Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-27
2001-02-06
Zitomer, Stephanie (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S810000, C536S023100, C536S024330
Reexamination Certificate
active
06183955
ABSTRACT:
The present invention relates to methods of screening pig nucleic acid to determine pig genotype with respect to coat colour, and to kits for use in carrying out such methods.
White is the predominant coat colour among European commercial pig breeds e.g. Large White and Landrace. However, there are a number of commercially important coloured breeds, demonstrating a number of colours and combinations. The Duroc, associated with meat tenderness, is red, the Pietrain, which is a heavily muscled animal which produces a very lean carcass, is spotted, and the prolific Chinese Meishan breed is black.
Coat colour is important to the pig breeding industry for a number of reasons. Firstly, in a number of markets there is a preference for white skinned meat. This is due to the fact that pork is often marketed with the skin still attached, and skins from coloured pigs, even if dehaired, can still exhibit coloured hair roots, which can lead to a negative perception by the consumer, since the surface of the meat may appear to be spotted by mould. It is necessary, therefore, in these markets, to remove the skin from such carcasses, entailing additional cost. For example, in the U.S., coloured carcasses are associated with approximately 1% of skin defects requiring dehairing and skinning to remove pigment. As a result of this, coloured pig carcasses are generally discounted.
Secondly, gross variation in the appearance (i.e. a range of coat colours) of pigs claimed to be genetically consistent for traits other than coat colour can lead to questions about the consistency and quality of the animals in the mind of pig-producing customers.
In addition, pig breeders would like to be able to be in a position to ensure consistency in breeding populations. Thus, breeders may wish to ensure that progeny produced by breeding crosses were always white. Alternatively, a breeder of Pietrain pigs may wish to ensure that breeding crosses always produced the characteristic Pietrain colouring. Traditional animal breeding practices have, in the past, been used to attempt to eliminate colour (other than white) from pig lines.
The gene determining whether the animal is coloured or the desired white is designated I (for inhibition of coat colour). The version of the gene preventing the expression of any colour (I) is dominant to that which allows colour to develop (i). Traditional selection for white animals has reduced the frequency of i, but it still remains in the population in white heterozygous carrier animals. These animals can only be identified when they produce coloured offspring through matings with other heterozygous animals. Only through a programme of test matings can heterozygotes be identified which would enable the recessive allele to be eliminated from a given population. Such a programme would be time-consuming and costly and as such is not cost effective. Thus, i/i animals will inevitably be produced.
In addition, the situation is complicated further by the existence of another allele of I called I
P
(I-patch). The I
P
allele is recessive to I but is dominant to i. Thus, animals which have the genotype I
P
/I
P
or I
P
/i will show patches of colour.
Using a reference family developed from crosses between the European wild pig and a large white breed (Swedish Yorkshire), the position of the I gene on the porcine genetic map has been determined. The gene is located on chromosome 8 in the pig, close to the genes for albumin and for the &agr;-subunit of platelet-derived growth factor (PDGFRA) (Johansson et al,
Genomics
14: 965-969 (1992)). The mouse genetic map includes a homologous region located on mouse chromosome 5. This region contains a number of genes playing a role in the determination of mouse coat colour, namely W (dominant white spotting), Ph (patch) and Rw (rump white). The mouse W gene has been shown to co-locate with the KIT gene and some mutant genotypes at the W locus are due to structural changes in the KIT gene (Chabot et al,
Nature
335: 88-89 (1988), Geissler et al,
Cell
55: 185-192 (1988) and Nocka et al,
EMBO Journal
9: 1805-1813 (1990)).
We have now found that the KIT gene in pigs is involved with coat colour determination. More particularly, we have found that the difference between I, or I
P
, and i is duplication of at least part of the KIT gene in the I or I
P
allele. This duplication can result in two or more copies of a particular region of the KIT gene being present.
Thus, this has allowed us to develop methods of distinguishing between the alleles I, I
P
and i, and thus for determining the genotype of individual pigs with respect to coat colour.
Therefore, in a first aspect, the present invention provides a method of determining the coat colour genotype of a pig which comprises:
(i) obtaining a sample of pig nucleic acid; and
(ii) analysing the nucleic acid obtained in (i) to determine whether duplication of all or part of the KIT gene is present
The presence of duplication in the KIT gene sequence indicates the presence of either the I or I
P
allele. In some pig populations it is known that the incidence of I
P
is low or indeed non-existent. In such populations determining the presence or absence of duplication will be sufficient to provide a reasonable degree of confidence concerning a particular pig's genotype. Thus, by means of simply determining the presence or absence of duplication of the KIT gene (either complete or part thereof), coat colour genotype of a particular pig can be determined with a reasonably high degree of certainty.
However, in other populations it will be necessary to distinguish between the presence of I and I
P
.
We have found that although both I and I
P
have a duplication in the KIT gene, only I and not I
P
exhibits a deletion in one of the duplicated regions. It is therefore possible to distinguish between these alleles on that basis.
Thus, the method may further comprise the step:
(iii) determining whether the duplication is due to the presence of I or I
P
.
Suitably, this determination is made by analysing for the presence or absence of a deletion in at least one of the duplicated regions.
Suitably, the method of the invention will be carried out on pig genomic DNA, although pig RNA may also be analysed to determine the presence or absence of duplication in the KIT gene.
There may be a number of effects on the production of RNA from this gene, resulting from the duplication of part of the DNA sequence. These could include the inhibition of the production of RNA, alteration of the level of synthesis of the RNA, alteration in the size or processing kinetics of the RNA or alteration in the distribution of RNA production throughout the body of the animal. There might also be effects on the production of RNA from other genes caused by epistatic effects of the duplication.
Preferably, the determination carried out in step (ii) involves the use of PCR techniques, using an appropriate pair of primers. PCR, or polymerase chain reaction, is a widely used procedure in which a defined region of a DNA molecule can be amplified in vitro using a thermostable version of the enzyme DNA polymerase. Two known sequences that flank the region to be amplified are selected and priming oligonucleotides synthesised to correspond to these regions. If the primers are located sufficiently close together on the same piece of DNA, the region between them will be amplified. A polymerase chain reaction consists of a number of cycles of amplification. Each cycle begins with a denaturation step, typically at 94° C., in which the two strands of the template DNA molecule are separated. The temperature is then dropped to a temperature at which the synthetic oligonucleotide primers can anneal to the template (typically 50-60° C.). Through the high concentration of primers relative to template, the primers anneal to the template before template-template hybrids form. The annealing temperature is chosen such that annealing only occurs to the complementary regions of DNA within the template, and not to other regions of imperfect complementarity. The temper
Andersson Leif
Moller Maria Johansson
Plastow Graham Stuart
Siggens Kenneth William
Wales Richard
Brobeck Phleger & Harrison LLP
Dalgety PLC
Zitomer Stephanie
LandOfFree
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