CN1361788A - Genetic Marker for meat quality, growth, carcass and reproductive traits in livestock - Google Patents

Abstract

Compositions and methods are provided for identifying polymorphisms associated with growth and reproductive traits in livestock.

Description

The genetic marker of domestic animal meat, growth, trunk and reproductive characteristic

Invention field

The present invention is the detection about hereditary difference relevant with growth, body composition and reproductive characteristic in the domestic animal generally.Clearer and more definite, the invention provides composition and the method for predicting some hereditary property relevant with metabolism with the steroid biosynthesizing.

Background of invention

In order to describe the situation in the relevant field of the present invention more fully, in this application, quote some works as proof, and in bracket, drawn the author's name, deliver time and magazine.The content of these works is hereby incorporated by.

Steroid hormone plays epochmaking effect in differentiation, growth, growth and the physiological function of most of animal tissuess.In all steroid hormone biosynthesizing, first and be that the step of speed limit is with cholesterol side chain nickase p450scc cholesterol to be converted into Vitarrine.The gene of coding P450scc is called CYP11a1.Cytochrome P450 is that one group of different monooxygenase that contains protoheme (is called CYP ' s; See Nelson etc., DNA Cell Biol. (1993) 12:1-51), the multiple oxidizing reaction of catalysis mainly is a steroid, but also comprises lipid acid and xenobiotic.CYP ' s is the richest at testis, ovary, placenta, suprarenal gland and liver expression.The most important steroid hormone that generates in these reproductive organ of testis, ovary and placenta is male sex hormone (as a testosterone), oestrogenic hormon (as estradiol) and progestogen (as progesterone).Most important steroid is mineralocorticoid (as aldosterone) and glucocorticosteroid (as hydrocortisone) in the suprarenal gland.

The boar meat that boils often has a kind of not good smell and unhappy taste, is called " pig stink smell " or " pig peculiar smell " usually, and this is to cause the major cause of extensively carrying out castrating in the production of pig.Cause a kind of important compound of pig peculiar smell, 5 α-androstenone (5 α-androstane-16 alkene-3 ketone) are synthetic in the testis of boar with other 16-androstene steroid, male sex hormone and oestrogenic hormon.During pubescence, Δ 16-androstene, the particularly 5 α-generation of androstenone (androstenone) in testis sharply increases.This causes accumulating of parts of body androstenone, particularly is accumulated in the lipid reserve and the submandibular salivary gland (SMG) of whole body, and the latter is contained the specific combination albumen of Δ 16-androstene.The concentration height correlation of Δ 16-androstene in the concentration of androstenone and other Δ 16-androstene and the fat among the SMG.Measure the Δ 16-androstene among the SMG, in fact as the measuring method that determines whether to exist the pig peculiar smell.Therefore, because this increase of Δ 16-androstene is castrated so that the peculiar smell in the meat drops to minimum young boar usually industrial.But, if can overcome the problem of pig peculiar smell, raise boar than raising hog economy many.Though the speed of weight increment of boar and hog is identical, the lipid content of the trunk of boar few 30%.Therefore, boar is more effective on production lean meat.In addition, boar utilizes feed ratio hog more effective (feed that the per unit body weight consumes few 10%).Because the capital cost that on behalf of live pig, feed produce, the raising boar is obtained pork and has the remarkable economical interests.

In the U.S., butcher 9,000 ten thousand pigs every year approximately, value is about hundred million.Feed is the major portion of live pig production cost, accounts for 70% of production cost.Therefore feed benefit raising 10% just can be saved 7% of total production cost., only consider male live pig in China, just can make the whole market save $3.35 hundred million.Castrate the very large economy loss that the productivity effect of losing constitutes global live pig industry because of carrying out.

Genotype and gene basis that the biosynthesizing of livestock sterol changes are identified that newborn egg product is of great use to producing high-quality meat.The purpose of this invention is to provide works and the method for identifying this hereditary change.

Summary of the invention

According to the present invention, provide the method for the evaluation hereditary change relevant with the steroid biosynthesizing.In one embodiment of the invention, whether the CYP11a1 DNA to tested object exists the polymorphism sign to measure.This class testing object is selected from important domestic animal kind, comprises pig, cow, and chicken and sheep, but be not limited thereto.According to the present invention, determine that some polymorphism of CYP11a1 gene is and increases growth that reproduction is relevant with the trunk feature.Therefore, provide some screening methods, can identify method with useful allelic tested object of CYP11a1.Can quicken to realize developing the breeding planning of the domestic animal with these improvement hereditary properties to the evaluation of this domestic animal.

Those skilled in the art knows, for the sequence difference of comparison nucleic acid molecule can adopt multiple technologies.This comprises: for instance, and restriction fragment length polymorphism analysis, heteroduple analysis, single-strand conformation polymorphism analysis, denatured gradient electrophoresis, and temperature gradient electrophoresis.

In a preferred embodiment of the invention, the CYP11a1 polymorphism is a kind of pvuii restriction fragment, and its mensuration comprises: identify the CYP11a1 gene in the separating obtained genetic material of tested object; Place the effect of a Restriction Enzyme to produce the restriction fragment of the different lengths of this gene down this gene; Form restricted pattern with electrophoresis or high pressure liquid chromatography (HPLC) separation limit fragment, relatively one be known as the given restriction fragment pattern of tested object CYP11a1 gene that has or do not have the expection sign.If the test of the mark of tested object is positive, such object can be considered to be included in raising in the works.If the marker gene type test result of tested object is not positive, other purposes can be rejected or be used for to this object from group so.

In a particularly preferred embodiment, tested object is a pig, and polymorphism is in 5 ' UTR of CYP11a1 gene, and Restriction Enzyme is SphI.Therefore, in this respect, one object of the present invention that Here it is: the method for a screening pig is provided, and testicular weight is low most probably in raising, the pig peculiar smell light, trunk is longer to determine which pig, rate of body weight gain improves, or weanling weight is higher, or selects to remove other pigs, and the allelotrope that these pigs have shows that testicular volume is bigger, the pig peculiar smell is strong, carcass length lowers, and rate of body weight gain is low, or weanling weight is lighter.The implication of " less testicular volume " described herein is the average that testicular volume is markedly inferior to certain colony.The implication of " the pig peculiar smell is light " described herein is the mean level (ML) that the pig peculiar smell significantly is lower than certain colony.The implication of " carcass length increase " described herein is that the mean level (ML) of the certain colony of carcass length has remarkable increase.The implication of " rate of body weight gain is higher " described herein is a rate of body weight gain significantly to be increased than the average of certain colony." heavier weanling weight " described herein implication is the average that weanling weight is higher than certain colony.Method of the present invention may further comprise the steps: the sample of 1) obtaining a genomic dna from pig on one's body; And 2) which kind of CYP11a1 allelotrope the analysis 1) genomic dna that is obtained determines to exist.In brief, obtain the sample of a genetic material on one's body from pig, analyze this sample then, determine whether there is a kind of polymorphism in the CYP11a1 gene, this polymorphism is and alleviates the pig peculiar smell, less testicular volume, and carcass length increases, higher rate of body weight gain, with and/or to increase weanling weight relevant.

In a most preferred embodiment, use primer and archaeal dna polymerase to be increased in the specific region of containing polymorphism in the gene, separate this gene.Then amplification region is separated with Restriction Enzyme digestion and to fragment.To fragment dyeing, or be marked at the primer or the ribonucleoside triphosphote that use in the amplification and just can manifest the RFLP image.

In another embodiment, the present invention includes a kind of method, be used for identifying genetic marker at a special group boar peculiar smell, testicular volume, carcass length, rate of body weight gain and/or weanling weight.Breed the male and female pigs of same kind or Hybrid or similar descending line, measure its feature, as the boar peculiar smell, testicular volume, carcass length, rate of body weight gain and/or weanling weight.Determine the polymorphism and related with features such as boar peculiar smell, testicular volume, carcass length, rate of body weight gain and/or weanling weights of the CYP11a1 gene of every pig.Preferably measure polymorphism with rflp analysis, most preferably: DNA cuts the restriction enzyme of restriction site with restriction enzyme SphI or other variantly.

Also provide certain methods, the linkage relationship between the allelotrope that the relevant DNA of the special allelotrope that can determine interchangeable DNA sign and the known specific gene relevant with certain feature (as the CYP11a1 gene of discussion herein) indicates.So just, can select pig indirectly, promptly select to have light boar peculiar smell probably, than micro-orchidia, carcass length increases, high rate of body weight gain, and/or the pig of higher weanling weight, or select removal may have high boar peculiar smell, big testis, short, the low rate of body weight gain of carcass length and/or the low pig of weanling weight, its method be by select to be positioned at the same karyomit(e) of CYP11a1 on the special allelotrope of replaceable sign, select some allelotrope with the CYP11a1 associated flag.

The present invention also comprises some test kits, be used for evaluation test object DNA sample and whether have the expection sign that is arranged in tested object CYP11a1 gene with the genetic material of understanding tested object, these signs can be indicated following hereditary feature, as boar peculiar smell (for pig), the testis size, carcass length, rate of body weight gain, and/or weanling weight.At least, when being tested object with the pig, test kit contains the reagent that can identify pig CYP11a1 gene pleiomorphism of one or more.Preferably, this reagent is the segmental Oligonucleolide primers that one group of pig CYP11a1 gene that can increase contains polymorphism.More preferably, test kit also comprises a kind of Restriction Enzyme that can cut at least one position of pig CYP11a1 gene.In the most preferred embodiment, Restriction Enzyme be SphI or a kind of can be at the Restriction Enzyme of this same recognition site cutting.

We provide to give a definition so that understand the present invention:

Noun " conform to " represent herein a kind of polynucleotide sequence with reference to all or part of homology of polynucleotide sequence, or a kind of peptide sequence is identical with the reference polypeptide sequence.In contrast, " complementation " implication herein is a complementary sequence and all or part of homology with reference to polynucleotide sequence.Illustrate, nucleotide sequence " TATAC " conforms to reference sequences " TATAC ", and with reference sequences " GTATA " complementation.Hybridization probe can be DNA or RNA, or can be with base special mode and the arbitrary synthetic nucleotide structure of complementary nucleic acid chains bonded.For example, comprise that the probe of peptide nucleic acid(PNA) is as mentioned below: Nielsen etc., Science 254:1497-1500 (1991).

What " chain " described is that gene, allelotrope, site or genetic marker have the tendency of heredity together owing to they are positioned on the same karyomit(e), can weigh with the recombination percent between two genes, allelotrope, site or genetic markers (also being recombination fraction or θ).Two physical locations of site on karyomit(e) are near more, and recombination fraction is just low more.Under the normal circumstances, during pleomorphism site and disease chain, recombination fraction can equal zero in measuring a Disease-causing gene, illustrate that disease and Disease-causing gene are always common hereditary.Under more rare situation, when a gene accounts for a very big sections in genome, then might observe in the pleomorphism site of gene one end and the reorganization of the other end origin cause of formation sudden change.But, if origin cause of formation sudden change is exactly tested and the chain polymorphism of disease, just can not observe reorganization.

" centimorgan " is a kind of unit of genetics distance chain between expression two genetic markers, allelotrope, gene or sites, and being equivalent in the reduction division incident in office has 1% reorganization probability between two marks or site.

The implication of " chain unbalance " or " allelotrope associating " is: special allelotrope, site, gene or a genetic marker and a preferential associating that is positioned at karyomit(e) specific allelotrope, site, gene or genetic marker nearby, its frequency is far above pressing arbitrary special allelic frequency in the colony of accidental expection.

" oligonucleotide " can be DNA or RNA, strand or double-stranded.Oligonucleotide can be naturally occurring or synthetic, but normally prepare with synthetic method.

Noun " primer " refers to the oligonucleotide that can play the starting point effect in DNA is synthetic, its condition that needs is: can induce with a nucleic acid chains complementary primer to prolong the synthetic of product, promptly exist four kinds of different nucleoside triphosphates and a kind of can polymerization reaction take place the factor (promptly, archaeal dna polymerase or reversed transcriptive enzyme), suitable buffer reagent and suitable temperature.Preferred primer is a single stranded oligonucleotide.The suitable length of a primer depends on the desired use to primer, but is generally 10～30 Nucleotide.The short primer molecule needs lower temperature so that form sufficiently stable hybridization complex with template usually.A primer needn't reflect the accurate sequence of template, can hybridize with template but must have enough complementarity.Noun " primer " indication can be more than one primer, particularly when there is ambident information the one or both ends of the target area of wanting to increase.For example, have a region list to reveal the quite polymorphism of level or sudden change in the colony, can prepare the mixture of some primers, variable sequence so just can increase.If desired, can labeled primer, mix a kind of mark that available spectrophotometry, photochemical method, biochemistry, immunochemistry or chemical process detect.For instance, useful mark comprises ³²P, fluorescence dye, the sub-reagent of cipher telegram, enzyme (as enzyme commonly used among the ELISA), vitamin H maybe can obtain the haptens and the albumen of corresponding antiserum(antisera) or monoclonal antibody.The mark zone can be used for " catching " primer, thereby helps primer or primer to prolong product, and the DNA as amplification is fixed on the solid support.

" 7 groups of the karyomit(e)s " of boar for example, is represented a tested object or family member's two of somatic karyomit(e) 7 copies or a copy in the germ line cell.The specific allelotrope of two copies of karyomit(e) 7 comprises the allelotrope that is positioned at or is close to the site of paying close attention to, and can be identical or different.7 groups of some parts that can comprise the karyomit(e) of collecting in karyomit(e) 7 libraries such as plasmid, yeast or the phage library 7 of karyomit(e).Referring to Sambrook etc., Molecular Cloning, the 2nd edition and Mandel etc., Science 258:103-108 (1992).

" penetrance " refers to the individuality that has dcc gene or polymorphism shows some characteristic symptoms owing to this hereditary change per-cent.Expressivity refer to feature the performance degree (as, slight, moderate or severe).

" polymorphism " refers to and have two or more convertible sequence or allelotrope by the heredity decision in colony.The polymorphism sign is the site of departing from this place.Preferred sign has at least two allelotrope, and the occurrence frequency of each is higher than 1%.A pleomorphism site may diminish to the difference of having only a base pair.Be applicable to that polymorphism sign of the present invention comprises restriction fragment length polymorphism, and variable number tandem repeat (VNTR ' s), hypervariable region, trinucleotide repeats, and tetranucleotide repeats, and other microsatellite sequence.

" restriction fragment length polymorphism " implication (RFLP) is a kind of variation of dna sequence dna, and it has changed the length of restriction fragment, is described in Am.J.Hum.Genet.32:314-331 (1980) as Botstein etc.Restriction fragment length polymorphism can produce or eliminate a restriction site, thereby has changed the length of restriction fragment.For example, dna sequence dna GAATTC has 6 bases, with its identification and the cleavage site of being construed as limiting property of complementary strand CTTAAG restriction endonuclease EcoRI.On the arbitrary chain of DNA, destroyed the EcoRI site with any that replaces 6 Nucleotide once different Nucleotide.This RFLP can detect with following method, and for example, amplification comprises the target sequence of polymorphism, with the sequence of EcoRI digest amplification, on sepharose or acrylamide gel reaction product is carried out size separation.If EcoRI Restriction Enzyme site unique in extension increasing sequence is exactly a pleomorphism site, the target sequence that comprises restriction site will demonstrate two fragments according to the pre-sizing of extension increasing sequence length.The target sequence that does not contain the Restriction Enzyme site only shows a fragment with extension increasing sequence length.Similarly, the probe of the also available adjacent domain of the detection of RFLP is detected the EcoRI digest of Southern engram DNA, and whether the EcoRI fragment that so just can be observed corresponding size exists.PFLP can cause because of point mutation, the latter can produce or destroy a Restriction Enzyme site, VNTR, dinucleotides tumor-necrosis factor glycoproteins, disappearance, repetition, or other can form or lose a Restriction Enzyme site, or changes the sequence variations of restriction fragment size and cause.

" variable number tandem repeat " (VNTR ' s) is the short sequence of nucleic acid, with head-to-tail mode arranged in series, and discovery in each is individual.As described in Wyman etc., Proc.Nat.Acad.Sci.77:6754-6758 (1980).In general, the VNTR sequence comprises the repetition of this sequence of the core sequence of at least 16 base pairs and variable number.In addition, in core sequence, also variation can be arranged, Jefferys etc., Nature 314:67-72 (1985).These sequences have the individuality of height, may be that each is individual unique.Therefore, VNTR can generate restriction fragment length polymorphism, can become a kind of amplified production difference sign based on size again.

" microsatellite sequence " comprises some sections at least about the DNA of 10 base pairs, connected by the short sequence of DNA (1-6 base pair) of variable number and repeats to form (Clemens etc., Am.J.Hum.Genet.49:951-960; 1991).Microsatellite sequence is distributed widely on the chromosomal DNA of body one by one usually.Number of iterations in specific vertical array has a great difference between individuality and individuality, therefore, microsatellite sequence can produce restriction fragment length polymorphism, can become a kind of amplified production difference sign based on size again.

The accompanying drawing summary

Fig. 1 describes the sequence of about 630 base pairs of 5 ' non-translational region of pig CYP11a1 gene (SEQ ID NO:1).Produce the PCR fragment with the DNA that carries from the pig testis sample.The primer is forward primer (SEQ ID NO:2) and reverse primer (SEQ ID NO:3).

Fig. 2 describes the polymorphism pattern of Sph1 digestion PCR product.Use the PCR condition of forward and reverse primer as follows: 94 ℃ 2 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ of 35 round-robin 1 minute, and last 72 ℃ 2 minutes.Sample is with SphI (New EnglandBiolabs) digestion, with separating 50V, 45 minutes under the 1.5% sepharose room temperature.The gel ethidium bromide staining.Swimming lane 1: lower molecular weight sign; Swimming lane 2: indigested PCR fragment; Swimming lane 3 and 7:CT genotype; Swimming lane 4-6:CC genotype.The 630bp PCR fragment of CC pig is digested the 450bp product, and the PCR fragment of CT pig is only partly digested, and shows to have T allelotrope, finds its restrictive fragment length polymerphism (RFLP) thus.

Fig. 3 describes the comparison of CC type boar and CT genotype boar submandibular salivary gland (SMG) Δ-16 androstene concentration.There are 5 to show that SMG Δ-16 androstene concentration is higher than the recommendation threshold value (55 μ g/g SMG) of identifying the peculiar smell trunk in 30 CC boars.Carry the allelic whole boars of T (n=20) and all be lower than the recommendation threshold value of boar peculiar smell.

Fig. 4 is a table, shows the difference of comparison CC boar and the viewed growth of CT boar, trunk and reproduction feature.The weight of testis, submaxillary gland and cowper gland is bigger, and the higher pig peculiar smell of CC boar of all indicating of SMG Δ-16 androstene concentration is higher.The amazing CC of being pig rate of body weight gain also increases by 5.9%, and trunk is also longer.

Fig. 5 shows the sequence of ox CYP11a1 gene, comprises 948 Nucleotide of 5 ' UTR.

Fig. 6 shows the sequence of chicken CYP11a1 gene, comprises 137 Nucleotide of 5 ' UTR.

Detailed Description Of The Invention

According to the present invention, we are provided for diagnosing the material and the method for the hereditary change of CYP11a1 gene relevant with steroid biosynthesizing unusual or that increase in the domestic animal.The variation of mouse CYP11a1 gene pleiomorphism is the reason of heredity difference during testosterone generates.The mouse CYP11a1 assignment of genes gene mapping is in karyomit(e) 9.This zone is collinear with pig karyomit(e) 7.

The major cause of boar peculiar smell is owing to there is Δ-16 steroid, androstenone, and it is one of multiple steroid that generates in the boar testis.Androstenone and metabolite thereof, as androstene alcohol by testicular secretion, the latent submandibular salivary gland (SMG) that is stored in.These steroid are discharged in the air by saliva in mating behavior, play a part sex pheromone, cause the emotionally behavior of sow (sow).Because Δ-16 steroid is highly lipophilic, androstenone also is stored in the body fat, and the existence of its high density constitutes the stink of pork, promptly so-called boar peculiar smell.

The concentration of androstenone is highly genetic in the fat.Identify fatty androstenone quantitative characteristic site (QTL) (little satellite sign SO 102), be positioned at swine leukocyte antigen mixture (SLA) zone of pig karyomit(e) 7.According to the present invention, determined one with the generation of androstenone with divide the pig peculiar smell relevant particular inheritance polymorphic sequence.

Have the quantitative characteristic site (QTL) of a fatty androstenone on the pig karyomit(e) 7, illustrate that the CYP11a1 of pig may be positioned on the karyomit(e) 7, and steroid synthetic rate-limiting enzyme may be the important control point that androsterone synthesizes and exist the boar peculiar smell.

In order to determine whether to exist the polymorphism that is associated with the compound that causes the boar peculiar smell, carried out a Study on Genome, (5 ' UTR) 2.4kb compares the pig CYP11a1 gene non-translational region that the boar of selecting is in advance organized.At first the genotype to 5 " high peculiar smell " and 5 " low peculiar smell " boars compares, by directly being checked order, the PCR product (uses ABIPrism 377, Nacleic Acid Facility, Penn State University BiotechnologyInstitute) is disclosed in and has a single nucleotide polymorphism (SNP) among whole 2.4kb 5 ' UTR.This SNP (CT allelotrope) only is found on one's body boar, Δ-16 steroid of lower concentration in its performance sialisterium, androstenone concentration height correlation in this tolerance and the fat.This polymorphism is made of-155 locational thymus pyrimidines (T) that are positioned at translation starting point or a cytosine(Cyt) (C).This polymorphic position is in the recognition site of a Restriction Enzyme, thereby when having T allelotrope, viewed restriction fragment length pattern will change after the specific limited enzymic digestion.Under this particular case, used Restriction Enzyme is Sphl (England Biolabs).Other Restriction Enzyme that can cut this identical DNA sequence also is available.With check Sphl the restrictive diges-tion product of CYP11a1 5 ' UTR is determined the allelic existence of T or lack as.No matter the allelic existence of T is homozygote (TT) or heterozygote (CT), is associated with low pig peculiar smell.What be associated with the allelic existence of CC is high pig peculiar smell, and the increase of testicular weight, cowper gland length and weight and submandibular salivary gland weight.In addition, having the allelic boar of CC shows rate of body weight gain improvement 5.9% and longer trunk is arranged.

Find that this polymorphism not only has the effect of reproduction feature also relevant with the increase of rate of body weight gain and carcass length, this illustrates multiple other g and D feature of this polymorphism influence.Thereby existing or lack this polymorphism also may be relevant with raise benefit and birth weight.This polymorphism is related with the reproduction feature, as testicular weight, and cowper gland length and weight, submaxillary gland weight, and Δ-16 steroid concentrations etc., all be the indication of the total effect of sexual gland steroid generation.

Data provided herein shows the existence of CYP11a1 polymorphism or lacks as may effect be arranged to other reproduction feature, ovulation rate for example, and the young size of nest is given milk, and fertility (comprise female, male the two).In addition, express and generate another place as the glucocorticosteroid of hydrocortisone because suprarenal gland is CYP11a1, this polymorphism also may be relevant with the disease reaction feature, because this category feature is regulated and control by adrenal steroid.

Another aspect of the present invention, this genetic marker also can be used for the allelotrope combination useful with the generation linked together of other genetic marker, or prevent to carry the tested object of bad combination with screening.These in the past the example of genes identified as tumor necrosis factor alpha (TNF α), CYP11a1, lactotropin (PRL), estrogen receptor (ER) and lactotropin acceptor (PRIR).Some example of little satellite sign that these identified in the past has: SO 064, and SO 102, and SO 078, and SO 158, and SO 066, and SW 304, and SW 1083, and SO 101, and SO 212.

Other polymorphism in the pig CYP11a1 gene can be identified with method of the present invention.This variation may occur in the non-translational region of gene, but also may and obtain identifying in intron and exon sequence in the translation district.One group of this class variation can cause or the variation of related male sex hormone function and phenotypic characteristic probably.In case identify such hereditary change, just might have the genotypic transgenic animal of desired CYP11a1 in order to generation with known technology with these or similarly change the quiding gene group.Data is also enlightened: other agricultural goes up that polymorphism in important species CYP11a1 homologous region may cause or related function and genotypic similar variation.

Invention more on the one hand, the corresponding C YP11a1 sequence of cow and chicken is provided.This information helps 5 ' UTR of ox or chicken CYP11a1 is carried out genome scanning, the polymorphism that is associated with exposure and growth, trunk characteristic and reproduction (comprise galactopoiesis and lay eggs).Implement the diagnostic kit of the inventive method

The present invention also comprises the test kit of the method for carrying out an invention.Test kit comprises a bottle, pipe or any container, wherein contains one or more and plants oligonucleotide, and it can be hybridized with a DNA sections, and this DNA sections is exactly CYP11a1 gene or the sections that is connected with this gene.Some test kit contains two kinds of such oligonucleotide, can be used as the primer of the chromosomal DNA sections that increases.Selecting the sections of amplification can be a CYP11a1 gene, and it comprises the site that known variation takes place.Some test kit comprises a pair of oligonucleotide to detect predetermined variation.For example, some test kit contains the oligonucleotide that is applicable to allele specific oligonucleotide oligonucleotide hybridization or allele specific oligonucleotide amplified hybridization.Test kit of the present invention also can comprise the composition of amplification system, comprises the PCR reaction material as damping fluid and heat-stabilised poly synthase.In other embodiments, test kit of the present invention can be united use with the amplification kit that the merchant sells, this class amplification kit can be available from GIBCO BRL (Gaithersburg, Md.) Stratagene (La Jolla, Calif.), Invitrogen (San Diego, Calif.), Schleicher ﹠amp; Schuell (Keene, N.H.), Boehringer Mannheim (Indianapolis, Ind.).Test kit can arbitrarily comprise positive or negative control reaction or mark, the molecular weight marker of gel electrophoresis etc.Test kit generally includes label or specification sheets, illustrate that test kit is used for the suitability that the biosynthesizing of diagnostics classes sterol changes, and how explanation uses oligonucleotide for this purpose.Noun " label " is generally used for comprising any material written or record, and these materials all were attached to or otherwise are with in diagnosis box in any time of production, transportation, sale or use.Mode 1. linkage analysises that carry out an invention

Determine the following method of chain employing between a polymorphism sign and a site relevant, promptly locate the polymorphism sign with particular phenotype, and observe they in the reduction division that information once is provided whether (for example) and high peculiar smell phenotype on the karyomit(e) be divided into from.Referring to, for example, Kerem etc., Science 245:1073-1080 (1989); Monaco etc., Nature 316:842 (1985); Yamoka etc., Neurology 40:222-226 (1990), and summary Rossiter etc., FASEB Journal 5:21-27 (1991).Single pedigree seldom comprises the enough reduction division that information is provided to provide definite chain, because family is less usually, sign may be not enough to provide information.For example, a sign may not be a polymorphism in specific family.

The affected sib pair analysis method of chain possibility mat is determined, is described in Terwilliger and Ott, Handbook of Human Genetic Linkage (Johns Hopkins, Md., 1994), and Ch 26.This method does not need penetrance or variation frequency are made supposition, and only need consider the data of smaller portions among all family members that determined phenotype and polymorphism sign (i.e. compatriot's pairing).Particularly, affected sib pair analysis method is calculated as every couple of affected compatriot the same allele variant of total (unanimity) or not total (inconsistent) every kind of polymorphism sign.Then,, probability can be calculated, viewed conforming compatriot can be obtained to the right ratio of nonconforming compatriot and chain without mark for each sign.

Describe as Thompson and Thompson: Genetics in Medicine, 5th ed, 1991, W.B.Saunders Company, Philadelphia, in linkage analysis, people have calculated under various possible θ values, (do not have and recombinate) the possible ratio (relative different) to θ=0.50 (random assignment) from θ=0.0.Like this, under a given θ value may ratio be: (if α site continuous data possibility under θ value)/(if the site does not connect, the possibility of data).Support Evidence for linkage usually with the log of this ratio ₁₀Represent, and " logarithm of difference " is called " lod branch ".For example, lod is divided into 5 expressions, 100,000: 1 difference, promptly viewed chain be not occurrent.

Adopt logarithm to make that the data of never collecting with family can be comprehensive with simple addition.Can utilize computer program to calculate the lod branch of different θ values.Available program comprises LIPED, and MLINK (Lathrop, Proc.Nat.Acad.Sci.81:3441-3446 (1984)).

To a concrete lod branch, can measure recombination fraction from mathematical table.Referring to Smith etc., " mathematical table that human genetics research worker uses " (Churchill, London, 1961) and Smith, Ann.Hum.Genet.32:127-150 (1968).θ value when the lod value is the highest is considered to the best-estimated of recombination fraction, " maximum possible estimation ".

Lod links to each other on the occasion of two sites of expression, and negative value represents that chain possibility (under this θ value) is lower than two disjunct possibilities in site.As usual, the comprehensive advantage logarithm be divided into+3 or higher (be equivalent to 1000: 1 above support chain) be considered to prove clearly that two sites link to each other.Similarly, as usual, negative lod branch-2 or be considered to more for a short time proves the chain of two sites that eliminating is compared definitely.If fully negative interlocking data is arranged, just site can be from a whole karyomit(e), or its part, to get rid of, this method is known as gets rid of the location.Just search is focused on then on the remaining chromosome position that is not excluded.General discussion about lod branch and linkage analysis sees also, as, T.Strachan, Chapter 4, " human genome mapping ", The Human Genome, 1992 BIOS ScientificPublishers Ltd.Oxford.

These data also can provide and make haplotype analysis.This analyzes selected individual interchromosomal allelotrope mark and makes that it is minimum separating needed recombination event number between the generation.But chain also mat is measured the relative possibility of the viewed separate data of any two marks is determined, these two signs its position when recombination fraction θ indicates disjunct situations corresponding to two herein, thereby independent separate.2.DNA separation and amplification

The sample separation of patient, propositus, tested object or family member's genomic dna comprises saliva, Stomatocyte from arbitrary suitable source, the hair root, blood, Cord blood, amniotic fluid, and other contains the suitable cell or the cell sample of harmless interphase nuclei or medium cell.Can obtain cell from the organ of solid tissue and fresh or preservation or from tissue sample or biopsy material.Can comprise in the sample some be not natively with biomaterial miscellaneous compound mutually, as preservatives, antithrombotics, damping fluid, fixing agent, nutrition agent, microbiotic etc.

The method of isolation of genomic DNA is described in from these different sources materials: as, Kirby, DNA Fingerprinting, An Introduction, W.H.Freeman ﹠amp; Co.New York (1992).Genomic dna can or inferiorly obtain for cell culture or from arbitrary aforementioned tissue sample deutero-transformation cell lines from former generation of cultivating.

The patient, the propositus, tested object or family member's RNA sample also can use.RNA is separable from the tissue of expressing the CYP11a1 gene, as described in (seing before) such as Sambrook.RNA can be a cell total rna, mRNA, poly A+RNA, or its any combination.For getting best result, RNA is a purifying, but also can be unpurified kytoplasm RNA.RNA can be reversed record and generate DNA, and the latter is again as the template of amplification, thereby PCR one group of special colony of cloning RNA transcript indirectly.Referring to: as Sambrook, see before, Sawasaki etc., PCR Technology the 8th chapter, see before (1992), and Berg etc., Hum Genet 85:655-658 (1990).3.PCR amplification

The most common amplification method is polymerase chain reaction (PCR), is described in U.S.Pat.Nos.4,683,195,4,683,202,4,965,188, and each is incorporated in this as a reference.If with the increase target area of hemocyte of PCR, the whole blood of heparinization should be infused in the valve tube of sealing, keeps separating with other sample, and is with clean gloves operation.For best result, blood sample should be after collection be handled immediately, if impossible, then should be kept at before use in 4 ℃ the sealed vessel.Cell in other physiological liquid also can be used to measure.When adopting any this class I liquid I, the cell in the liquid should be separated from liquid component with centrifuging.

Tissue should tentatively be pulverized in a 5mm culture dish with an aseptic disposable pocket knife and aseptic syringe needle (or 2 pocket knife).The method of removing deparaffnize from tissue slice is described in the multiple professional handbook well known to those skilled in the art.

Will be with one section target nucleic acid sequence of pcr amplification in a sample, this section sequence must be that the composition of amplification system can be near obtaining.A method of separating target DNA is slightly to carry, and this is to bigger sample of great use.In brief, the mononuclearcell in the samples such as chorionic villus cell of the amniocyte in blood, the amniotic fluid, cultivation is by separating by the standard method layering on aseptic Ficoll-Hypaque gradient liquid.Before extracting DNA, collection interval cell is also given a baby a bath on the third day after its birth inferior in aseptic phosphate-buffered saline.If the DNA of test peripheral blood lymphocyte, osmotic shock (sedimentation cell was handled 10 seconds with distilled water) is carried out in suggestion,, then washes 2 times if can see residual cells in first washing back again.This can prevent the restraining effect of hemochrome gene pairs PCR reaction in the oxyphorase.If after collecting sample, do not carry out the PCR test immediately, every part 10 ⁶Cell is precipitable to be collected in the aseptic Eppendorf pipe, and the throw out of doing is chilled in-20 ℃ until use.

Cell is suspended in the damping fluid (10 again ⁶Karyocyte/100 μ l), the damping fluid composition is: 50mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl ₂, 0.5%Tween 20, and 0.5%NP40 also adds the Proteinase K of 100 μ g/ml.After hatching 2 hours under 56 ℃, with cell be heated to 95 ℃ 10 minutes with inactivated proteases K, and move into immediately in the wet ice (quenching).If also have big aggregation to exist, then need in this same damping fluid, to carry out the digestion in another cycle.10 μ l of this extract are used for amplification.

When using tissue, when for example the culturing cell of chorionic villus cell or fusion extracted DNA, the amount of above-mentioned damping fluid and Proteinase K can be made change by the size of tissue sample.Extract 50 °-60 ℃ the insulation 4-10 hour then 95 ℃ 10 minutes with inactivated proteases.In long insulating process, after about 4 hours, should add fresh Proteinase K again by primary concentration.

When sample only contained small amounts of cells, extracting method can be by hereinafter carrying out: Higuchi, " simple and easy preparation promptly is used for the sample of PCR ", and PCR Technology, Ehrlich, H.A. (ed), Stockon Press, New York is hereby incorporated by.PCR can be used for the increasing target area of karyomit(e) 7 in the minute quantity cell (1000-5000), each colony that these cells can be cultivated from medullary cell and peripheral blood.Cells in sample is suspended in the molten born of the same parents' damping fluid of 20 μ l PCR (10mM Tris-HCl (pH 8.3), 50mM KCl, 2.5mM MgCl ₂, 0.1mg/ml gelatin, 0.45%NP40,0.45% polysorbas20), freezing until use.When preparing to carry out PCR, the cell in the molten born of the same parents' damping fluid of PCR adds 0.6 μ l Proteinase K (2mg/ml).Sample is warmed to about 60 ℃, is incubated 1 hour.Sample is heated to when stopping to digest 95 ℃ 10 minutes with inactivated proteases K, then in cooled on ice.

It is salting-out process that extraction DNA is used for the PCR simpler method, and this method is revised by laxative remedy: Miller etc., Nucleic Acids Res.16:1215 (1988) is hereby incorporated by.Mononuclearcell separates on the Ficoll-Hypeque gradient.The cell resuspending is in the molten born of the same parents' damping fluid of 3ml (10mM Tris-HCl, 400mM NaCl, 2mM Na ₂EDTA, pH8.2).Proteinase K solution from the 20mg/ml of 50 μ l to cell and the 20%SDS solution of 150 μ l, 37 ℃ of incubated overnight then of adding.The jolting test tube can improve the digestion of sample in insulating process.If the digestion of Proteinase K is also insufficient after the incubated overnight (still can see fragment), add the 20mg/ml Proteinase K solution of 50 μ l again, mixing in solution, 37 ℃ of insulations are after a night on a gentle jolting or universal stage.After treating digestion fully, in sample, add 1ml 6MNaCl solution, the violent mixing.The solution that obtains centrifugal 15 minutes with 3000rpm.Contain the cell protein that precipitates in the precipitation, and contain DNA in the supernatant.Supernatant is moved into one to be contained in the 15ml test tube of 4ml Virahol.The test tube content is mixed gently, mix and form the DNA precipitation of white up to water with alcohol mutually fully.Take out the DNA precipitation, immerse 70% spirituous solution and mixing gently.The DNA precipitation is taken out dry air from alcohol.The DNA precipitation is inserted distilled water, and dissolving.

The test kit that the extraction high-molecular-weight DNA is used for PCR comprises: genome separating kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Ind.), genomic dna separation system (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA purification kit (Schleicher ﹠amp; Schuell, Keene, N.H.), the DNA extraction test kit (Stratagene, La Jolla, Calif.), the Turbogen separating kit (Invitrogen, San Diego, Calif.), etc.Before implementing method of the present invention, it generally is suitable using these test kit purify DNAs according to the specification sheets of manufacturer.

The concentration of the DNA that mensuration is extracted and purity can be diluted the absorption of sample at 260nm and 280nm with the spectrophotometric analysis portion.After having extracted DNA, just can carry out pcr amplification.Each round-robin the first step of PCR comprises separates the nucleic acid duplex that is formed by primer extension.After in case chain is separated, next step of PCR comprise make isolating chain with at the primer hybridization of target sequence flank.Primer extension then forms the complementary copy of target chain.For carrying out the pcr amplification of success, primer is design like this: each primer just in time make along the position of a duplex sequence hybridization from a primer synthetic extension products with the template that can become another primer extension after template (complement) is separated.Sex change, hybridization and the loop cycle that extends repeat many times until the amount that can obtain required amplification of nucleic acid.

In a useful especially pcr amplification embodiment, the separation of chain be by reaction is heated to sufficiently high temperature, continue time enough make the duplex sex change and can not cause that the irreversible denaturation of polysaccharase realizes (see U.S.Pat.No.4,965,188, be hereby incorporated by).Typical thermally denature comprises temperature and the time from the several seconds to several minutes scopes from 80 ℃ to 105 ℃ of scopes.But, the separation of chain also can be adopted arbitrary suitable denaturation method, comprises physics, method chemistry or zymetology.It is available to induce chain to separate, and for example, helicase maybe can show the active enzyme that untwists.For example, the PecA enzyme just has the activity of untwisting in the presence of ATP.With the suitable reaction condition of helicase disengaging latch be in the art know (referring to Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63-67; And Radding, 1982, Ann.Rev.Genetics 16:405-436, each piece of writing is hereby incorporated by).

The primer extension that template relies among the PCR is to be subjected to polymerizing agent catalytic, needs four kinds of deoxyribonucleotide triphosphoric acids (dATP, dGTP, dCTP, and dTTP) typically of q.s, and reaction medium is by suitable salt, and metallic cation and pH buffer system are formed.Suitable polymerizing agent is the DNA synthetic enzyme that the energy catalytic templating relies on.In some cases, to encode be a kind of proteic part of cell expressing at least in the target area.In the case, mRNA can be used to the target area of increasing.Perhaps, available PCR generates a cDNA library for further amplification from RNA, and the starting template of primer extension is RNA.Being suitable for from the polymerizing agent of the synthetic complementary copy of RNA template DNA (cDNA) sequence is reversed transcriptive enzyme (RT), as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or thermus thermophilus (Tth) archaeal dna polymerase, PerkinElmer Cetus, Inc. sell a kind of heat-stablely has an active archaeal dna polymerase of reverse transcription.Usually, the first time of geneome RNA template after initial reverse transcription step, degradation in the denaturing step only stayed dna profiling.The suitable polysaccharase that is used for dna profiling comprises: for example, and e. coli dna polymerase I or its Klenow fragment, T4 archaeal dna polymerase, Tth polysaccharase, and Taq polysaccharase, a kind of thermostability archaeal dna polymerase of separating from the thermus aquaticus, can be from Perkin Elmer Cetus, Inc buys.This a kind of enzyme in back is widely used in the amplification and the sequencing of nucleic acid.The reaction conditions that uses the Taq polysaccharase is described in Gelfand in dawn known in the art, and 1989, PCR Technology sees before.4. allele specific pcr

Allele specific pcr can show that there is or lacks difference aspect a kind of variation or the polymorphism in karyomit(e) 7 target areas.The pcr amplification primer of selecting only combines with certain allelotrope of target sequence.Like this, for example, amplified production is just by 7 groups of generations of the karyomit(e) that contains the primer binding sequence, and 7 groups of karyomit(e)s that do not contain the primer binding sequence just not generate amplified production.This method is described in Gibbs, Nucleic Acid Res.17:12427-2448 (1989).5. allele specific oligonucleotide screening method

Further diagnosis examination method is used allele specific oligonucleotide oligonucleotide examination method (ASO), as described in Saiki etc., and Nature 324:163-166 (1986).Produced corresponding to specific allelotrope, had the oligonucleotide of one or more base-pair mismatch.ASO examination method detects the target gene group or the pcr amplified dna and the mispairing that does not suddenly change between oligonucleotide of variation, show oligonucleotide binding ratio sudden change oligonucleotide in conjunction with reduction.Oligonucleotide probe can be designed to like this: under low stringency, can all combine with allelic two kinds of polymorphic forms, but its corresponding allelotrope combination under the tight degree of height.Perhaps, the stringency condition can be designed to obtain reactions in two fens basically under this condition, promptly corresponding to the ASO of CYP11a1 genovariation form can with this equipotential gene recombination, and do not hybridize with wild-type allele.6. the allelotrope detection method of ligase enzyme mediation

The DNA target area of a tested object can be detected with target area unaffected and affected family member with the allelotrope of ligase enzyme mediation and compare.Referring to Landegren etc., Science241:1077-1080 (1988).Ligase enzyme also can be used for detecting the point mutation in the ligase enzyme amplified reaction, sees Wu etc., Genomics 4:560-569 (1989).Ligase enzyme amplified reaction (LAR) utilizes the amplification of specific dna sequence, uses the sequential turnover of the ligation of template dependence, as Wu, see before, and Barany is described Proc.Nat.Acad.Sci.88:189-193 (1990).7. denaturing gradient gel electrophoresis

Amplified production with the PCR gained can be analyzed with denaturing gradient gel.The different sequence-dependent character of unwinding according to DNA in the solution can be identified different allelotrope with electrophoretic migration.Temperature raise or the condition of sex change under, dna molecular is called the territory of unwinding at sections, in unwind.Each territory of unwinding is worked in coordination with down in a melting temperature(Tm) clear and definite, that base is special (Tm) and is unwind.The length in territory of unwinding is at least 20 base pairs, also may reach hundreds of base pairs.

Different and difference that produce can be measured with polyacrylamide gel electrophoresis between allelotrope based on the sequence specific territory of unwinding, see Erlich ed., PCR Technology, the 7th chapter, DNA cloning principle and application, W.H.Freeman and Co.New York (1992), its content is hereby incorporated by.

In general, intend increasing with the target area of denaturing gradient gel electrophoresis analysis PCR primer with the target area flank.PCR product after the amplification imposes on the polyacrylamide gel of linear denatured gradient, see Myers etc., Meth.Enzymol.155:501-527 (1986), and Myers etc., Genomic Analysis, A Practical Approach, K.Davies Ed.IRL PressLimited, Oxford, pp.95-139 (1988), its content is hereby incorporated by.The temperature that electrophoresis system is kept is a little less than the unwind Tm in territory of target sequence.

In another kind of denaturing gradient gel electrophoresis method, can connect one section GC Nucleotide earlier on the target sequence, be called the GC clip, as described in Erlich (seing before) the 7th chapter.Being preferably in the GC clip at least 80% Nucleotide is guanine or cytosine(Cyt).Preferably the length of GC clip is at least 30 bases.This method is suitable for having the target sequence of high Tm especially.

Usually, the target area pcr amplification, as indicated above.In the oligonucleotide PCR primer one be with GC clip district at 5 ' end, this district has 30 bases that are rich in the GC sequence at least, is incorporated into 5 of target area ' end when amplification.Gel electrophoresis is being carried out under the denatured gradient condition as described above in target area after the amplification of gained.The dna fragmentation of the difference of the single base that changed can move to different positions in gel, available ethidium bromide staining is observed.8. temperature gradient gel elec-trophoresis (TGGE)

The principle of temperature gradient gel elec-trophoresis (TGGE) (TGGE) is identical with denaturing gradient gel electrophoresis, and just denatured gradient is to be generated rather than generated by chemical denaturant concentration difference by the temperature difference.The TGGE of standard uses a kind of electrophoresis apparatus, and its thermograde distributes along electrophoresis path.When sample is containing in the gel of chemical denaturant of same concentration when mobile, they have run into the temperature that increases.Another kind of TGGE method, sequential temperature gradient gel elec-trophoresis (TGGE) (TTGE or tTGGE) adopts the monoblock running gel constantly to promote method of temperature and reaches same result.When sample was mobile in gel, the temperature of monoblock gel was increasing, and made them run into the temperature that is increasing when sample moves through gel.The preparation of sample comprises the pcr amplification that mixes the GC clip, and all identical with denaturing gradient gel electrophoresis to the observation of resultant.9. single-strand conformation polymorphism analysis

Target sequence on the CYP11a1 site or allelotrope can be differentiated with the single-strand conformation polymorphism analysis method, this method is identified the base difference according to the variation of the electrophoretic migration of strand PCR product, as described in Orita etc.: Proc.Nat.Acad.Sci.86:2766-2770 (1989).Generate amplification PCR products as stated above, heating or other method make it sex change, form single-stranded amplification product.Single-chain nucleic acid may refolding be shaped as secondary structure, and this part ground depends on base sequence.Therefore, the electrophoretic mobility of single-stranded amplification product can be found the base sequence difference between allelotrope or target sequence.10. the chemistry of mispairing or zymetology cutting

Difference between target sequence is also available to be detected the right difference chemical chop of base mismatch, as described in Grompe etc.: Am.J.Hum.Genet.48:212-222 (1991).Another kind method, the difference between target sequence can be with cutting detect to the right zymetology of base mismatch, as described in Nelson etc.: Nature Genetics 4:11-18 (1993).Briefly, the genetics material of taking from patient or affected family member can be used to generate the allos hybrid dna duplex of no mispairing.The implication of " allos hybridization " used herein is in the composition of a dna double coiled strand, and the chain of DNA is from a people, patient normally, and the 2nd DNA chain be from another person, normally affected or unaffected family member.The positive of the allos crossbred of no mispairing is selected can determine little insertion, disappearance or may change related other polymorphism with male hormone metabolism.11. the DNA of non-PCR diagnosis

To the link to each other evaluation of dna sequence dna of patient and family member's CYP11a1,, also can not need amplification step based on the polymorphism analysis that comprises restriction fragment length polymorphism.Hybridization probe is oligonucleotide normally, and it is by combining in pairs with all or part of complementary base of a target nucleic acid.The stringency that depends on hybridization conditions, probe combines with the target sequence that lacks complete complementarity usually.Preferably probe is carried out direct or indirect mark, thus the existence of measuring probe or lack as the time, the existence of target sequence or lack as also being predicted.Direct labelling method comprises isotopic labeling, as using ³²P or ³⁵S.The indirect labelling method comprises: fluorescence labels, the biotin composite that can combine with avidin or Streptavidin, or polypeptide or albumen label.The visual detection method comprises photoluminescence, texas Red, and rhodamine and derivative thereof, " red white " dye well 3,3 ', 5,5 '-tetramethyl benzidine, fluorescein and derivative thereof, red sulphonyl, Umbelliferone etc., or use horseradish peroxidase, alkaline phosphatase etc.

Hybridization probe comprise any can with the nucleotide sequence of the pig karyomit(e) hybridization that has CYP11a1, thereby can determine the genetic marker that links mutually with CYP11a1, comprising: a kind of restriction fragment length polymorphism, a hypervariable region, repetition composition, or VNTR.Hybridization probe can be a gene or a suitable analogue.In addition, Shi Yi hybridization probe comprises that exon fragment or known locations are in the part of the gene or the cDNA of karyomit(e) appropriate area.

The preferred series connection recross probe that is used for purposes of the present invention is to discern segmental probe in a small amount in a distinguished point under high stringency hybridization conditions, or discerns the segmental probe of larger amt under low stringent condition on this site.

We provide following illustration with explanation embodiment of the present invention.But attempt by no means this invention is limited.

Example I

The genetic marker of meat, growth, trunk and the reproduction feature of reflection pig

According to the present invention, meat improvement and the growth of identifying and characterized a kind of and pig improve relevant genetic marker with the trunk feature.Material and method below in the practice of routine I, having used.

The testis tissue sample adopts 50 boars in knob gram state, and the growth of these pigs, trunk and pig peculiar smell data are collected in advance.Boar was weaned during ages in about 10 weeks, specified fence, raised " raising-finish " diet with standard, was about 120kg to final body weight.Boar kills with electric shock, in the laboratory bloodletting of Penn State University meat.Collect testis, cowper gland and submandibular salivary gland, repair only, weigh then.Trunk is weighed, cool overnight.Gather standard trunk take off data next day, such as carcass length, either side of the small of the back area, fat thickness and meat texture.

The mensuration of submandibular salivary gland Δ-16-androstene is adopted the method (J.Animal Sci.69:1092-1100,1991) of Squires development.In brief, submandibular salivary gland is repaiied only, food processors (Cusinart) chopping is got the tissue of 1 gram chopping and is inserted the 50ml test tube.Add methyl alcohol (5ml), mixture was played homogenate 30 seconds with Polytron.Sample centrifugal 5 minutes with 2800rpm.In the 3ml supernatant, add 3ml distilled water, vortex mixed.Add hexane 6ml and extract Δ-16-androstene.With the mixture vortex mixed, and left standstill 5 minutes, make it phase-splitting.Change the 3ml organic phase over to a glass culture tube, extract water-bath under nitrogen (30 ℃) drying.Add developer (0.5ml 0.5% resorcyl aldehyde Glacial acetic acid liquid adds 0.5ml 5% vitriolic Glacial acetic acid liquid).Test tube placed 95 ℃ of heating plates 10 minutes.Purple taking place, be the indication that has Δ-16-androstene, draws 100 μ l test fluid and be added in the hole of 96 hole microwell plates and measure.Measure light absorption under near the several wavelength the maximum light absorption (593nm) of known Δ-16 androstene, and with contain 5 α-androstane-16-alkene-3 β-alcohol (in the submandibular salivary gland main Δ-16-androstene) standard testing solution relatively.The typical curve made from standard test solution is determined the concentration of Δ-16-androstene.

ANOVA is adopted in data analysis, uses the GLM method (1990) of SAS.

Get the testis tissue sample from (20 ℃) 10 boars of refrigeration: 5 Δ-16 androstenes (high boar peculiar smell), 5 Δ-16-androstenes (low boar peculiar smell) with minimum concentration with maximum concentration.Extract DNA with the protease K digesting method.About 50mg testis tissue is wrapped in the aluminium foil, and is freezing in liquid nitrogen.Then sample is pulverized, got about 20mg and insert micro-centrifuge tube, add 0.5ml digestion damping fluid (50mM Tris, pH8.5; 1mM EDTA; 0.5%Tween 20; 200 μ g/ml Proteinase Ks (Gibco Life Technologies, Grand Island, NY)).Proteinase K is stored in-20 ℃ of (20mg/ml Proteinase Ks with storage liquid; 1mM Tris-HCl, pH7.5; 20mM calcium chloride and 5% glycerine).Sample is suspended from the digestion damping fluid, inserts 55 ℃ of water-baths, 3 hours.With sample centrifugal 1 minute with 13000g, insert the heating plate, 95 ℃, 10 minutes.Take out sample, be stored in-20 ℃, until analysis.

Obtain 4 groups of primers, be equivalent to each about 600bp, be equivalent to 5 ' UTR (sequence derives from Urban etc., J.Biol.Chem.269:25761-25769,1994) of the 2.4kb of pig CYP11a1 gene altogether.Every group of primer is to each starting PCR reaction of 10 dna profilings.PCR is undertaken by following program:

1. 2 minutes, 94C.

2. 1 minute, 94C.

3. 1 minute, 55C.

4. 1 minute, 72C.

5. 35 circulations are to (2).

6. 2 minutes, 72C.

7. remain on 5C.

10 * damping fluid (w/MgCl is used in reaction ₂); DNTP ' s (10nmol); Primer CYPscc Forl (20pmol); Primer CYPscc Revl (20pmol); The Taq polysaccharase; Distilled water and dna profiling (the protease K digesting sample of dilution in 1: 10, about 100ng).

The agarose gel electrophoresis analysis of PCR product cuts out～band of 600bp from sepharose then, with QIAquick gel extraction kit purifying (QIAGEN Inc., Valencia CA).The nucleotide sequence of each is all used ABI Prism Model 377Sequencer (Perkin Elmer in 40 PCR products, CA) carry out forward and reverse mensuration, be determined at Penn StateNucleic Acid Facility, PSU Biotechnology Institute carries out.

The sequence of PCR product is arranged by hand, checks the difference between 10 animals.Compare with reported sequence (Urban etc., 1994, the same), 37 place's differences are arranged in the sample, and have only a base pair between this treated animal, variation to be arranged.In (155 bases before initiation site ATG codon) on-155 positions, 6 samples contain cytosine(Cyt) (CC), and 4 is polymorphic; Be that they have cytosine(Cyt) and born of the same parents' gland pyrimidine the two (CT), show the heterozygosity of this base pair.Especially meaningfully all 5 high peculiar smell pig samples all are the CC genotype, and have 4 to be the CT genotype in 5 low peculiar smell pig samples.

This polymorphic position is in a Restriction Enzyme recognition site, thereby the allelic existence of T just can change observed restriction fragment length pattern after the specific limited enzymic digestion.Under this particular case, used Restriction Enzyme is SphI (New England Biolabs).The allelic existence of T or scarce as measuring with the restriction fragment length polymorphism analysis comprises the restrictive diges-tion thing of checking CYP11a1 5 ' UTR with SphI in the DNA sample of 50 pigs of full group.Gel example sample is seen Fig. 2.The allelic existence of T is no matter be that (TT) that isozygoty or (CT) of heterozygosis link mutually with low pig peculiar smell.The allelic existence of CC is accompanied by high pig peculiar smell, and testicular weight increases cowper gland length and weight increase and the increase of submandibular salivary gland weight.See Fig. 3 and table 4.Have the allelic pig of CC in addition and show rate of body weight gain improvement 5.9%, relatively large lean meat is arranged, prove, and find out the tendency that has lipid content lower by measuring the back backfat degree of depth as long trunk.Having the allelic pig of CC, also to trend towards when butchering in institute's blood sampling serum testosterone concentration higher.

Record of production to the lineal female relatives (dam and compatriot) of these boars carries out retrospective analysis, show with have that relevant female of the allelic boar of T trends towards having bigger nest son (+.31 pig/nest) and the young weanling weight of nest higher (+4.27kg).Therefore, this kind polymorphism be it seems the feature that can bring useful fertility of sow and productivity.

Example II

The genetic marker of meat, growth, trunk and the reproduction feature of reflection cow and chicken

The evaluation of the CYP11a1 polymorphism of pig and the feature that feature is illustrated the corresponding polymorphism of improving reproduction and trunk feature that has promoted ox are illustrated.Fig. 5 provides the sequence of ox CYP11a1.The amplification ox is that the sequence of one group of CYP11a1 5 ' UTR autoploid suitable primer is as follows: justice is arranged: 5 '-GCAGATGTCCCTGGTGATTC-3 '; Antisense: 5 '-TGAACGGAGGGGAAGCC-3 '.

The feature of carrying out to the ox CYP11a1 sequence of amplification and corresponding hereditary property is illustrated as described in preamble herein pig CYP11a1 gene is gone.

Fig. 6 describes the CYP11a1 gene of chicken.For the chicken CYP11a1 sequence that Genbank is provided is determined its hereditary change in longer a 5 ' UTR, the cDNA sequence that Fig. 6 provides is used as the basis of carrying out the terminal rapid amplifying of 5 ' cDNA, and the test kit that uses contains the cDNA that carries out RACE for preparing from chicken as Clontech.The clone who obtains through this RACE approach produces genetics change relevant with the trunk characteristic with improving reproduction among 5 of chicken CYP11a1 sequence ' end 5 ' UTR for further analysis.Genetic polymorphism of Jian Dinging and variation are within the scope of the present invention like this.Suitable scheme about operation RACE is seen Current Protocols ofMolecular Biology, J.Wiley and Sons, and Inc.1998, the 15.6.9 chapter, its whole contents is hereby incorporated by.

The present invention is not limited to above specifically described embodiment, can have some change and modify under the situation of not leaving appended claim scope.

Sequence table

＜110〉Pennsylvania Research Foundation (The Penn State Research Foundation)

＜120〉genetic marker of domestic animal meat, growth, trunk and reproductive characteristic

<130>PSU-99-2102

<140>PCT/US00/13168

<141>2000-05-15

<150>60/134，180

<151>1999-05-13

<160>7

<170>FastSEQ?for?Windows?Version?3.0

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<220>

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＜223〉polymorphic site N=C or T

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<210>2

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＜213〉artificial sequence

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＜223〉composition sequence

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＜213〉artificial sequence

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＜223〉composition sequence

<400>3catgctgccc?ctaccactgc?tactct 26

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<212>DNA

＜213〉ox (Bos taurus)

<400>4gcagatgtcc?ctggtgattc?ctgaaacagg?ccctctgttt?aaattcttca?gcagttagag 60ggaaggtcaa?tttttcccaa?ggcttttggg?ctttgattgt?tttcattttt?aaattatctg 120cattctaaag?agatattttg?ggtggcagat?tttgctctcc?tacaggactt?tgtctaggag 180acggctctca?ggccagctcc?gacgactgtt?ccaaagaagt?aagggaaagc?tagggtttat 240atcaatcttt?ttttttgctg?ggagaagggg?gatgaacatg?tagtcaaaca?taaaaagatc 300actgctaatc?ccaaacaaca?gacacctcaa?gtgaatggtt?ttagtgtttt?tctatatatg 360ttgtttagtc?actaagtcct?gtccgactct?tttgcgactc?catagactgt?agcccaccaa 420gctcctctgt?ccatgggatt?tttctaggca?agaatactgg?agtgggttgc?catttccttc 480tccctgggat?cttcctaacc?caaggactga?acccttgtct?cctgcattgc?aggtggattt 540tttaccgact?gagccaccag?ggaagttatg?tgtgcaagaa?tccggggtca?tggaaatttt 600cccttagata?tacatcgtat?ctagggacca?gtacaatgca?aatgcttcct?gtttttcttc 660atcctgaagt?ctcctcaggg?tgcattgagg?gagggagtcc?cctcaggtgg?gtgaccacag 720tggctgacgc?ttgatgttgt?agaactggaa?tgatgggtta?cattctttcg?tttacagtac 780tgagtctggg?aggagctgtg?tgggctggag?tcagccggag?gaggctgacc?gccctgtcag 840cttctcactt?agccttgagc?tggtgattat?aagctgggtc?ccagggtccc?agggccagag 900tcacctgctg?cagtacgagc?agagacagca?gcagctgtgg?gggcagcatg?ctagcaaggg 960ggcttcccct?ccgttcagcc?ctggtcaaag?cctgcccacc?catcctgagc?tcagtggggg 1020agggctgggg?ccaccacagg?gtgggcactg?gagagggagc?tggcatctcc?acaaagaccc 1080ctcgccccta?cagtgagatc?ccctcccctg?gtgacaatgg?ctggcttaac?ctctaccatt 1140tctggaggga?gaagggctca?cagagaatcc?actttcgcca?catcgagaac?ttccagaagt 1200atggccccat?ttacaggtaa?gcctggcagg?aggattgggg?ctggcgggat?agggaagcct 1260gtggtggccc?cctccctgaa?aggtctgccc?tccccttcca?ggctctggtt?cacctctgac 1320tttatttctt?cctgcctggc?ggtggcagga?gtagagttaa?tgcttcccag?acagtgggtt 1380cacttcccag?ccctgaggcc?tcaacagtcc?ccgggctcta?cacccttaga?aactttgggg 1440aggtggggag?gcccaagaaa?ataagccccg?g 1471

<210>5

<211>1820

<212>DNA

＜213〉chicken (Gallus gallus)

<400>5cttttttcgg?ttgtaccttt?gtctctgtac?agatattttg?taatatatta?aaaacaaaac 60ctactgagct?cctcgccttg?agcccaggat?tcagggataa?gagcgaggtc?gccccggccg 120tgcgccgccc?tgctcccatg?ctctccaggg?ctgcacccat?agcgggcagc?tttcaggcat 180gccgctgtgc?cggagggatc?ccagccctcg?cgggggtcca?ctacccattg?cccagctcct 240cgggagctcg?gcctttcgac?caggtgccgg?gtgaatggag?agcgggttgg?ctcaacctgt 300accacttctg?gaaggaggga?ggcttccaca?acgtgcacaa?catcatggcc?agcaagttcc 360agcgctttgg?gcccatctac?agggagaagt?tgggtgtcta?cgagagcgtg?aatatcatca 420gcccccgcga?tgcggccacg?ctcttcaagt?cagaggggat?gctgcccgag?cgcttcagcg 480tgcccccatg?ggtggcatac?cgtgactacc?gcaacaagcc?ctacggcgtg?ctcctcaaga 540caggggaggc?ctggcgctcg?gaccgcctga?ccctgaacaa?ggaggtgctg?tcgccgcagg 600tggtggacag?cttcgtgccc?ttgctggacc?aggtgagcca?ggactttttg?cggcgggcac 660gggcgcaggt?ccagcagagc?ggccgggagc?gctggacggc?cgacttcagc?cacgagctct 720tccgctttgc?cttggagtct?gtgtgccacg?tgctgtatgg?ggaacgcctg?gggctgctgc 780aggactttgt?ggacccagag?gcacagcagt?tcatcgacgc?cgtcaccctc?atgttccaca 840ccacctcccc?catgctctac?gtgccacccg?ccctgctccg?ccacctcaac?accaagacat 900ggcgtgacca?cgtgcatgct?tgggatgcca?tcttcacaca?ggctgacaaa?tgtatccaaa 960acgtttaccg?ggacatccgg?ctgcaacgca?agagcaccga?ggagcacacg?ggcatcctct 1020tcagcctcct?tgtgcaggac?aagctgcccc?tggatgacat?caaggccagc?gtcaccgaga 1080tgatggcggg?cggcgtggac?acgacttcca?tgactctgca?atgggccatg?ctggagctgg 1140cacgatcccc?gggcatccag?gagcggctgc?gggcagaggt?gctggcagcc?aagcaggagg 1200cacaggggga?cagggtgaag?atgctgaaga?gcatccgact?gctcaaagcc?gccatcaagg 1260agactctcag?gctgcacccg?gtggcggtga?cgctgcagag?gtacaccaca?caggaggtca 1320tcctgcagga?ctaccgcatc?ccccccaaga?cgctggtgca?ggttggtctc?tacgccatgg 1380gacgagaccc?tgaggtcttc?cccaagccgg?agcagttcaa?ccctgagcgc?tggctggtga 1440tgggctccaa?gcacttcaag?ggactgagct?ttgggtttgg?gccacggcag?tgtctgggtc 1500gtcgcatcgc?cgagctggag?atgcagctct?tcctcatgca?catcctggag?aactttaaga 1560tcgaaaccaa?gcgggcggtg?gaagttggga?ccaagttcga?cctcattctt?gtccctgaaa 1620aacccatcta?cctgagactg?cggcccctcc?agccccagga?gtgacatggg?gtgtccccag 1680ttggtcccag?cttggggaca?cctccatcag?ctcagcgcat?tcagccttgg?ctccagccct 1740tcttacgcca?tgggggagat?ggctgccccc?ttcccatttt?cttcgcctct?gatttgctct 1800gtaatttctg?caccaaaagc 1820

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉composition sequence

<400>6gcagatgtcc?ctggtgattc 20

<210>7

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉composition sequence

<400>7tgaacggagg?ggaagcc 17

Claims (32)

1. the method for a filler test object, in order to identify those have preferably probably grow, growth, reproduction and trunk feature, as rate of body weight gain, carcass length, or the object of the young size of nest, comprise: obtain the sample of genetic stocks from tested object, measure the existence of the polymorphism in the CYP11a1 gene, and the latter is relevant with the young size of rate of body weight gain, carcass length and nest.

2. the method for claim 1, wherein said determination step is selected from next group of methods: restriction fragment length polymorphism (RFLP) is analyzed, heteroduple analysis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel elec-trophoresis (TGGE) (TGGE).

3. the process of claim 1 wherein that described mensuration exists the step of described polymorphism may further comprise the steps: with a kind of this genetic stocks of digestion with restriction enzyme that can at least one position, cut the CYP11a1 gene; Separating digesting gained fragment; Detect the restricted pattern that this fragment generates; This pattern and second kind of pattern with the CYP11a1 gene of this restriction enzyme gained are compared, and second kind of restricted pattern described herein increases with rate of body weight gain, carcass length increases and the young size increase of nest is associated.

4. the process of claim 1 wherein that described tested object is selected from pig, cow and chicken.

5. the method for claim 3, wherein said Restriction Enzyme is SphI, described tested object is a pig.

6. the method for claim 3, wherein said separation is gel electrophoresis.

7. the method for claim 3, the step of wherein said more restricted pattern comprises according to size identifies specific fragment, and more described segmental size.

8. the method for claim 5 also comprises this step: before digestion step, and the amount of amplification pig CYP11a1 gene, or it contains the amount of the part of this polymorphism.

9. the method for claim 3, wherein said restriction site is positioned at the non-translational region of CYP11a1 gene.

10. the method for claim 7, wherein said amplification may further comprise the steps: selection can be increased and be contained the forward and the reverse sequence primer in a zone of polymorphism restriction site in the pig CYP11a1 gene.

11. the method for claim 10, wherein said forward and reverse primer length between 10～50 Nucleotide, and are selected from SEQ ID NO:1.

12. the method for claim 10, wherein said forward primer are SEQ ID NO:2, described reverse primer is SEQ ID NO:3.

13. the method for claim 6, the step of wherein said detection clip size comprises: separate described fragment with gel electrophoresis in the presence of the contrast dna fragmentation that known dimensions is arranged; The fragment of having separated is contacted with probe, and probe and fragment hybridization form probe-fragment mixture; Determine separated segmental size by the segmental existence of detection probes.

14. an evaluation may further comprise the steps about the method for the genetics sign of pig growth rate, carcass length, the young size of nest or pig peculiar smell: breed same kind or Hybrid or derived from the male and female pigs of similar genetic pedigree; Determine the existence of growth rate, carcass length, filial generation quantity or pig peculiar smell; Measure the existence of polymorphism in every pig CYP11a1 gene; Find out growth rate, carcass length, filial generation quantity or pig peculiar smell exist with described polymorphism between related, identify a kind of polymorphism thus about these characteristics.

15. the method for claim 14 further comprises: select to be predicted as according to described sign have better growth rate, longer trunk, the young size of nest increases or the pig of low pig peculiar smell is raised step.

16. the method for claim 14, wherein said analysis comprise the DNA with Restriction Enzyme Sphl digestion pcr amplification.

17. the method for claim 12, wherein the polymorphism that is associated with growth rate, carcass length, the young size of nest or pig peculiar smell detects with forward and reverse primer, and these primers comprise at least 4 continuous bases in SEQNOS:2 and 3.

18. a test kit of estimating the pig DNA sample comprises: in a container, a kind of reagent of identifying pig CYP11a1 gene pleiomorphism.

19. the test kit of claim 18, reagent wherein are a kind of amplification pig CYP11a1 gene or its segmental primer.

20. the test kit of claim 18 also comprises a kind of archaeal dna polymerase, a kind of Restriction Enzyme that cuts at least one position of pig CYP11a1 gene; And the forward and the reverse primer that contain the zone of a polymorphic site in the pig CYP11a1 gene that can increase.

21. be used for measuring a kind of primer that there is polymorphism Sphl site in pig CYP11a1 gene, wherein this primer comprises a sequence from SEQ ID NO:2 and SEQ ID NO:3.

22. with young size of growth rate, carcass length, nest and the related a kind of genetic marker of pig peculiar smell of pig, this sign comprises the polymorphism in the pig CYP11a1 gene.

23. the genetic marker in the claim 22, wherein said polymorphism are Sphl restriction sites.

24. the sign of claim 22, wherein said polymorphic position is in 5 ' non-translational region of pig CYP11a1 gene.

25. the section of DNA sequence in pig CYP11a1 gene 5 ' non-translational region, this sequence is made up of SEQID NO:1.

26. design be used for increasing a kind of primer of pig CYP11a1 gene polymorphism Sphl restriction site, wherein this primer is from 4 among the SEQ ID NO:1 or more base continuously.

27. design be used for increasing a kind of primer of pig CYP11a1 gene polymorphism Sphl restriction site, wherein this primer is a kind of reverse primer that generates from SEQ ID NO:1.

28. a kind of method of screening pig, in order to determine that those more may have the pig of the growth rate that increases, longer trunk, big nest son, higher pig peculiar smell, and/or those lessly may show the pig that increases growth rate, longer trunk, big nest son or higher pig peculiar smell, the method includes the steps of: the allelotrope of determining the existing CYP11a1 gene of pig; Determine the allelotrope of other sign of the gene of young size of known effect growth rate, carcass length, nest or pig peculiar smell; Selection has allelic desirably combined animal, and gets rid of the animal that those have bad combination.

27. the method for claim 28 wherein comprises the allelic mensuration of CYP11a1 and measures at least one allelic existence, this equipotential gene indicates with at least one, with DNA that CYP11a1 directly or indirectly links to each other and is associated.

30. the method for claim 28, wherein the DNA sign is a little satellite.

31. the method for claim 28, wherein the DNA sign is SO 064, and SO 102, SO078, and SO 158, and SO 066, and SW 304, and SW 1083, SO 101 or SO 212.

32. the method for claim 28, wherein sign is selected from following group: tumor necrosis factor alpha (TFN α), CYP11a1, lactotropin (PPL), estrogen receptor (ER) and lactotropin acceptor (PRLR).

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