2005

A mechanobullous skin disorder was identified in the progeny of a 3-y-old Friesian-Jersey crossbred bull. The condition presented as loss of skin and mucosa from contact areas and inflammation. Examination of skin samples under light microscopy revealed separation of the epidermis from the dermis. Electron microscopic analysis refined the site of cleavage to above the basement membrane involving lysis of basal keratinocytes. These observations were consistent with the simplex form of epidermolysis bullosa (EB) in humans. Candidate genes based on human gene mutations were assessed, resulting in keratin 5 being identified as the most likely candidate gene. The sequence of bovine keratin 5 was established and sequencing led to identification of a G to A substitution in all affected animals. This mutation leads to an amino acid change of glutamic acid to lysine in the final E (478) of the KLLEGE motif of the protein. The sire carried a de novo mutation and was mosaic, explaining his asymptomatic status and the less than expected frequency of affected offspring. Remarkably, the same mutation has been previously described in EB simplex in humans.

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Molecular Dissection of a Quantitative Trait Locus:

A Phenylalanine-to-Tyrosine Substitution in the Transmembrane Domain of the Bovine Growth Hormone Receptor Is Associated With a Major Effect on Milk Yield and Composition

Authors: Sarah Blott, Jong-Joo Kim, Sirja Moisio, Anne Schmidt-Küntzel, Anne Cornet, Paulette Berzi, Nadine Cambisano, Christine Ford, Bernard Grisart, Dave Johnson, Latifa Karim, Patricia Simon, Russell Snell, Richard Spelman, Jerry Wong, Johanna Vilkki, Michel Georges, Frédéric Farnir, and Wouter Coppieters

Genetics 163: 253-266, January 2003

We herein report on our efforts to improve the mapping resolution of a QTL with major effect on milk yield and composition that was previously mapped to bovine chromosome 20. By using a denser chromosome 20 marker map and by exploiting linkage disequilibrium using two distinct approaches, we provide strong evidence that a chromosome segment including the gene coding for the growth hormone receptor accounts for at least part of the chromosome 20 QTL effect. By sequencing individuals with known QTL genotype, we identify an F to Y substitution in the transmembrane domain of the growth hormone receptor gene that is associated with a strong effect on milk yield and composition in the general population.
© 2003 by the Genetics Society of America.

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Characterization of the DGAT1 Gene in the New Zealand Dairy Population

Authors: R. J. Spelman, C. A. Ford, P. McElhinney, G. C. Gregory, and R. G. Snell

Journal of Dairy Science 85: 3514-3517, 2002

Recently, DGAT1 was identified as the gene that underlies the QTL for bovine milk production on chromosome 14. This study investigated the effect of the reported polymorphism in three dairy breeds in New Zealand. Statistically significant results were identified for milk fat, milk protein, and volume for Jersey and Holstein-Friesian breeds, and only milk volume for Ayrshires. The average allele substitution effects were 2 to 3 kg of protein and 120 to 130l milk for both the Jersey and Holstein-Friesian breeds. For milk fat, the average allele substitution effect was 6 kg for Holstein-Friesians and 3 kg for Jerseys. In all breeds, where the polymorphism increased milk fat yield, it decreased milk protein yield and milk volume.
© 2002, by the American Dairy Science Association. All rights reserved.

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Positional Candidate Cloning of a QTL in Dairy Cattle:

Identification of a Missense Mutation in the Bovine DGAT1 Gene with Major Effect on Milk Yield and Composition

Authors: Bernard Grisart, Wouter Coppieters, Frédéric Farnir, Latifa Karim, Christine Ford, Paulette Berzi, Nadine Cambisano, Myriam Mni, Suzanne Reid, Patricia Simon, Richard Spelman, Michel Georges, and Russell Snell

Genome Research 12(2): 222-231, February 2002

We recently mapped a quantitative trait locus (QTL) with a major effect on milk compositionparticularly fat contentto the centromeric end of bovine chromosome 14. We subsequently exploited linkage disequilibrium to refine the map position of this QTL to a 3-cM chromosome interval bounded by microsatellite markers BULGE13 and BULGE09. We herein report the positional candidate cloning of this QTL, involving

• the construction of a BAC contig spanning the corresponding marker interval,
• the demonstration that a very strong candidate gene, acylCoA:diacylglycerol acyltransferase (DGAT1), maps to that contig, and
• the identification of a nonconservative K232A substitution in the DGAT1 gene with a major effect on milk fat content and other milk characteristics.

©2002 by Cold Spring Harbor Laboratory Press

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