Genetic marker for superior milk products in dairy cattle

An assay for a genetic marker associated with increased milk production is disclosed. Also disclosed are kits for use in connection with the assay and breeding methods that use the assay. The assay centers on finding a genetic marker in a bovine cell (e.g. in the DNA of the cell). The presence of the marker is confirmed by exposing a gene sequence from the cell to a restriction enzyme so as to yield gene fragments of varying lengths. During a separation step there is a separation of some of the fragments from others (such as by using electrophoresis), and there is then a hybridization of a plurality of probes that contain a portion of bovine prolactin sequence to the separated fragments. The probe is radiolabelled. Then, there is a comparison of the results of the hybridization with the hybridization results for a gene sequence known to either have the marker or not have the marker. The assay appears to be of greatest utility in connection with the Carlin-M Ivanhoe Bell Holstein family.

BACKGROUND OF THE INVENTION 
A. Field Of The Invention 
The present invention relates to recombinant DNA technology. More 
specifically it relates to a means of determining from restriction 
fragment hybridization patterns whether a gene polymorphism associated 
with improved milk production is present in a bovine cell. 
B. Description Of The Art 
With the competitive pressures that the dairy industry is facing, there has 
been significant interest in breeding and selecting dairy cattle which 
have improved milk production characteristics. Significant improvements 
have been achieved using standard breeding techniques in which progeny are 
studied. Their production results are then used to guide further breeding. 
One particularly successful family (from a milk production standpoint) is 
the Holstein line deriving from Carlin-M Ivanhoe Bell (registration number 
1667366, Holstein-Friesian Association, Brattleboro, Vt.). It has been 
estimated that currently more than 25% of the highest total performance 
index Holstein bulls in the U.S. are progeny of this individual. 
Unfortunately, such standard techniques require years to evaluate the true 
genetic value by progeny testing each bull. During progeny testing, many 
cows must be bred and give birth to offspring. The females must be raised, 
bred, allowed to give birth and, finally milked for a minimum length of 
time. The costs of confirming that a particular bull has superior genetics 
is therefore very high. 
Given the problems involved in using standard selection techniques, some 
have tried to improve milk production by locating genes that express 
proteins important to milk production, cloning them, and then adding 
commercially produced amounts of these proteins to feeds, drugs, and the 
like. Various bovine genes have in fact been shown to express proteins 
that are important for the control of mammary growth, lactogenesis, and/or 
lactation. One of these, bovine prolactin, is approximately 10 kilobases 
(kb) in length. See S. Camper et al., 3 DNA 237-249 (1984). Unfortunately, 
there has been significant political and regulatory resistance to the 
introduction of such methods. 
Various other research has discovered that polymorphisms (change in the 
genetic code) can be associated with recognizable differences in 
restriction fragment lengths of certain portions of the human genome. This 
has been of value in creating an assay for certain genetic diseases in 
humans. See e.g. D. Botstein et al., 32 Am. J. Human Gene. 314-331 (1980). 
Polymorphisms which do not affect amino acid composition have been reported 
adjacent to the bovine prolactin gene. These bovine prolactin studies have 
generally focused on differences around these loci between breeds or among 
individuals of an undetermined relationship. To date, applicants are 
unaware of anyone else having successfully located any polymorphism 
associated with a bovine gene which is indicative of improved milk 
production. 
Thus, it can be seen that a need exists for a means of more efficiently 
selecting and breeding cattle for the trait of improved milk production. 
SUMMARY OF THE INVENTION 
In one embodiment, there is provided an assay for the presence in a bovine 
gene sequence of a genetic marker that is located within 1.5 kb of a 
bovine prolactin coding exon in the sequence. The marker is indicative of 
an inheritable trait of increased milk production in progeny. 
The assay involves exposing the gene sequence to a restriction enzyme (e.g. 
Ava II) so as to yield gene fragments of varying lengths; then separating 
at least some of the fragments from others (e.g. using electrophoresis); 
then hybridizing a plurality of probes (e.g. radio-labelled cDNA probes) 
that contain a portion of a bovine prolactin gene sequence to the 
separated fragments; and then comparing the results of the hybridization 
with assay results for a bovine gene sequence known to have the marker or 
a bovine gene sequence known not to have the marker. The preferred bovine 
gene sequence is from a Holstein Carlin-M Ivanhoe Bell cell or its 
progeny. 
In another embodiment, the invention provides a kit for assaying for the 
presence in a bovine gene sequence of a genetic marker that is located 
within 1.5 kb of a bovine prolactin coding exon in the sequence, the 
marker being indicative of an inheritable trait of increased milk 
production in progeny. The kit has a probe containing a portion of a 
bovine prolactin gene sequence, and also a bovine gene sequence known to 
contain said marker. The probe is preferably a cDNA sequence of a portion 
of bovine prolactin and the probe can be radio-labelled. 
The gene sequence containing the marker is preferably a sequence contained 
in the cell of ATCC 40573, or its progeny, or sequences derived from 
either. The kit may also contain a restriction enzyme such as Ava II. 
In another embodiment there is a breeding method whereby one conducts an 
assay of the above type on a plurality of gene sequences from different 
bovine cells to be selected from, and one then drops out of the breeding 
program at least one of the cells (or its progeny) that do not contain the 
marker. 
It will be appreciated that the present invention can reduce the number of 
animals selected to achieve the same goal and reduce breeding costs: 
1. Young bull calves can be tested before entry into sire programs. Those 
without the marker would be selected not to be continued in the program. 
2. Daughters of bulls in this family who are being considered as mates 
could be tested. Those that are of an especially elite type AA (as 
described below) could be selected as preferable because they increase the 
chances for the elite marker being passed along. 
3. When the line goes to the commercial stage, daughters could be tested at 
birth. Those not having the marker could be culled, and those having it 
could be used for milk production. 
4. The screening process could be used to lower the number of bulls needed 
to be tested to maintain the same selection advantages as exist today. 
It should be appreciated that the marker gene provides information as a 
supplement to other traditional tools for selection. However, in cases of 
equal pedigree merit, the marker will help distinguish the lines, and thus 
lead to substantial improvements at much lower cost, and much more 
quickly. In the analyses conducted thus far, it appears that the marker, 
all other things being equal, is associated with a significant improvement 
in milk production in the Carlin-M Ivanhoe Bell family. 
Thus, the objects of the present invention include: 
(a) providing an assay of the above kind for the presence of a genetic 
marker associated with improved milk production traits; 
(b) providing a kit of the above kind to be used in connection with such 
assays; 
(c) providing a breeding method of the above kind for using such assays; 
(d) producing cattle by using breeding methods of the above kind; and 
(e) providing such assays, kits, and methods so as to save time and money. 
These and still other objects and advantages of the present invention will 
be apparent from the description which follows. In this description, the 
preferred embodiments of the invention will be described with reference to 
the accompanying drawings. These embodiments do not represent the full 
scope of the invention. Rather, the invention may be employed in other 
embodiments. Reference should therefore be made to the claims to interpret 
the breadth of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
We followed the following general steps: 
1. Extraction Of DNA: Semen from commercially available Holstein bulls (or 
other bovine cells) provided the source of the DNA to be tested. 
Spermatozoa were then treated so that their DNA would be released from the 
cell and concentrated in relatively pure form. 
2. Digestion And Fragment Separation: A restriction enzyme (preferably Ava 
II) which recognizes a sequence in double stranded DNA near bovine 
prolactin was used to cleave the DNA. DNA fragments were separated by 
electrophoresis in agarose gels against standards of known size. The gels 
were stained with ethidium bromide and photographed. Fragments of DNA were 
transferred to nylon membranes. 
3. Hybridization: Blots were hybridized in high stringency conditions to 
bovine prolactin cDNA that had been radio-labelled by nick-translation. 
Blots were washed with solutions of decreasing salt concentration to 
remove any nonspecifically bound probe. Blots bound to the labelled probe 
were exposed to autoradiography film with intensifying screens for three 
to five days at low temperature. 
4. Correlation To Data On Milk Production Of Progeny: Three genotypes were 
discovered by analysis of hybridization patterns of a particular family of 
sons. 
1. Extraction 
Semen from commercially available registered U.S. Holstein progeny bulls of 
the Carlin-M Ivanhoe Bell family provided the source material from which 
genomic DNA was isolated. DNA was extracted from sperm using a procedure 
modified from E. Borenfreund et al., 297 Nature 1375-77 (1961). Briefly, 
frozen 0.5 ml artificial insemination units containing approximately 
30.times.10.sup.6 sperm/unit were allowed to thaw at room temperature. The 
thawed semen was then treated with 2-mercaptoethanol in 10 mM Tris, pH 
8.0, 100 mM NaCl, 50 mM EDTA and 0.25% sodium dodecyl sulfate (SDS) and 
incubated at 53.degree. to 55.degree. C. for 30 minutes. Semen samples 
were cooled in an ice bath for 10 minutes prior to addition of proteinase 
K to a final concentration of 200 .mu.g/ml and incubation continued at 
37.degree. C. for 3 hours. DNA was extracted sequentially with phenol 
followed by phenol:chloroform: isoamyl alcohol (25:24:1) and finally 
chloroform:isoamyl alcohol (24:1). 
Following extraction, the DNA was precipitated with an equal volume of cold 
isopropanol (-20.degree. C.). The DNA was removed by spooling it onto a 
polyethelene pipet tip and air dried before being dissolved in 10 mM Tris, 
pH 7.5, containing 1 mM EDTA and 50 mM NaCl. 
Concentrations of DNA in each sample were estimated by their optical 
density at 260 nm. Samples were stored at 4.degree. C. 
2. DIGESTION/SEATION 
Prior to enzymatic digestion of the DNA, 15 .mu.g of isolated genomic DNA 
were dialyzed against a buffer compatible with the restriction 
endonuclease employed. The preferred restriction enzyme to digest the 
sperm genomic DNA from subsets of bulls was Ava II (New England BioLabs, 
Beverly, Massachusetts). DNA samples were digested in accordance with the 
enzyme manufacturer's standard recommendations for at least six hours. 
The resulting DNA fragments were separated by electrophoresis in agarose 
gels (1.0% agarose) in 40 mM Tris, 20 mM NaCl, 20 mM acetic acid and 2 mM 
EDTA. After completion of electrophoresis, gels were stained with ethidium 
bromide (2 .mu.g/ml) and photographed using UV light. Transfer to DNA to 
Hybond-N membrane (Amersham, Arlington Heights, Ill.) was accomplished 
using the manufacturer's recommended modified Southern blotting method. 
See generally E.M. Southern, 98 J. Mol. Biol. 503-517 (1975). 
DNA fragments were crosslinked to the nylon membrane by baking for 2 hours 
at 80.degree. C. followed by a two minute exposure to 300 NM UV light from 
a transilluninator (Fotodyne, New Berlin, Wis.). 
3. Hybridization 
Blots were preybridized on the membrane in 5x SSPE (0.9 M NaCl, 25 nM 
sodium phosphate, pH 7.4 and 2.5 mM EDTA) 0.4% SDS, 50% deionized 
formamide, 5x Denhardts (0.1% each of bovine serum albumin, Ficoll, and 
polyvinylpyrrolidone), and denatured herring sperm (50.mu.g/ml) at 
42.degree. C. for 6 hours. 
A plasmid containing a portion of bovine prolactin cDNA (pBPRL27) was 
radio-labelled by nick-translation. See e.g. P. Rigby et al., 113 J. Mol 
Biol. 237-251 (1977). pBPRL27 with the restriction endonuclease employed. 
The preferred restriction enzyme to digest the sperm genomic DNA from 
subsets of bulls was Ava II (New England BioLabs, Beverly, Mass.). DNA 
samples were digested in accordance with the enzyme manufacturer's 
standard recommendations for at least six hours. 
The resulting DNA fragments were separated by electrophoresis in agarose 
gels (1.0% agarose) in 40 mM Tris, 20 mM NaCl, 20 mM acetic acid and 2 mM 
EDTA. After completion of electrophoresis, gels were stained with ethidium 
bromide (2 .mu.g/ml) and photographed using UV light. Transfer to DNA to 
Hybond-N membrane (Amersham, Arlington Heights, Ill.) was accomplished 
using the manufacturer's recommended modified Southern blotting method. 
See generally E.M. Southern, 98 J. Mol. Biol. 503-517 (1975). 
DNA fragments were crosslinked to the nylon membrane by baking for 2 hours 
at 80.degree. C. followed by a two minute exposure to 300 NM UV light from 
a transilluninator (Fotodyne, New Berlin, Wis.). 
3. Hybridization 
Blots were prehubridized on the membrane in 5x SSPE (0.9 M NaCl, 25 nM 
sodium phosphate, pH 7.4 and 2.5 mM EDTA) 0.4% SDS, 50% deionized 
formamide, 5x Denhardts (0.1% each of bovine serum albumin, Ficoll, and 
polyvinylpyrrolidone), and denatured herring sperm (50.mu.g/ml) at 
42.degree. C. for 6 hours. 
A plasmid containing a portion of bovine prolactin cDNA (pBPRL27) was 
radio-labelled by nick-translation. See e.g. P. Rigby et al., 113 J. Mol 
Biol. 237-251 (1977). pBPRL27 is deposited with the American Type Culture 
Collection, Rockville, Md., as ATCC No. 40574. Samples from the deposit 
are available in accordance with U.S. patent law requirements upon 
issuance of the patent and the requirements of any applicable foreign 
patent laws. No patent license is intended by such availability. Another 
plasmid cDNA which could be used for this purpose is (pBPRL72) from N. L. 
Sasavage, et al. 257 J. Biol. Chem. 678-681 (1982). 
The labelled probe was added with fresh hybridization solution and the 
incubation continued for 36 hours. Blots were washed twice with 2x SSC 
(300 mM NaCl and 30 mM Na citrate, pH 7.0) at 65.degree. C. for 15 
minutes, followed by 2x SSC and 0.1% SDS at 65.degree. C. for 30 minutes. 
The final wash was at high stringency (0.lx SSC at 65.degree. C. for 10 
minutes). Blots were exposed to Kodak XAR-5 film with intensifying screens 
for 3 to 5 days at -80.degree. C. The probe was removed according to the 
manufacturer's recommendations for multiple probing of blots. 
4. Correlation Analysis 
The resulting hybridization patterns were analyzed. Three types were 
identified. The patterns of these types (labelled AA, AB, and BB, 
respectively) are shown in FIG. 1 as possible offspring of type AB. 
Analysis of family lines shows that sons of the AA type all carry the A 
allele, and sons of the BB type are certain of not carrying the marker. 
Sons of the AB type would be a mixture of those carrying either the B from 
the sire or A from the sire. 
A statistical model was then formulated to test for differences in 
predicted genetic values for milk production traits between those carrying 
A from the sire versus those carrying B from the sire. Results of the 
analysis revealed a statistically significant higher genetic transmitting 
value for milk yield from the sons in this family who carried the A 
marker. 
FIG. 1 shows that the "most preferred" AA pattern shows fragments of about 
1.15 kb, but not one at about 1.35. The second most preferred AB pattern 
has lines at 1.35 and 1.15. The 1.15 fragment is missing in the third 
(undesired) pattern. 
Applicant has deposit a bovine sperm cell 14H9689 of type AB with the 
American Type Culture Collection, Rockville, Md., as ATCC No. 40573. 
Samples from the deposit are available in accordance with U.S. patent law 
requirements upon issuance of the patent and the requirements of any 
applicable foreign patent laws. No patent license is intended by such 
availability. It will be appreciated that one skilled in the art can use 
this "known" to confirm the location of the key fragments' hybridization 
pattern. 
It should be understood that the above description deals with a preferred 
embodiment of the invention, and that many other embodiments are within 
the scope of the invention. For example, the invention should work with 
other types of bovine cells that contain DNA (other than just sperm). In 
this regard, it should be applicable to other cells types. 
Also, while Ava II restriction fragments associated with prolactin have 
been chosen as a model system, other restriction enzymes when used with 
prolactin (or prolactin adjacent) probes may also yield characteristic 
hybridization patterns, that can be compared to knowns developed using the 
Ava II patterns. Moreover, while the primary utility of the invention is 
for Carlin-M Ivanhoe Bell progeny, the principles of the invention may 
also apply to other Holstein families. 
Also, it should be noted that the presence of the marker is a statistical 
indication of improved production. Thus, breeders will also want to 
continue to use their standard breeding techniques when this marker is 
used. This marker does not replace such techniques. It supplements them.