CN101603086B - Molecular marking method for indicating and identifying chicken abdominal fat weight by using ACC alpha gene - Google Patents
Molecular marking method for indicating and identifying chicken abdominal fat weight by using ACC alpha gene Download PDFInfo
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Abstract
本发明提供了一种用ACCα基因预示和鉴定鸡腹脂量的分子标记方法。根据鸡ACCα基因序列设计一对引物；利用该引物对鸡的基因组DNA进行PCR扩增，并利用限制性内切酶Mwo I消化扩增的PCR产物。7周龄实验结果表明具有AA基因型个体的腹脂重和腹脂率分别比GG基因型个体腹脂重和腹脂率高6.03克和0.25％。本发明操作简单、费用低、精确度高，可进行快速检测。利用本发明的分子标记方法对鸡腹脂性状进行选择，不仅为鸡育种工作中标记辅助选择提供了一个更为有效、简便易行的分子标记方法，同时也为鸡的腹脂性状改良提供了一种有效的分子标记育种手段，从而可以加速低脂肉鸡的育种进程。
The invention provides a molecular marker method for predicting and identifying chicken abdominal fat mass with ACCα gene. A pair of primers were designed according to the sequence of the chicken ACCα gene; the primers were used to carry out PCR amplification on chicken genomic DNA, and the amplified PCR product was digested with restriction endonuclease Mwo I. Experimental results at 7 weeks showed that the abdominal fat weight and abdominal fat rate of individuals with AA genotype were 6.03 grams and 0.25% higher than those of individuals with GG genotype. The invention has the advantages of simple operation, low cost, high precision and rapid detection. Utilizing the molecular marker method of the present invention to select chicken abdominal fat traits not only provides a more effective and convenient molecular marker method for marker-assisted selection in chicken breeding work, but also provides a new method for improving the abdominal fat traits of chickens. An effective molecular marker breeding method can accelerate the breeding process of low-fat broiler chickens.
Description
(1) technical field
The invention belongs to animal molecular genetics field, particularly a kind of method that adopts the Markers for Detection chicken abdomen fat content.
(2) background technology
Fryer is through various countries breeder seed selection for many years, and its production performance has had and significantly improves.When fryer early growth speed was obviously improved, a series of problem demanding prompt solutions had also appearred in aspects such as fryer self physiological fitness.Fast large-scale fryer body fat (especially abdomen fat) is accumulated and has too much been become special distinct issues.It is many unfavorable that broiler chicken internal deposition excess fat has: (1) obviously reduces feed efficiency, because the fatty tissue of sedimentation unit weight consumes the triple energy than the lean meat of sedimentation unit weight more; (2) reduced the ratio of trunk lean meat, thereby reduced the output that cuts meat fatty tissue; (3) processor and human consumer abandon a big chunk (abdomen fat pad, muscular stomach are fatty, crop is spoken in a low voice fat and mesentery fat etc.) of these fat of broiler chicken internal deposition on every side, this has not only increased processor and human consumer's burden, and increased lipid content in refuse and the treating water, thereby contaminate environment.In view of this, broiler chicken internal deposition excess fat causes conspicuous financial loss will for the producer, processor and human consumer.Meat kind chicken overfertilization also will have a strong impact on laying rate, rate of fertilization and hatching rate simultaneously, and the generation of meeting induced lipolysis liver syndromes, thereby has strengthened the death rate of laying period.Therefore, control fat is too much accumulated in that chicken is intravital, further improves the feed efficiency of fryer and carcase quality and is China and be badly in need of the significant problem researched and solved.
(acetyl-CoA carboxylase ACC) belongs to intermolecular carboxylation transfer family member to acetyl-CoA carboxylase.Acetyl-CoA carboxylase has two kinds of analogss: acetyl-CoA carboxylase α (ACC α) and acetyl-CoA carboxylase β (ACC β).ACC can generate malonyl CoA by the catalysis acetyl-CoA, is the speed limit regulatory enzyme of lipogenesis.Malonyl CoA is as longer chain fatty acid synthetic precursor, in lipid acid synthetic as the donor of 2 carbosilane units, and in oxidation of fatty acids,, can also come ingesting and energy balance is regulated and control by regulating hypothalamus as a kind of signaling molecule simultaneously as the regulatory factor of plastosome shuttle system.
In Mammals, the function of two types ACC genes there has been research more deeply.ACC α and ACC β lay respectively on kytoplasm and the mitochondrial membrane, and the malonyl CoA that is produced by their catalysis is positioned at two relatively independent zones, can not mix.The malonyl CoA that is arranged in kytoplasm is used to carry out the de novo synthesis of lipid acid, be positioned at the activity that near the malonyl CoA of plastosome is then regulated carnitine palmitoyltransferase I, thereby regulation and control are to oxidation of fatty acids (Abu-Elheiga, 2005).Therefore, the synthetic and oxidising process of lipid acid is not simultaneous.In birds and mammiferous lipogenesis tissue, ACC α gene transcription level is subjected to nutrition and hormone dual regulation.The transcriptional level that is in ACC α in its liver of chicken of starvation is very low, and ACC α gene transcription level can improve 11 times (Hillgartner etc., 1996) when the Hi CHO of feeding, low-fat feed.During vitro culture chicken embryo of former generation liver cell, add activated form T3 (triiodothyronine), can make ACC α gene transcription efficient improve 5-7 doubly (Zhang etc., 2001).Mooney etc. (2008) find when why the research isoamyleneguanidine can cause mouse to lose weight, its main path is the activity that isoamyleneguanidine can suppress ACC, the activity of ACC α and ACC β reduces can make lipogenesis reduce, and Fatty Acid Oxidation is accelerated simultaneously, thereby influences the weight character of mouse.People such as Liang (2006) studies show that, ACC α has important effect in metabolism of fat, and diseases such as ACC α dysfunction can cause fat, type ii diabetes have significance with ACC α as relative diseases such as the target gene of medicine treatment obesities.After Abu-Elheiga etc. (2005) pointed out to knock out rat ACC α gene, rat produced the embryonic death phenomenon, and ACC α is extremely important to embryo's early development as can be seen.
People such as Takai (1987) isolate ACC α in the liver organization of chicken.ACC α gene is positioned at No. 19 karyomit(e)s of chicken, CDS total length 6974bp, and dna sequence dna is made up of 50 exons and 49 introns, total length 92409bp, 2324 amino acid of encoding.The aminoacid sequence of chicken, mouse, people's ACC α is closely similar, and its homology is about 90%.In Mammals, ACC α gene is mainly expressed in liver and fatty tissue.The ACC α gene that studies show that of this seminar all has expression in chicken abdomen fat, liver, spleen, glandular stomach, muscular stomach.
(3) summary of the invention
The object of the present invention is to provide a kind of Protocols in Molecular Biology, promptly adopt the method for PCR-RFLP to detect G → A variant sites in the chicken ACC α gene extron 19, simple to operate, expense is low, tolerance range is high, that can carry out rapid detection is a kind of with the indication of ACC α gene with identify the molecule marking method of chicken abdomen fat content.
The object of the present invention is achieved like this:
1, designs a pair of primer G2292AF and G2292AR according to chicken ACC α gene order, wherein downstream primer 3 ' holds last bases G to be artificial design base (not with the genome sequence pairing), and purpose is in order to produce a mandatory restriction enzyme site near mutating alkali yl:
G2292AF：5’-TAG?GTG?GAT?GGT?TGG?GCT?TGA-3’
G2292AR：5’-CCC?CAT?CCT?CCA?CCA?CAT?G-3’
2, utilize this primer that the genomic dna of chicken is carried out pcr amplification, pcr amplified fragment length is 186bp;
3, utilize restriction enzyme Mwo I digest amplification PCR products;
4, functional quality is carried out electrophoretic separation than 14% polyacrylamide gel to postdigestive PCR product, detects the G → A single base mutation in the ACC α gene extron 19;
5, when variant sites is bases G in the chicken ACC α gene extron 19, can produce the restriction enzyme site (GCTTATC/CAGC) of restriction enzyme Mwo I, can produce 2 fragments (165bp, 21bp) behind the Mwo I digestion PCR product, with its called after GG genotype; When this site is base A (GCTTATCCAAC), then there is not Mwo I restriction enzyme site, produces 1 fragment (186bp) behind the Mwo I digestion PCR product, its called after AA genotype; When this site is the G/A heterozygosis, can produce 3 fragments (186bp, 165bp, 21bp) behind the Mwo I digestion PCR product, (the 21bp fragment that produces after wherein enzyme is cut is run out of gel with its called after AG genotype, do not show this fragment among the glue figure, utilize 186bp, 165bp two bar segment that this mutational site somatotype is not influenced the result).
The present invention also comprises some technical characterictics like this:
1, the method for described analyzing gene polymorphism is the genotype of classifying by the sudden change that detects base.
2, the described position that is used for the analyzing gene polymorphism is an exon.
3, the described position that is used for the analyzing gene polymorphism is the exon district of the primer amplification represented by G2292AF and G2292AR.
4, the described site that is used for analyzing polymorphism is selected from the base mutation of the G → A of the 464th of the following sequence of chicken ACC α gene.
1 GATGACGTGT?GGTCACTTGT?AAGAGCCCCA?GTACTCTGAC?CATGCAGTTA?GCTGAACCAA
61 TGTGCAGGTT?ATTTAGGTGG?GCTCAGATTC?CCATTTCCTA?AAGCTTCATG?GTTTGTGATT
121?TAAAATATGT?GCCAGAGGAG?ATTTAGATTG?GATACCAGAA?AGAATTTATT?CAAAGGAGAG
181?GTAGTTAGGT?GTTGTAACAG?ACTGCCCAGG?GAAGTGGTAG?TATTCTGCTA?CCTGGGAACA
241?TTTAAGAGAT?GTGGACGTGG?TGCGTAGGGA?TGTGATTTAG?CAGTGGACTT?GTCATGTTAG
301?GTGGATGGTT?GGGCTTGATG?GTCCTGAAAG?TCTTTTGCAA?CCTAATTCCG?TTCTATCTGT
361?TATTTCCTAG?GTATCGCATC?ACTATAGGTA?ACAAGACCTG?TGTGTTTGAA?AAGGAAAATG
421?ATCCTTCTAT?TCTGCGCTCA?CCTTCGGCTG?GGAAGCTTAT?CCAGTATGTG?GTGGAGGATG
5, described chicken abdomen fat content refers to the heavy and abdomen fat rate of abdomen fat of chicken.
6, the described restriction enzyme that is used to digest the PCR product is Mwo I.
7, the polyacrylamide gel quality during described PCR-RFLP analyzes is 14% than concentration.
The present invention has designed the primer of a pair of mandatory restriction enzyme site, adopts the method for PCR-RFLP to detect G → A variant sites in the chicken ACC α gene extron 19, and is simple to operate, expense is low, tolerance range is high, can carry out rapid detection.The present invention designs a pair of primer according to chicken ACC α gene order; Utilize this primer that the genomic dna of chicken is carried out pcr amplification, and utilize restriction enzyme Mwo I digest amplification PCR products, use 14% polyacrylamide gel that postdigestive PCR product is carried out electrophoretic separation then, detect the single nucleotide variation site of a G → A in the ACC α gene extron 19; Homozygous and heterozygous is named to two kinds in this site of chicken ACC α gene respectively.7 age in week experimental result show the heavy and abdomen fat rate of the abdomen fat with AA genotype individuality heavy and high 6.03 grams of abdomen fat rate and 0.25% than the individual abdomen fat of GG genotype respectively.Utilize molecule marking method of the present invention that chicken ventral fat character is selected, not only provide more effective, a simple and easy to do molecule marking method for marker assisted selection in the chicken breeding work, also improve simultaneously a kind of effective molecular marker breeding means are provided, thereby can quicken the breeding process of low fat fryer for the ventral fat character of chicken.
Experimental results show that the abdomen fat with AA genotype individuality weighs and the abdomen fat rate utmost point is significantly higher than the heavy and abdomen fat rate (P＜0.01) of the abdomen fat with GG genotype chicken.In the colony that the present invention studied, have heavy high 6.03 grams of abdomen fat of the abdomen fat anharmonic ratio GG genotype individuality of AA genotype individuality; It is higher by 0.25% than the abdomen fat rate of GG genotype individuality to have the abdomen fat rate of AA genotype individuality.
(4) description of drawings
Fig. 1 is that ACC α gene G2292A restriction enzyme site is analyzed collection of illustrative plates.
(5) embodiment
The invention will be further described below in conjunction with specific embodiment:
One, experiment material
1. laboratory animal and property determination
The high and low fat of Northeast Agricultural University be the 8th from generation to generation, the 9th from generation to generation, the tenth from generation to generation, eleventh generation resource colony, chosen 374,362,624 and 477 chickens respectively; Wing venous blood collection during 7 ages in week, the oxalate anti-freezing; Butcher after the weighing live-weight, the weighing carcass is heavy, abdomen fat heavily waits proterties.
2. medicine and enzyme
Tutofusin tris (Tris), Sigma ChemicaLs Co; The saturated phenol of Tris, Beijing ancient cooking vessel state biotech development center; Proteinase K (Proteinase K), MMERCK Co; DL 2000, dNTP (dATP; DTTP; DCTP; DGTP), the Taq enzyme, the precious biotech firm in Dalian; Agarose (Agarose), acrylamide, methylene diacrylamide, the white company in Yuanping City.
3. key instrument
PTC-200PCR instrument (PERKIN ELMER), Biometra grads PCR instrument, the multi-functional imaging system of UVP, ULtrospec 1000 ultraviolet spectrophotometers, BECKMAN refrigerated centrifuge, MiLLi-Q ultrapure water instrument, DYY-III2 voltage stabilizing electrophoresis apparatus and supporting electrophoresis chamber.
Two, experimental technique
1. the preparation of damping fluid and common agents
1M TrisCl:121.14g Tris alkali is dissolved in the 800ml distilled water, with hydrochloric acid adjust pH to 8.0, is settled to 1000ml, autoclaving.
TE damping fluid: 10mM TrisCl, 1mM EDTA, pH8.0, autoclaving.
20 * SET damping fluid: 3MNaCl, 1M TrisCl (pH 8.0), 20mM EDTA (pH 8.0), autoclaving.
5 * tbe buffer liquid: 54g Tris alkali, 27.5g boric acid, 20ml 0.5M EDTA (pH8.0) adds water to 1L.
50 * TAE damping fluid: 242g Tris alkali, the 57.1ml glacial acetic acid, 100ml 0.5MEDTA (pH8.0) adds water to 1L.
1M TrisCl:121.14g Tris alkali is dissolved in the 800ml distilled water, with hydrochloric acid adjust pH to 8.0, is settled to 1000ml, autoclaving.
0.5M EDTA:186.1g EDTA is dissolved in the 800ml distilled water, with NaOH adjust pH to 8.0, is settled to 1000ml, autoclaving.
3M NaAc (pH5.2): 408.1g NaAc3H
2O is dissolved in the 800ml distilled water, transfers pH to 7.0 with glacial acetic acid, is settled to 1000ml.
200ml silver dye liquor: NH
3H2O 2ml; 3.6%NaOH 4.2ml; 20%AgNO
33.6ml, add deionized water to 200ml.
The 200ml liquid that develops the color: 1% Trisodium Citrate 1ml; Formaldehyde 100ul; Add deionized water to 200ml.
Fowl blood lysate liquid: 10mM TrisCl (pH8.0), 0.1M EDTA (pH8.0), 0.5%SDS.
2. primer design is with synthetic
Following primer is synthetic by Shanghai Bo Ya biotech firm:
G2292AF：5’-TAG?GTG?GAT?GGT?TGG?GCT?TGA-3’
G2292AR：5’-CCC?CAT?CCT?CCA?CCA?CAT?G-3’
3. a small amount of of chicken genomic dna is extracted
Method one:
(1) get 20 μ l anticoagulated bloods, add 500 μ l fowl lysates, adding Proteinase K to final concentration is 100-200 μ g/ml, and 55 ℃ of digestion of mixing 12hr no longer includes the heavy-gravity agglomerate in solution.
(2) solution is cooled to room temperature, adds 5M NaCl to final concentration 1.5M, mixing 10min.Add equal-volume phenol/chloroform, put upside down centrifuge tube mixing 10min repeatedly.
(3) 12,000rpm, the centrifugal 10min of room temperature.Get supernatant, add equal-volume chloroform mixing 10min.
(4) 12,000rpm, the centrifugal 10min of room temperature.Get 2 times of volume dehydrated alcohols of supernatant deposit D NA.
(5) DNA is chosen be put in the 1.5ml centrifuge tube, wash 1 time with 70% ethanol.
(6) 7,500rpm, the centrifugal 5min of room temperature abandons supernatant.
(7) (attention can not be too dried) after the DNA drying is dissolved among the 200 μ l TE.
Method two:
(1) adding of 20 μ l whole bloods is equipped with in the 1.5ml centrifuge tube of 700 μ l, 1 * SET, gently mixing.
(2) add Proteinase K (10mg/ml) to the SDS of final concentration 100-200 μ g/ μ l and 10% to 0.5%, 55 ℃ of digestion of final concentration 12h.
(3) after waiting to digest fully, add the saturated phenol of isopyknic Tris, put upside down back and forth, make its mixing
The centrifugal 10min of (4) 12,000rpm carefully moves into the upper strata water in another centrifuge tube with cutting off most advanced and sophisticated suction nozzle, discards organic phase.Repeat third and fourth step once.
(5) add isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution (volume ratio is 24: 23: 1) to aqueous phase, mix 10min.12,000rpm., centrifugal 10min shifts out water to another centrifuge tube.
(6) add isopyknic chloroform, primary isoamyl alcohol mixed solution (23: 1) to aqueous phase, put upside down back and forth and mix 10min, 12,000rpm, centrifugal 10min shifts out water to another centrifuge tube.
(7) (3M pH5.2) and the dehydrated alcohol of 2 times of volumes, puts upside down deposit D NA back and forth to add 1/10 volume NaAc to aqueous phase.
(8) DNA is chosen be put in the 1.5ml centrifuge tube, wash 1 time with 70% ethanol.
The centrifugal 5min of (9) 7,500rpm.Carefully outwell ethanol in the pipe, will be upside down on the filter paper, allow ethanol flow to end, place air drying.
(10) TE of adding 200 μ l puts the dissolving DNA that spends the night in 50 ℃ of water-baths.Be stored in after the dissolving-20 ℃ standby.
4.PCR reaction
(1) be that template is carried out pcr amplification with chicken DNA, comprise following solution or reagent in the 10uL reaction system:
10×PCR?reaction?buffer 1μL
DNTP Mixture (each 2.5mM) 0.8 μ L
Primer 1 (10 μ M) 0.1 μ L
Primer 2 (10 μ M) 0.1 μ L
EX-Taq(5U/μL) 0.1μL
Deionized water 6.9 μ L;
Genomic dna (50ng/ μ L) 1.0 μ L
(2) carry out the PCR reaction with above-mentioned solution mixing and by following condition.
94 ℃ of sex change 7min; 94 ℃ of 30sec, 56 ℃ of 30sec, 72 ℃ of 30sec, 33 circulations; 72 ℃ are extended 7min.
(3) after reaction finishes, get PCR reaction solution (3 μ L) and carry out agarose gel electrophoresis, detect the PCR product, 4 ℃ of preservations.
5.PCR-RFLP reaction system and condition
It is as follows that enzyme is cut system:
Mwo I restriction endonuclease 10U/ μ L 0.8 μ L
10×Buffer： 1μL
PCR?Production 0.8μL
Add deionized water to 10 μ L
With above-mentioned reaction solution mixing, digestion is 4 hours in 37 ℃ of water-baths.
6. polyacrylamide gel analysis
With quality than concentration be 14%, volume is 25ml, thickness is that the glue of 0.1cm is example.
(1) cleans sheet glass that glue uses and clean, after the oven dry, with the slit between 0.8% agarose closed glass plate and adhesive tape with distilled water flushing.
(2) in the 100ml beaker, add 30% acrylamide 11.7ml, 5 * TBE 5ml, 10% ammonium persulphate 0.175ml, TEMED8 μ l, deionized water 8.1ml, rapid encapsulating behind the mixing.
(3) stop encapsulating when watering extremely from sheet glass upper edge 0.1cm, insert comb, room temperature is gathered and half an hour, 4 ℃ of preservations of unnecessary acrylamide.At any time observe the gel polymerisation situation, and add acrylamide.
(4) gel polymerisation good after, add 1 * TBE to electrophoresis chamber, use the irrigation with syringe well.
(5) prerunning 10min prepares point sample simultaneously.
(6) get 10 μ l enzymic digestion products and place the PCR pipe, add sample Buffer mixing in the non-sex change of 5-6 μ l, use the microsyringe point sample.
(7) 180 volts, electrophoresis 3-4h.
7. cma staining method
(1) electrophoresis is shut electrophoresis apparatus after finishing, and emits electrophoresis liquid, carefully takes off gel, places 70% ethanol, and the water-bath oscillator slowly shakes up fixedly 10-15min.(ethanol uses up and can reclaim).
(2) distilled water is washed glue 2 times, and each 2min removes residual ethanol.
(3) with 200ml staining fluid dyeing 30min.
(4) distilled water is washed glue 3 times, each 2min.
(5) with the colour developing of 200ml colour developing liquid, about 10-30min when the intensity of being with as DNA is suitable, outwells colour developing liquid.
(6) deionized water is washed unnecessary colour developing liquid off, and preservative film is sealed, scanography or preservation.
8. statistical model is set up
Characteristics according to resource colony make up suitable statistical model:
①Y＝μ+G+L+GE+G*L+G*GE+F(L)+D(F，L)+BW
7+e
Y is the character observation value, μ is colony's average, G is the genotype fixed effect, and L is the strain fixed effect, and GE is the generation effect, G*L be genotype and strain make effect mutually, the G*GE genotype with from generation to generation make effect mutually, F (L) be the stochastic effect of the interior family of strain, D (F, L) be the stochastic effect of hen in family and the strain, BW
7As the covariance variable, e is a residual value shi.Use the JMP of statistical software 4.0 (SAS Institute Inc., Cary, the NC) degree of correlation between check genotype and proterties, and the least square average of estimation proterties.
Three, experiment conclusion
The polymorphism of chicken ACC α gene and the high and low fat two-way choice of fryer strain the 8th, nine, ten, ten be merging (hereinafter to be referred as " merging from generation to generation ") ventral fat character relevant from generation to generation of four generations altogether
In conjunction with Fig. 1, utilize primer of the present invention (G2292AF and G2292AF) that the genomic dna of the high and low fat two-way choice of fryer strain chicken is carried out pcr amplification, and utilize Mwo I digestion with restriction enzyme PCR product, carry out volume ratio 14% polyacrylamide gel electrophoresis analysis then.Detect 3 kinds of genotype altogether, when variant sites is bases G in the chicken ACC α gene extron 19, the restriction enzyme site (GCTTATC/CAGC) of restriction enzyme Mwo I can be produced, 2 fragments (165bp, 21bp) can be produced behind the MwoI digestion PCR product, its called after GG genotype; When this site is base A (GCTTATCCAAC), then there is not Mwo I restriction enzyme site, produces 1 fragment (186bp) behind the Mwo I digestion PCR product, its called after AA genotype; When this site is the G/A heterozygosis, can produce 3 fragments (186bp, 165bp, 21bp) behind the Mwo I digestion PCR product, (the 21bp fragment that produces after wherein enzyme is cut is run out of gel with its called after AG genotype, do not show this fragment among the glue figure, utilize 186bp, 165bp two bar segment that this mutational site somatotype is not influenced the result).
3 kinds of genotype that ACC α gene G2292A site is produced with the abdomen fat that merges 1837 individualities from generation to generation heavy and abdomen fat rate carry out the least square analysis, the result show genotype to the abdomen fat of chicken weigh, abdomen fat rate has utmost point remarkably influenced (P＜0.01) (table 1).
Table 1 merges the least square analytical results (P value) of ventral fat character from generation to generation
Carry out multiple comparisons to merging from generation to generation the least square average of chicken ventral fat character between 3 kinds of genotype, the result shows that the heavy and abdomen fat rate utmost point of the abdomen fat of AA genotype individuality is significantly higher than the abdomen fat weight and the abdomen fat rate (P＜0.01) of AG type and GG type individuality; Heavy and the abdomen fat rate of the abdomen fat of AA genotype individuality is than abdomen fat heavy and high respectively 3.88 grams of abdomen fat rate and 0.14% of AG genotype individuality; Heavy and the abdomen fat rate of the abdomen fat of AA genotype individuality is than heavy and high respectively 6.03 gram and 0.25% (tables 2) of abdomen fat rate of abdomen fat of GG genotype individuality.
Table 2ACC α gene different genotype is combined the influence of chicken ventral fat character from generation to generation
During the average comparison same row do not have same letter person's difference extremely significantly (
A-BP＜0.01)
A: additive effect, a=(GG-AA)/2
D: dominant effect, d=AG-(AA+GG)/2
D: degree of dominance D=d/a
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