Iowa State University Patents

Iowa State University Research Foundation, Inc.

3-8-1994

Association of bovine mitochondrial DNA with traits of economic importance Donald C. Beitz Iowa State University, [email protected]

Albert E. Freeman Iowa State University

Michael M. Schutz Iowa State University, [email protected]

Gary L. Lindberg Iowa State University

Follow this and additional works at: http://lib.dr.iastate.edu/patents Part of the Agriculture Commons, and the Animal Sciences Commons Recommended Citation Beitz, Donald C.; Freeman, Albert E.; Schutz, Michael M.; and Lindberg, Gary L., "Association of bovine mitochondrial DNA with traits of economic importance" (1994). Iowa State University Patents. Paper 112. http://lib.dr.iastate.edu/patents/112

This Patent is brought to you for free and open access by the Iowa State University Research Foundation, Inc. at Digital Repository @ Iowa State University. It has been accepted for inclusion in Iowa State University Patents by an authorized administrator of Digital Repository @ Iowa State University. For more information, please contact [email protected].

Illllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll US005292639A

United States Patent [191

[11] Patent Number:

Beitz et al.

[45]

[54] ASSOCIATION OF BOVINE

Date of Patent:

5,292,639 Mar. 8, 1994

DNA From a Single Animal” Biochimica et Biophysica

MITOCHONDRIAL DNA WITH TRAITS OF ECONOMIC IMPORTANCE

Acta, 565 (1979) 22-32. King, et al. “Mapping of Control Elements in the Dis

[75] Inventors: Donald C. Beitz; Albert E. Freeman, both of Ames, Iowa; Michael M. Schutz, Laurel, Md.; Gary L. Lindberg, Ames, Iowa

placement Loop Region of Bovine Mitochondrial DNA” The Journal of Biological Chemistry, vol. 262,

[73] Assignee: Iowa State University Research Foundation, Inc., Ames, Iowa

[21] Appl. No.: 872,265 [22] Filed:

Apr. 24, 1992

Continuation-impart of Ser. No. 398,898, Aug. 28, 1989.

[51]

Int. Cl.5 ...................... .. C12Q 1/08; C12P 19/34;

[52]

US. Cl. ..................................... .. 435/6; 536/ 18.7;

[58]

Field of Search ........................ .. 435/6, 172.3, 91;

C07_H 5/04; C07H 17/00

536/23.1; 536/24.3; 536/25.4; 935/78 436/501, 614; 536/27, 18.7, 23.1, 24.3, 25.4; 935/3, 9, 63, 78

[56]

References Cited

PUBLICATIONS Laipis, et al. “Unequal Partitioning of Bovine Mito

chondrial Genotypes Among Siblings” Proc. Natl. Acad. Sci. U.S.A., vol. 85 pp. 8107-8110, Nov. 1988.

Anderson, et al. “Complete Sequence of Bovine Mito chondrial DNA”, J. Mol. Biol. 156, 683-717 (1982).

Ashley, et al. “Rapid Segregation of Heteroplasmic Bovine Mitochondria” Nucleic Acids Research, vol. 17, No. 18, pp. 7325-7331 (1989).

Structure Depends on Growth State in Bovine Cells”

The Journal ofBiological Chemistry vol. 262, No. 13, pp. 6214-6220 (1987). Sanger, et al. “DNA Sequencing With Chain-Ter minating Inhibitors” Proc. Natl. Acad. Sci USA, vol. 74, No. 12, pp. 5463-5467 (1977).

Related US. Application Data [63]

No. 13, pp. 6204-6213 (1987). King et al., “Mitochondrial DNA Displacement Loop

,

Hauswirth, et al. “Heterogeneous Mitochondrial DNA D-Loop Sequences in Bovine Tissue” Cell, vol. 37,

1001-1007 (1984). Laipis, et al. “A Physical Map of Bovine Mitochondrial

Anderson et al. J. Mol. Biol. 156:683 (1982).

Lapiz et al. Biochem. Bioph. Act. 565:20 (1979). Lapiz et al. P.N.A.S. 85:8107 (1988). King et al. J. of Biol. Chem.: 262 (13) 6214 (1987). King et al. J. of Biol. Chem. 262(13): 6204 (1987). Ashley et al. Nucleic Acids Res. 17(18):7325 (1989). Primary Examiner-Christopher S. F. Low Assistant Examiner-Miguel Escallon Attorney, Agent, or Firm-Zarley, McKee, Thomte, Voorhees & Sease

[57] ABSTRACT A method of genetically evaluating animals by using mitochondrial DNA is disclosed. Polymorphisms in mitochondrial DNA are detected by isolating, frag menting, and sequencing the DNA. The restriction patterns and nucleotide sequences of mitochondrial DNA of different animals are correlated to expressed traits in the animals. This may be con?rmed by compar ing results to expression of the trait in maternal lineages of animals. Further, effects of maternal lineages are

determined by partitioning maternal genetic variation from nuclear variation.

16 Claims, No Drawings

1

5,292,639

2

ASSOCIATION OF BOVINE MITOCl-IONDRIAL DNA WITH TRAITS OF ECONOMIC IMPORTANCE

DNA has been developed so that fragments in the mito chondrial DNA may be associated with phenotypic expression as a particular trait in the animal. This dis covery has been applied speci?cally to a herd of dairy

CROSS REFERENCE TO A RELATED APPLICATION

cattle by associating the presence of particular nucleo tide substitutions in the D-loop region of mitochondrial DNA (mtDNA) with increased volume productions of

This application is a continuation-in-part of an earlier

milk and with greater milkfat content. Other associa

?led, commonly assigned application, Beitz et al, Ser. 1 o tions are also made for important economic traits such No. 07/398,898, ?led Aug. 28, 1989 and entitled, as days open and certain health traits such as mastitis METHOD OF GENETIC EVALUATION OF ANI MALS BY USING MlTOCI-IONDRIAL DNA.

BACKGROUND OF THE INVENTION In genetically evaluating animals, researchers use theory based on nuclear genes. Higher organisms have a nucleus within their cells that contains nuclear genetic material. This has been the source of genetic material

and reproductive problems. Mitochondrial lineage in?uences on health differ ences of cattle have not been examined. Much work associating human diseases to mitochondrial DNA se

quence differences has been reported. Keams-Sayres Syndrome (K88) and Leber’s hereditary optic neuropa thy (LHON) are examples of such diseases. In fact,

for scientists in analyzing genetic background and in

LHON has been shown to be correlated with a single

elles within the cells that have their own genetic makeup, one of which includes the mitochondria with

histidine in NADH dehydrogenase subunit 4 gene of mtDNA. Such mtDNA nucleotide sequence substitu tions may affect health and other production traits in

heritance of animals. However, there are other organ 20 guanine-adenine transition that converts an arginine to a its own DNA.

cattle. Until now, it was widely held that essentially no Molecular variation in bovine mtDNA has been dem variation existed in mitochondrial DNA. This invention 25 relates to the ?nding that there is variation, or polymor onstrated through RFLP analysis and comparison of

phism, within mitochondrial DNA, and these polymor

nucleotide sequences. Displacement loop (D-loop) se

phisms may be used to evaluate inheritable traits of quences of mtDNA from 36 distinct registered maternal animals. lineages available for this study were previously com The use of mitochondrial DNA is a signi?cant ad 30 pared Lindberg, G. L. 1989. Sequence heterogenity of vancement in the science of animal breeding. Current bovine mitochondrial DNA. Ph.D Dissertation, Iowa genetic improvement programs are based solely on use State University, Lib. Ames. University Micro?lm of nuclear genes. Classical animal breeding methods order No. DA-90l4925. Ann Arbor, Mich. Fifty-one have been developed ?rst by using mass selection and sequence differences were located, including 48 single then by progressing to evaluation of lineages, pedigrees,

selection index, and best linear unbiased prediction methods. Progeny testing is the breeding method used most often in cattle. All methods are based on use of

base pair (bp) substitutions, one 9-bp deletion, and two variable length poly G-C runs. Where possible, D-loops

from two or more animals of the same maternal lineage

were sequenced to verify accuracy of mtDNA isolation nuclear genetic material. Mitochondrial genes are inher ited maternally. That is, they are transmitted from a 40 and nucleotide sequencing and to con?rm constancy of mtDNA within maternal lineages, supra. female to both sexes of progeny; males do not transmit The D-loop region of mtDNA does not code for any mitochondrial genes to their progeny. Thus, in a prog known gene products; hence, sequence polymorphisms eny test, bull mothers pass their maternal genetic mate rial to their sons, but these sons do not transmit this there would not alter speci?c protein chain subunits.

genetic material to their daughters. Both nuclear and 45 Promoters for transcription of both heavy and light mitochondrial genetic material contributes to the pro strands of mtDNA as well as the origin of heavy strand duction records of bull mothers, but, because only nu replication, however, lie within the D-loop. Thus, se clear material is transmitted through their sons to his daughters, the bull mothers may be over- or under

evaluated, depending upon the bull mother’s mitochon drial contribution. If the magnitude of the cytoplasmic or mitochondrial effect is known, the pedigree estimate of the bull mother’s contribution can be adjusted appro

priately. Milk volume and percentage of milk fat and milk 55

protein (two constituents of milk, along with lactose and minerals) are economically the most important traits of dairy cattle. Reproduction and health of cattle, however, merit attention as well, because the interval between successive calvings and health costs also deter mine pro?tability of dairy cows. Effects of cytoplasmic

quence differences in the mtDNA D-loop may alter transcription or replication rates. Moreover, such D loop polymorphisms may serve as markers of differ ences elsewhere on the mtDNA genome in coding re

gions of genes that are associated with phenotypic ex

pression of traits. The primary objects of the invention are, therefore, to evaluate animals genetically 'by using their mitochon drial DNA by phenotypic analyses of differences be tween maternal lineages. Another object of the invention is to use polymor phism in mitochondrial DNA of animals to associate the absence or presence of single or several markers with

inheritance on reproductive measures have been shown

traits of economic importance including health trait

for number of days open (days from calving to next conception), days from calving to ?rst detected estrus,

importance in animals.

and preparing speci?c fragments of the mitochondrial

nomic and health importance.

'

A further object of the invention is to determine the - ?rst service conception rate, and number of services. 65 sequence of nucleotides in mitochondrial DNA frag ments and associate presence or absence of speci?c It has now been found that there are polymorphisms nucleotide sequences of fragments with traits of eco in mitochondrial DNA and that a method of isolating

5,292,639

3

4

A still further object is to associate polymorphism in mitochondrial DNA with its expression in the maternal lineage of an animal.

mitochondrial DNA from each animal. Cloning in

A further object is to provide for improved genetic evaluation of animals by phenotypically determining

cally this segment to a vector by commonly used meth ods such as those described by Hackett, et al., P. B., Fuchs, J. A., and Messing, J. W. (An Introduction to

volves segmenting the mitochondrial DNA by using restriction endonucleases and then ligating enzymati

the relative worth of maternal lineages for economic traits. Another object of the invention is to predictably isolate ef?cient milk producers and lineages of milk

Recombinant DNA Techniques. (Benjamin/Cummings

producers from dairy herds by evaluation of polymor phism in mitochondrial DNA. Other objects of the invention will become apparent in the following disclosure.

cells for propagation and ampli?cation of the recombi nant plasmid. This will yield an amount of the targeted mitochondrial DNA sufficient for restriction fragment

Publishing Co., 2nd Ed. 1988). The recombinant con structs then are introduced into the Escherichia coli

length polymorphism analysis.

SUMMARY OF THE INVENTION Another method used in one laboratory for ampli?ca 15 tion of mitochondrial DNA is by the polymerase chain The invention relates to the use of cytoplasmic ge

netic material, speci?cally the mitochondrial DNA, to

reaction (PCR). We have isolated, ampli?ed by the

genetically evaluate an animal. The mitochondrial -DNA of an animal is isolated, fragmented and se

the PCR-ampli?ed mitochondrial DNA. Even though

PCR, and then determined the nucleotide sequence of

quenced, and polymorphism within the mitochondrial 20 this procedure allows one to obtain nucleotide sequence data, only the previously described cloning procedure DNA is detected. This polymorphism is then correlated allows one to permanently store the mitochondrial DNA from test animals.

with the presence or absence of a desired trait. This

relationship may be further con?rmed by comparing the lineage of animals showing polymorphisms with lineages not showing the polymorphisms. Additionally, values of maternal lineages can be determined on a

phenotypic basis to improve accuracy of bull mother selection and choice of females as donors for embryo

transplants. DETAILED DESCRIPTION OF THE INVENTION Mitochondria are the source of cytoplasmic genetic information. Mitochondria carry multiple copies of a circular genome that is replicated and expressed within

Following cloning, or using directly isolated mito 25

chondrial DNA, the mitochondrial DNA then is frag

mented. One method by which this may occur is by digesting the mitochondrial DNA with selected restric tion enzymes. A number of different kind of enzymes are employed to obtain an adequate screening. These 30 enzymes will fragment the DNA at particular selected

sites, thereby resulting in fragments of known compo nents. The restriction endonucleases are chosen to cause

multiple cleavages within the target DNA and to create

an optimal population of differently sized cleavage frag ments capable of being resolved by gel electrophoresis.

the organelle and is inherited maternally. It is the only

The number of restriction endonucleases employed will

genetic element known to be inherited cytoplasmically

vary depending upon the size of the population to be in mammals. Mitochondrial DNA in animals codes for screened and, in the example, below reflects the use of 13 polypeptides that have been identi?ed as components of the ATP synthesis system and codes for all of the 40 at least 10 restriction endonucleases. Restriction digests are conducted according to the speci?cation of the transfer RNAs used in mitochondrial protein synthesis. supplier. The use of restriction endonucleases to mea The only non-coding portion of the genome is a region of approximately 900 base pairs that is referred to as the sure mitochondrial DNA sequence relatedness is

displacement loop or "D-loop”. This D-loop is in volved in the control of transcription and replication of

45

known and has been discussed, for example, by Avise, J. C., Lansman, R. A., and Shade, R.0., "The Use of Re

mitochondrial DNA. Variation in mitochondrial DNA will affect the size of the mitochondrial DNA popula tion in the cell, abundance of mitochondrial DNA gene

striction Endonucleases to Measure Mitochondrial

transcripts and translation products, and mitochondrial oxidative energy transduction capacity.

donucleases that are useful in characterizing bovine

As discussed more fully below, this invention in

volves the discovery that polymorphisms within the mitochondrial DNA of mammals can be associated with expressed traits in mammals.

,

DNA Sequence Relatedness to Natural Populations”, Genetics 92: 279 (1979). Examples of restriction en mitochondria are listed below. These are only exam

ples; other endonucleases can be used. In general, these will produce between 5 and 25 fragments that are larger

than 100 base pairs. See. e.g., Anderson, S., deBruyjn,

Mitochondrial DNA is ?rst obtained from the blood 55 M. H., Coulson, A. R., Eperon, I. C., Sanger, F. and Young, I. G., J. Mol. Biol., 156:683 (1982). Although by of the animal. A variety of methods exist for isolating using 20 different enzymes, a screen could be extended mitochondrial DNA, such as Lindberg, G. L., Koehler, to cover about 220 recognition sites, far less extensive C. M., May?eld, J. M., Myers, A. M., and Beitz, D. C. screens such as those discussed below in the example 1992 Biochem. Genet. 30:27, and incorporated herein by reference. One procedure is described in more detail 60 may be employed with satisfactory results. in the example below. TABLE 1 After isolating the mitochondrial DNA, it then can be Restriction Endonucleases

cloned, and typically this is done in Escherichia coli Accl cells. This cloning must be done because the quantity of DNA required is usually more than can be practically 65 Ahalll Banll collected from blood samples, and also because individ Bbvl ual dairy cattle are not maintained permanently in the Bsrnl herd. This cloning allows for a permanent source of

EcoRII

(10)‘ (l l) (12) (10) (1o) (9)

Fokl Haelll Hincll Hpall l-lphl Sau3A

(21) (29) (1o) (16) (19) (l9)

Sau96 Seal ScrFl SfaNl Spel SspI

(l2) (9) (l4) (2 l)

(s) (l4)

Stul (9) Styl (l0) Taql (21) Tthl ll (24) Xbal (a) Shol (5)

5,292,639

5 Restriction Endonucleases

Fnu4Hl

6

1308 kg of milk per cow per lactation when these data were analyzed.

TABLE l-continued

Ancestral pedigrees of registered foundation females

(15)

in the herd were tracked backward through the H01

‘The number in parenthts'u is the number of expected fragments from the complete mtDNA that will be larger than 100 base pairs.

stein-Friesian Herd Book. Eighty-one distinct maternal lineages were de?ned by convergence of maternal pedi grees after 1885. It is possible that these lineages would have been found to converge to fewer lineages had

Fragments resulting from digestion with restriction endonucleases are then subjected to agarose gel electro phoresis. This process is widely known to those skilled in molecular biology. Brie?y, the fragments are ?rst

registration records been kept prior to importation of these cows from Europe in about 1885.

Thirty-six maternal lineages had surviving members

end-labeled with radioactive nucleotides and then are

in the herd when samples for nucleotide sequencing

placed in a gel of a substance which provides a homoge nous matrix, for example, agarose or polyacrylamide.

were collected. These lineages had from one to six

purchased foundation females in the herd. Nucleotide

The gel is permeated with a buffered aqueous solution, and the fragments move in an electrical ?eld towards

sequence polymorphism data were obtained and all

the positive pole, depending on their net charge. This analysis will reveal nucleotide sequence heterogenity at random sites throughout the bovine mitochondrial

cows within the same maternal lineage that were ever in the herd were assumed to have identical mtDNA. Be

DNA. This is useful in analysis of the mitochondrial DNA.

.

cause of the among-lineage emphasis of the comparisons in the clonal nature of propagation of mitochondrial

20 DNA, each lineage was represented by at least two

Any observed polymorphism can be veri?ed by se

quencing the nucleotides involved in the polymorphism and observing sequence heterogeneity. Sequencing nu cleotides of DNA can be performed by the dideoxy method described by Sanger, et al. 1977. [Spanger, F., S. Nicklen, and A. R. Coulson. “DNA Sequencing With Chain-Terminating Inhibitors”, Proc. Natl. Sci.

cows.

Isolation of Mitochondrial DNA Total leukocytes were isolated from 400 ml of antico

agulated blood, obtained by jugular venipuncture, by low-speed centrifugation after erythrocyte lysis with

140 mM ammonium chloride, pH 7.4. Leukocytes were lysed with 1% wt/vol Triton X-lOO in the presence of polymorphism then is compared with the phenotypic 1% wt/vol sodium dodecyl sulfate. Nuclei and cell expression of a trait. Obviously, there are many meth 30 membranes were separated by the cytosolic fraction by ods for associating a particular trait with the sequenced centrifugation at 12,000Xg for 5 min. Soluble proteins DNA. The concept is to compare the maternal lines were extracted from the resulting supernatant with 1:1 that contain or do not contain the particular genetic phenolzchloroform, and nucleic acids were precipitated marker. If, for example, of 38 cow lines analyzed, 28 from the aqueous phase with 3 vol. ethanol. This proce have the marker and the rest do not, one would then dure yielded a mixture of supercoiled and relaxed cova attempt to associate a particular trait with the presence lently closed and nicked circular mitochondrial DNA or absence of that marker. This is then con?rmed by molecules, RNA and only trace amounts of nuclear USA. 74:5463 (1977)]. The presence or absence of a

looking at the phenotypic expression of the various

lines. An animal model, which is a mixed linear model, is 40

DNA contaminants (Koehler et al., 1988 as cited ear

lier).

used to estimate the effects due to maternal lineages.

Selection and Cloning The region of the mitochondrial DNA molecule that dom (not repeatable) and are simultaneously estimated. includes the D-loop, which is markedly variable across The ?xed effects have properties of Best Linear Unbi mammalian species and in which mutations had been 45 ased Estimates, and the random effects are Best Linear detected previously in Holstein cattle, was chosen as Unbiased Predictions. In this case maternal lineages the target of the screen. The segment of the mitochon have been classified as ?xed when lineages were deter drial DNA molecule from the PstI restriction endonu mined by tracing cows to their origin in the Holstein, clease cleavage site at nucleotide (nt) 15,738 to the SstI Friesian Herdbook of America, and also classi?ed mo 50 cleavage site at at 3,684 was excised and ligated enzy lecular polymorphism or sequences as ?xed. matically into the multiple cloning site of the 3.2 kilo DESCRIPTION OF EXPERIMENTAL ANIMALS base phagemid cloning vector pUCll8. This segment constituted one fourth of the mitochondrial DNA mole Cows in this study were from a selection experiment cule. The recombinant insert DNA was 4,284 base pairs founded at Iowa State University in 1968. Heifers for (bp) long and included a 52-bp tRNAP'o gene, the 910 this herd were purchased form 38 Holstein breeders bp D-loop region, the 66-bpPm gene, the 954-bp 12S throughout Iowa to keep the herd as genetically broad rRNA gene, the 66-bp tRNAVaI gene, the 1,570-bp 16S based as possible. Cows were bred arti?cially to sires rRNA gene, the 74-bp tRNALe" gene and a 592-bp from commercial arti?cial insemination organizations, portion of the ?rst mitochondrially coded subunit of the allowing a continuous in?ux of nuclear genes. Frequen cies of bovine lymphocyte antigen phenotypes were 60 nicotinamide adenine dinucleotide dehydrogenase com plex (ND 1 gene). similar to frequencies in the US Holstein population, Recombinant constructs were introduced into Esch meaning that these nuclear genes are likely representa erichia coli TG-l cells (K12; hsdD5, supE, thi, [lac tive of the entire US. Holstein population. Females Effects are classi?ed as either ?xed (repeatable) or ran

pro]/f:traD36, proA+B+, lacI‘I, lacZ M15) by the were assigned to groups and arti?cially mated to bulls with either high or average estimated additive genetic 65 calcium-heat shock method (Mandel, M. J. and Higa, transmitting ability for milk yield. Females born in each

A., “Calcium Dependent Bacteriophage DNA Infec

group were mated to new bulls chosen for that group,

tion,” J. Mol. Biol, 53:154 (1970)). Transformation was con?rmed by insertional inactivation of vector alpha

thus forming divergent selection lines that differed by

5,292,639

7 complementation

of

isopropyl-B-D-thiogalac

8 TABLE 3-continued

tropyranoside-induced B-galactosidase activity in am

picillin-resistant colonies. Ampli?ed recombinant plas

Traitl

mids were isolated from 50-rnl Luria broth cultures of

FAT %

transformed bacteria by alkaline lysis (Birnboim, H. C. and Doly, J., “A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucleic

P > F .001

‘For lbbt'tvillitlll, is: Table 2.

EXAMPLES OF RELATING MOLECULAR EVIDENCE TO TRAITS OF ECONOMIC IMPORTANCE Table 4 has location, type, and frequency of the 17

Acids Res., 7:1513 (1979)) to yield an amount of the target mitochondrial DNA sufficient for restriction

fragment length polymorphism (RFLP) analyses. EXAMPLE OF ESTIMATION OF MATERNAL LINEAGE EFFECTS

most common sequence polymorphisms of the mtDNA

D-loop in this herd of Holstein dairy cattle. Only those polymorphisms occurring in at least 4 percent of cows

' Cows in our reaeach herd were traced by maternal

in the herd are listed, because information on markers occurring in a very small number of cows would not be

lineages'to foundation femals in the Holstein-Friesian I-Ierdbook to establish maternal lineages. Solution for maternal lineages were obtained from the following animal model.

F L82

statistically informative. Transitions at bp 169 and 216 occurred in 80 and 84 percent of cattle, respectively. The probable explanation is that the cow originally 20 sequenced, Anderson, S., M. H. L. deBruijn, A. R. Coulson, I. C. Eperow, F. Sanger, and I. G. Young 1982, Complete sequence of bovine mitochondrial DNA. J. Mol. Biol. 156:683, had the rarer genotype at those two sites. From 30 to 608 cows were polymorphic

Effects in the model are as previously de?ned except

L1 is the "high” or "average" sire selection line; 25 at individual bp sites of the least and most frequent G(L)m:] is the mth sire birthyear group nested in the 1th mtDNA D-loop sequence difference, respectively. selection line; PE,I is the permanent environmental ef Production records of all cows in the 36 maternal fect of the nth animal with a record; and A,I is composed lineages with known mtDNA D-loop sequences were of breeding values of sire and dam and a segregation considered. Milk and fat yield records adjusted to a effect and is the additive genetic effect of the nth ani 30 uniform age and lactation length, or mature equivalent mal. (ME), basis were obtained. Percentages in milk of fat Because animal models are usually large, conven and solids-not-fat (SNF) which is total solids in milk less tional tests of signi?cance, requiring elements of vari fat in milk, were known for each record of each cow. ance-covariance matrices and, hence,'direct inversion, Up to seven production records were used for individ are often not feasible. A test of signi?cance using mixed 35 ual cows. mode] conjugate normal equations was developed to Because mitochondria play an extensive role in en test signi?cance of maternal lineage effects. Variance ergy metabolism, mtDNA polymorphism may alter ratios for random effects in the model were calculated energy content in milk. Fat, protein, and lactose are the from results of our variance component estimation. carriers of energy in milk; however, information was Maternal lineages were evaluated as ?xed effects in this 4-0 complete since i968 only for fat and solids-not-fat,

animal model including random animal and permanent environment effects. Ranges of maternal lineage esti mates were 2934 kg milk, 154 kg fat, and 0.907 percent

which combines protein, lactose, and minerals. Net energy concentration in milk, which is based on fat and SNF, was calculated in terms of kilocalories per kilo

fat. (Table 1)Matemal lineages signi?cantly affected kg fat and fat percentage. (Table 3) Maternal lineages also

45

affected calculated net energy of milk, but were not

megacalories.

apparently important for solids-not-fat yield or concen

To evaluate the effect of mtDNA D-loop sequence

tration. (not shown in Tables)

polymorphism, each cow was assigned a value of l if polymorphic or 0 if not polymorphic with respect to the ?rst published mtDNA sequence at each of the 17 loca

TABLE 2 Ranges of estimates or solutions of maternal

tions considered. Each production trait was analyzed individually with the following animal model:

lineage effects from animal model analysis Maternal

Lineage Range

Trait MEMILKZ MEFAT3 FAT %‘

‘7,1

2934 154

55

1514 59

.907

.393

where Yij-knp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record; it is an overall mean; YS,~ is the effect common to all cows calving in year-season; X); is the effect com mon to cows in either the high or average selection line;

‘with herd pbenotypic mime deviation.

111mm quivlk‘nl, SOS-day milk yield. ‘Mature equivalent, JOS-day milk in yield. ‘Milk fat percentage.

TABLE 3 Tim‘

gram of milk and multiplied by lactation milk yield to approximate lactation energy production in terms of

F

P > F

Fixed

MEMILK

1.17

.1811

sire

MEFAT

1.47

.017

groups Westell groups

FAT % MEMILK MEFAT

1.71 1.09 1.39

.(XJl .308 .035

31 to [317 are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms; PE,I is permanent environmental effect common to all 65 records of cow ii; an is effect of animal n and is com

posed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect; and e; is a random residual.

5,292,639

9

10

Regression of production traits on mtDNA D-loop polymorphism was of primary interest. Effects of the overall mean, year-season of calving, parity, and selec

whether effects of D-loop polymorphisms were speci?c

tion line were treated as ?xed effects in the mixed model

sequence polymorphism being present differs among bp

to account for explainable environmental background. Additive genetic covariances among related individuals were incorporated in this model. Permanent environ

sites was tested versus the null hypothesis that the effect of the presence of a polymorphism at one site equals the effect at any other site. Effects of presence of D-loop polymorphism at different sites were signi?cant for milk

An overall test of signi?cance was used to determine

for individual locations. That the effect of a D-loop

ment and additive genetic effects were treated as ran

dom and have properties of Best Linear Unbiased Pre

fat yield (P >F 1 = .05 +1’ > t a .10.

more and 30.9 kg less fat included In their phenotypic estimates. Thus, bull mothers would have been over and

under estimated, respectively.

TABLE 6 Regression of reproduction and health costs on sequence

40

polymorphisms in the mtDNA D-Loop and overall

The dam to cow pathway has traditionally been se

means of reproduction and health cost means in a herd of dairy cattle

lected least intensely. However, new developments in

reproductive technology and embryo manipulation

ReproLocation in

seem poised to make this pathway of selection more

viable. Differences in mtDNA could be incorporated 45 D-LOOP into embryo transfer breeding programs to better

choose donor and recipient females to produce replace ment heifers. Current cloning techniques require nu clear transplantation into an enucleated ovum without

regard to cytoplasmic content. Potential exists for using 50 mtDNA sequence polymorphism to identify ova of

females with inferior nuclear genetics in superior mtDNA background as candidates for enucleation and a subsequent introduction of nuclei with greater genetic

potential.

55 TABLE 4 location, type, and frequency of seventeen most common sequence polymorphisms of mtDNA D—loops in a herd of dairy cattle

Location in

polymorphic

D~Loop“

event

8 106 169 216

GA T-C AG Var. length

Fre-

quency in D-Loop‘

06' G~A

16247

Overall mean”

Poly morphic

Fre

event

quency

.07 .14 .80 .84

16058 16074 16085 16111

C-T T-C T-C A-C'

.12 .07 .05 .04

16113

T-C

.11

.46 .14

16141 16230

T-C GT

.11 .06

G~C run

363 16022

Location

8 106 169 216 16022 16049 16057 16058 16074 16085 16111 16113 16141 16230 16231

Days open

(d) -31.7 0.5 14.6‘'' —0.5 —13.4 16.1 9.7 28.3 + 3.7 -36.3' —14.1 —5.5 —11.7 —4.4 —16.0 — 8.2

135 ‘ (72)

Number of Breedings

(n) —.73 .13 .34 .12 —.12 .28 .14 .68 .34 —.99' —.64 —.63 —.36 —.02 —.53

duction Costs

Total Mammry Costs

Health Costs

(5)

(3)

(3)

—6.12 —2.66 6.054‘ .07 -3.89 .88 —2.90 2.44 6.28 —12.82+ —4.06 —1.64 —3.01 —2.04 -3.53

24.79+ 1614+ .84 -9.70+ 9.77 4.58 —9.31 2.94 13.56 —2.31 —8.94 1.18 —15.67'* 13.10 —4.81

10.22 21.28+ 11.47 —14.54‘'' 1.83 17.94 -15.93 18.44 25.93+ 19.70 —11.19 —2.24 —19.58 12.12 -11.30

.02

2.47

2.61 (2.24)

38.73 (34.49)

3.19

19.07 (43.8)

7.71

77.67 (60.14)

IOverall standard deviations are in paranthaaes. ‘p > t i .05. "'p > t i .10.

65

Mitochondrial DNA (mtDNA) displacement-loop (D-Loop sequence polymorphism information from 36 maternal lineages was evaluated. Of 17 base pair substi tutions evaluated, several were signi?cantly associated

13

5,292,639

14

with milk, fat, and solids-not-fat-production. Another

essentially of: a mutation event at D-loop position 8,

marked a large impact on fat percentage and net energy concentration. Positive and negative effects on all pro duction traits were observed. One base pair substitution was related to a large favorable decrease in days open,

106, 169, 216, 363, 16022, 10649, 16057, 16058, 16074, 16085, 16111, 16113, 16141, 16230, 16231, or 16247. 3. The method of claim 1 wherein said milk produc tion traits include total output of milk, fat content of milk, solids-not-fat content of milk, and energy concen tration in milk.

number of breedings, and reproductive costs. Maternal lineage groups de?ned by several methods of classi?cation using mtDNA sequence characteristics

4. The method of claim 1 wherein said reproduction ef?ciency includes number of days open and number of

’ were evaluated with animal models. Groups de?ned as

breedings.

those maternal lineages with or without base pair substi tution at nucleotide 169 accounted for increased milk fat

5. The method of claim 1 wherein the genetic D

and estimated milk energy production. Clustering the 36 maternal lineages using 17 mtDNA D-loop sequence differences produced groups with signi?cant effect on fat percentage and energy concentration. This method allows one to employ analysis of the mitochondrial DNA to predict which animals differ in traits of economic importance. Thus, for example a

Loop mitochondrial marker comprises the presence of one or more polymorphisms, said polymorphisms se

lected from the group consisting essentially of: an ade 15 nine at position 8, a cytosine at position 106, a guanine at position 169, a guanine at position 363, an adenine at position 16022, a thymine at position 16049, an adenine at position 16057, a thymine at position 16058, a cyto sine at position 16074, a cytosine at position 16085, a single Adenine to Guanine transition at bp 169 relates to increased milk production as measured by volume and 20 cytosine at position 16111, a cytosine at position 16113, a cytosine at position 16141, a thymine at position lowered milk fat; polymorphism at 16074 has a large 16230, a thymine at position 16231, and a thymine at positive effect on milk, fat and SNF; polymorphism at position 16247. bp 16058 is evidence of increased days open and repro 6. The method of claim 1 wherein said marker is a ductive costs. This provides a new source of genetic material to use in evaluating inheritance in an animal. 25 guanine at position 169 and is indicative of increased Mitochondrial in?uences on a dam’s production are not

milk production.

transmitted through her male offspring to his progeny, and such cytoplasmic effects currently are included in an estimate of the dam’s transmitting ability, which may

cytosine at position 16074 and is indicative of decreased milk production, solids-non-fat yield, and lactation en

7. The method of claim 1 wherein said marker is a

cause the dam’s transmitting ability to be over- or un 30 ergy and lowered fat content. 8. The method of claim 1 wherein said marker is a

der-estimated for the purpose of predicting her son’s progeny test. Knowledge of the dam’s maternal effect

thymine at position 16231 and is indicative of decreased milk fat content, solids-non-fat yield, and lactation en

allows adjusting her transmitting ability to accurately

ergy. 9. The method of claim 1 wherein said marker is

predict her son’s progeny test. This method provides a

basis for exploitation of potential gains from selection

selected from the group consisting of: a thymine at position 16058, an adenine at position 16085, a thymine at position 16230, and a thymine at position at 16247 and cows with good maternal traits as herd replacements. is indicative of increased fat percentage and energy Even small differences in feed ef?ciency can be ex ploited and would have major economic importance 40 concentration of milk. 10. The method of claim 1 wherein said marker is a when applied to the dairy cattle population. By way of cytosine at position 106 or position 16074 and is indica another example, feed costs are 40 to 50% of dairy tive of increased total health costs. production expenses, and even if only a 5% increase in 11. The method of claim 1 wherein said marker is a feed ef?ciency resulted from application of mitochon guanine at position 169 is indicative of increased days drial DNA to selective practices, a savings of over 3 open. billion dollars annually could be realized by the dairy 12. The method of claim 1 wherein a cytosine at industry in the United States. position 16085 is indicative of increased days open (days Other potential applications include the possibility of between calving and conception) and number of breed combining and transplanting superior mitochondrial

from more ef?cient maternal genotypes based upon

cytoplasmic differences. It is also possible to identify

DNA with superior nucleic DNA, resulting in animals which predictably will carry an inheritable desired trait,

such as high milk production. Another application is choice of cows from the best

maternal lineages (cytoplasmically or mitochondrically based), as donors for embryo transplants. The practice of embryo transplantation is used extensively in the

dairy industry. Thus, it is apparent that the invention accomplishes at least all of its objectives. What is claimed is: 1. A method of evaluating the material mitochondrial phenotypic contribution to economic traits of milk pro duction and reproduction ef?ciency of a dairy cow

50

ings to achieve conception. 13. Genetic mitochondrial D-Loop markers for milk

production and reproductive ef?ciency traits in dairy cattle, said markers comprising the group consisting essentially of: a transition at mitochondrial D-loop posi

tion 8, 106, 169, 16022, 16049, 16057, 16058, 16074, 16085, 16113, 16141, 16230, 16231, 16247, 363 or 16111. 14. The markers of claim 13 wherein said marker is associated with fat content of milk, solids-non-fat con tent of milk, total milk production, lactation energy, number of days open, number of breedings. 15. The markers of claim 13 wherein said markers occur in at least 4%.

.

16. A method of evaluating inheritable milk produc

comprising:

tion and reproduction efficiency traits in dairy cattle by

markers in the mitochondrial D-loop of said cow. 2. The method of claim 1 wherein said genetic mito chondrial marker is selected from the group consisting

clear effects, said method comprising:

assaying for the presence of one or more genetic 65 partitioning effects of mitochondrial lineages from nu

isolating mitochondrial DNA from a group of said dairy cows,

15

5,292,639

16

fragzjlmng md sequcncmg sud m'tochondnal

chondrial DNA sequences with a milk production

comparing said mitochondrial DNA of said group of dm) cows t°.kn°wn mnochondn? ‘FIRMS to

phcnotypic trait of said dairy cow, and selecting said cattle for favorable breeding features.

detect nucleotide sequence polymorphism,

5

determining the presence of said polymorphic mito-

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UNITED STATES PATENT AND TRADEMARK OFFICE

CERTIFICATE OF CORRECTION PATENT NO.

I

5, 292, 639

DATED

:

March 8, 1994

INVENTOHS) 2

Beitz et a1.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 13 line 61, cancel "material" and substitute ——maternal—— .

Signed and Sealed this

Nineteenth Day of July, 1994

BRUCE LEHMAN

Arresting Officer

Commissioner of Patents and Trademarks