CN102747153B - Millet waxy gene cosegregation molecular marker and detection method thereof - Google Patents
Millet waxy gene cosegregation molecular marker and detection method thereof Download PDFInfo
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Abstract
The present invention relates to a millet waxy gene cosegregation molecular marker and a detection method thereof, which are used for utilization of millet waxy resource and breeding of a novel waxy variety. The molecular marker is an InDel molecular marker, and an amplification primer is a waxy-TSI2 / int1 primer combination and a waxy-int1-1F / 4R primer combination.
Description
Technical field
The molecular marker identification method that the invention provides millet IV type waxy gene, belongs to molecular genetics field, is exclusively used in the Molecular Identification of the IV type waxy gene type of millet.
Background technology
Millet, claims millet after shelling, millet essential substance is starch, and content is in 75% left and right, and general kind amylose content is more than 12.8%, and waxy kind amylose content is between 1.24%-7%.Semen setariae grain is little, and look yellowish is or deep yellow, and quality is harder, and Semen setariae starch molecule amount is less more than 20 times than common millet, and edible digestibility is higher more than 20% than common millet, and it also has higher viscosity and good palatability, and finished product have fragrant and sweet taste.The Semen setariae starch of heat treatment has higher bulging force and the transparency, and these excellent characteristics are given the value of Semen setariae preciousness and purposes widely.
Millet is a kind of nutritious food, and Semen setariae is also cited as " king of nutrition " and classifies " protective foods " as abroad.Often edible millet has important effect to Protein requirement, amino acid whose metabolic balance.The effect of Semen setariae mainly contains 1. because of its rich vitamin B1, B12 etc., has the effect of the raw sore of the maldigestion of preventing and bicker; 2. there is the effect that prevents general stomach, vomiting; 3. also there is the function of nourishing YIN and benefiting blood, can make the physique of puerpera's cold of insufficiency type be taken good care of, help them to regain one's strength; 4. Semen setariae has the wrinkle of alleviating, color spot, Pigmented effect.For this reason, Semen setariae has unlimited processing potentiality to be exploited, to pulling China's characteristic millet industry development to have important effect.
Michelia floribunda is the waxy Spiked Millet Resources of high-quality, but other economical character is very poor.Yungu No.1 system obtains the improved seeds of state award for inventions, stalk property quality.The Ji wound No. 1 (seed selection of this seminar) being formed by the seed selection of Yungu No.1 * (1066A sterile material * Michelia floribunda) F4 composite hybridization, it is the waxy kind of high yield, high-quality, its amylose content (accounting for sample dry weight) only 1.22%, in the glutinous matter kind of millet, content, in minimum level, shows that it is high glutinous matter kind at home.Made at present the millet processed finished products that Semen setariae wine, Semen setariae vinegar, Semen setariae breast, sticky cake etc. have glutinous matter characteristic.
The Breeding Process that No. 1, Ji wound is gone through 15 years, and molecular mark technology is by target gene is selected fast and accurately, can provide very effective instrument for breeding.If served as a mark with the difference between target gene again, can effectively avoid the problem of molecule marker and the chain exchange of target gene, can select in seedling stage simultaneously, not high to the specification of quality of DNA with round pcr, and practicality is very strong, thereby greatly shortened breeding cycle, also improved efficiency of selection.For this reason, this has been researched and developed and can carry out to glutinous matter proterties the codominant marker of genotype identification, for setting up the technical system of millet waxy gene evaluation, lays a good foundation, and can greatly improve the glutinous matter breeding process of millet, promotes millet industrialized development.
Summary of the invention
The object of the invention is: the molecule marking method that provides a kind of glutinous matter millet to differentiate, utilize the Applied economy phenotypic marker of the PCR-based of these research and development, can effectively from millet, identify the glutinous matter millet containing IV type waxy gene, for the utilization of the glutinous matter resource of millet and the seed selection of glutinous matter kind, described in be labeled as waxy-TSI2/int1 and waxy-int1-1F/4R.
The present invention relates to a kind of molecule marking method of differentiating millet waxy gene, it is characterized in that:
With the codominance InDel molecule marker primer of two pairs of specific amplified millet waxy genes, difference called after waxy-TSI2/int1 and waxy-int1-1F/4R:
Waxy-TSI2/int1 upstream primer: 5 ' GAAGTGGAGGATACTGATGGG 3 '
Waxy-TSI2/int1 downstream primer: 5 ' GGATTTGACTGCATGAAAATG 3 '
Waxy-int1-1F/4R upstream primer: 5 ' CCGTTTCGGTCGGTAGGAAG3 '
Waxy-int1-1F/4R downstream primer: 5 ' AGTGGGTTCGATGTTTGAAC3 '
The DNA of amplification millet, if waxy-TSI2/int1 combination of primers can amplify the amplified fragments of 984 bp, and waxy-int1-1F/4R combination of primers can not amplify the amplified fragments of 540 bp, this material is the glutinous matter millet containing IV type waxy gene.
The InDel mark of differentiating glutinous matter millet provided by the present invention, obtains by the following method:
1) from Foxtail Millet Germplasm Resources, select the glutinous millet Michelia floribunda of high-quality, by itself and stalk matter millet variety Yungu No.1 preparation reciprocal cross F
1.Results reciprocal cross F
1after selfing again, build the F of waxy gene
2segregating population, F
2the F that ties
3seed is through iodine staining, and the segregation ratio of stalk matter individual plant and glutinous matter individual plant meets 3 ︰ 1, and the waxy by stealthy Dominant gene of Michelia floribunda is described.
2) the Waxy genes encoding particle condensation type amylosynthease (GBSS1) of millet, determines that the amylose starch in millet endosperm and pollen is synthetic.Starch in common millet (WxWx) seed is comprised of amylose starch and amylopectin.In glutinous millet (wxwx) seed, contain on a small quantity or not containing amylose starch, almost all by amylopectin, formed.Because different transposons insert the different loci of GBSS1 gene order, Waxy transgenation becomes various allelotrope, weaken or eliminated the expression of GBSS1 gene, the enzyme of its proteins encoded particle condensation type amylosynthease is lived and affected between boundary in 5% to 95%, thereby form glutinous matter (waxy) or low starch content (low-amylose) proterties.4 pairs of primer sequences of the glutinous matter type of differentiation of having reported according to document (Mol Gen Genomics, 2005,274:131 – 140), we synthesize these 4 pairs of primers, and sequence is as follows:
Ex1/ex2 primer nucleic acid base sequence:
Upstream primer: 5 ' TGCAAGCCAGTGACGGATCGACGACGACAC3 '
Downstream primer: 5 ' ATGCCGGTGACCAGCGTGGAGGGCTAGCTA3 '
Ex2int2/ex4r primer nucleic acid base sequence:
Upstream primer: 5 ' CATGGCCGTAAGTCCCATCGATCGATCATC3 '
Downstream primer: 5 ' TAGCAGTGGAAGAACCTCACCCTCTCGTAC3 '
M5/R7 primer nucleic acid base sequence:
Upstream primer: 5 ' GGACGTCAGCGAGTGGGACC3 '
Downstream primer: 5 ' ACGAGTCCACCGGTGGACGC3 '
M7/R10 primer nucleic acid base sequence:
Upstream primer: 5 ' CACCAGCCGCTTCGAGCCCT3 '
Downstream primer: 5 ' GCCGGCGTGCCGACCACCTT3 '
As shown in Figure 1, wherein, only have the genomic dna amplified production different (other 3 group primer extension products all identical) of ex1/ex2 combination of primers to the glutinous matter Michelia floribunda of millet male parent and maternal stalk matter Yungu No.1, the former amplified fragments is in 5 kb left and right, and the latter's amplified fragments is 828 bp.By confirmation that male parent PCR product is partly checked order, amplified fragments sequence and gi|62318482|dbj|AB210215.1| Setaria italica GBSS1 gene, transposable element TSI-2 in intron 1 mates completely, illustrate that Michelia floribunda belongs to IV type waxy gene, be that the interior unit 1 that is inserted into GBSS1 gene by TSI-2 transposon forms, TSI-2 transposon size is 5251 bp.Utilize first 1 sequence in transposon TSI-2 sequence and GBSS1, design a pair of InDel labeled primer waxy-TSI2/int1, can identify the glutinous matter of IV type isozygoty recessive and heterozygosis dominant gene type; Utilize interior unit 1 sequence at TSI-2 insertion point place, designed again a pair of InDel labeled primer waxy-int1-1F/4R, can differentiate that obstructing matter isozygotys and heterozygous genes type, two pairs of primers are used the interference that can get rid of heterozygous genes type in IV type waxy gene type simultaneously.Two pairs of InDel labeled primer sequences are as follows,
The primer nucleic acid base sequence of waxy-TSI2/int1 mark is:
Upstream primer: 5 ' GAAGTGGAGGATACTGATGGG 3 '
Downstream primer: 5 ' GGATTTGACTGCATGAAAATG 3 '
The primer nucleic acid base sequence of waxy-int1-1F/4R mark is:
Upstream primer: 5 ' CCGTTTCGGTCGGTAGGAAG3 '
Downstream primer: 5 ' AGTGGGTTCGATGTTTGAAC3 '
Waxy-TSI2/int1 labeled primer can amplify 984 bp bands in Michelia floribunda, there is no amplified band in female parent; Waxy-int1-1F/4R labeled primer can amplify 540 bp bands in maternal Yungu No.1, there is no amplified band in male parent.If use these two groups of InDel labeled primers to detect millet DNA, waxy-TSI2/int1 primer can amplify 984 bp bands, waxy-int1-1F/4R primer can not amplify band, illustrates that this millet is the waxy material that contains the IV type waxy gene that isozygotys, and refers to table 1.In addition, two pairs of mark amplifications one have a nothing, have increased reliability and the accuracy of result.
Table 1: the genotype of codominance InDel mark and phenotype list
Note: "+" expressive notation primer can amplify band; "-" expressive notation primer can not amplify band.
3) utilize CTAB method to extract 400 parts of F of Michelia floribunda and Yungu No.1
2the genomic dna of each individual plant of colony, utilize two groups of InDel labeled primers to carry out PCR, 1.2% agarose gel electrophoresis analysis for amplified production, we find this mark and glutinous matter proterties be divided into from, can be to glutinous matter recessive gene type, stalk matter is isozygotied and heterozygosis dominant gene type is accurately differentiated.Marker genetype is WxWx, F
3in seed endosperm, starch is met iodine and is all deepened blueness; Marker genetype is Wxwx, and seed starch is met iodine and become indigo plant or red-brown, and ratio is 3 ︰ 1; Marker genetype is wxwx, and seed starch is met iodine and all become red-brown, in full accord with Michelia floribunda performance.Marker genetype WxWx:Wxwx:wxwx meets the segregation ratio of 1:2:1.
4) utilize this mark to analyze 100 parts of millet materials collecting, only have 2 parts of material use waxy-TSI2/int1 labeled primers can expand the amplified fragments of 984 bp, and utilize waxy-int1-1F/4R labeled primer there is no amplified production, and the seed endosperm of these 2 parts of materials detects and all becomes red-brown through iodine liquid, illustrate that these 2 parts of materials belong to glutinous material, and be to be controlled by IV type waxy gene.Visible, this marker combination can accurately be differentiated IV type waxy gene.
The glutinous matter of amplification IV type and stalk matter heterozygous genes type millet DNA, mark waxy-TSI2/int1 can expand the amplified fragments of 984 bp, amplification stalk matter (comprise and isozygotying and heterozygous genes type) millet DNA, mark waxy-int1-1F/4R can expand the amplified fragments of 540 bp.Because this mark is the codominant marker designing based on IV type waxy gene, can carry out genotype identification, can differentiate glutinous matter homozygous genotype and stalk matter heterozygous genes type.
The molecule marking method of the glutinous matter millet of discriminating IV type that this institute provides, tool has the following advantages:
By molecule marker of the present invention, obtained first in the world the codominant marker that millet IV type waxy gene type is differentiated.
Molecule marker of the present invention can identify the glutinous matter millet of IV type from millet, and accurately and reliably, it is convenient to identify.This marker detection fast, economical and effectively, not affected by environment.Utilize this mark can the glutinous material of IV type in Spiked Millet Resources be identified exactly and be distinguished.
Accompanying drawing explanation
Fig. 1: the agarose gel electrophoresis result of 4 different primers combination (ex1 and ex2, ex2int2 and ex4r, M5 and R7, M7 and R10) GBSS1 gene fragments that increase in Parent; (M is DL2000 marker, and P1 is male parent Michelia floribunda, and P2 is maternal Yungu No.1)
Fig. 2: labeled primer waxy-TSI2/int1 and waxy-int1-1F/4R are to " Michelia floribunda/Yungu No.1 " F
2colony's amplification agarose gel electrophoresis result; (M is DL2000 marker, and P1 is male parent Michelia floribunda, and P2 is maternal Yungu No.1, and 1-14 is F
2the part individual plant of colony, upper plate is waxy-TSI2/int1 detected result, lower plate is waxy-int1-1F/4R detected result)
Fig. 3: labeled primer waxy-TSI2/int1 and waxy-int1-1F/4R are to 31 parts of millet material amplification agarose gel electrophoresis results.The totally 2 parts of glutinous material banding patterns in 1-2 road is consistent, contains IV type waxy gene; The totally 14 parts of stalk material banding patterns in 3-16 road is consistent; The totally 11 parts of glutinous material banding patterns in 17-27 road is consistent, and the totally 2 parts of glutinous material banding patterns in 28-29 road is consistent, all not containing IV type waxy gene; The totally 2 parts of impure material banding patterns in 30-31 road is consistent, is all heterozygote genotype; (M is DL2000 marker, and 1-2 and 17-29 are glutinous matter millet, and 3-16 is stalk matter millet, and 30-31 is impure millet, and upper plate is waxy-TSI2/int1 detected result, and lower plate is waxy-int1-1F/4R detected result)
Specific implementation method (is noted: it is not quite identical that the content that accompanying drawing shows seems the content done with embodiment, allows both be consistent as far as possible.In addition, at embodiment 1, determined after molecule marker and phenotypic cognation, need to verify described method, current verification method so much when also can sample size more, can be by adopting the form of form to list detected result and the corresponding phenotypic overall number of molecule marker, so that set up more intuitively molecule marker and phenotypic cognation.）
In embodiment, method therefor is ordinary method if no special instructions below.
1, for examination material
Utilize the waxy resource Michelia floribunda of millet high-quality and stalk matter kind Yungu No.1, with and hybrid F
1, F
2colony.
2, glutinous matter macroscopical identification
Every part of material is got 15 of ripe seeds at random, is placed in respectively little mortar and smashs to pieces with pestle, and (2 g KI are placed in 1 mL water, add 1 g I after dissolving to add Wagner's reagent
2, concussion, to dissolving completely, is supplied distilled water to 300 mL, in Brown Glass Brown glass bottles and jars only, preserves, and the used time is diluted 10 times) 30 mL, pestle mixes sample and solution, observes the variation of starch color in endosperm.
3, glutinous cytoplasmic inheritance is analyzed colony
Within 2006, utilize the glutinous matter resource of millet Michelia floribunda, with stalk matter kind Yungu No.1 preparation reciprocal cross cross-fertilize seed F
1, plantation F in 2007
1and gather in the crops F
2seed, plantation quadrature F in 2008
1the F in source
2colony's 400 strains, and before the phase, get blade to extract genomic dna at 5 leaves, and gather in the crops F
3seed.Utilize I
2-KI solution carries out color reaction to starch in seed endosperm, and iodine is met amylose starch and deepened blueness, otherwise becomes red-brown.Reciprocal cross F
1offspring F
2seed endosperm is met iodine and is occurred that mazarine is separated with red-brown phenotype, F
3seed endosperm color occurs that mazarine, with henna separated, comprises 3 kinds of situations: 15 grain endosperms all become blue, all become red-brown, or part blue portion red-brown, and blueness is obviously more than red-brown.F
3the endosperm character of seed is by F
2genotype determines, occurs that the individual plant number of mazarine endosperm and the ratio of red-brown endosperm individual plant number meet 3:1, illustrates that glutinous confrontation stalk matter is the recessive character by Dominant gene.
Table 2:F
2segregating population obstructs glutinous phenotype statistics
4, DNA extraction
The CTAB method providing with reference to people such as Gill is extracted millet leaves genomic DNA, and makes suitable modification, and concrete steps are as follows:
(1) 1-1.5 g millet blade is used to liquid nitrogen grinding powdered in precooling mortar.
(2) by powder transfer in 1.5 mL centrifuge tubes, add CTAB Extraction buffer 650mL(in advance 65 ℃ of water-baths), shake powder be dispersed in extracting solution.
(3) 65 ℃ of water-bath 40 min, put upside down therebetween and mix sample 3-4 time.
(4) be cooled to after room temperature, add isopyknic Lv Fang ︰ primary isoamyl alcohol (24:1), mix gently, 4 ℃ of 12 000 centrifugal 15 min of rpm.
(5) get supernatant, proceed in new 1.5 mL centrifuge tubes, add isopyknic Lv Fang ︰ primary isoamyl alcohol (24:1), mix gently, 4 ℃ of 12 000 centrifugal 15 min of rpm.
(6) get supernatant, proceed in new 1.5 mL centrifuge tubes, add the Virahol of equal-volume (650mL)-20 ℃ precooling, shake up ,-20 ℃ of free sedimentation 30 min.
(7) 4 ℃ of centrifugal 15 min of 12000 rpm.
(8) abandon supernatant, 75% ethanol washing and precipitating 1-2 time.
(9) abandon 75% ethanol, after DNA precipitation is air-dry, be dissolved in 200mL TE(1/10), 4 ℃ save backup.
Agarose gel electrophoresis with 0.8% detects DNA sample quality.
5, design of primers and PCR reaction
Makoto Kawase etc. (Mol Gen Genomics, 2005,274:131 – 140) think that waxy gene waxy is inserted into after gene GBSS1 due to transposon, thereby cause the low expression of gene or do not express, weaken or loss of function causes.According to the transposon type of inserting and the difference of insertion point, waxy gene can be divided into 7 types.4 pairs of primer sequences of the glutinous matter type of differentiation providing according to document, we synthesize these 4 pairs of primers, and sequence is as follows:
Ex1/ex2 primer nucleic acid base sequence:
Upstream primer: 5 ' TGCAAGCCAGTGACGGATCGACGACGACAC3 '
Downstream primer: 5 ' ATGCCGGTGACCAGCGTGGAGGGCTAGCTA3 '
Ex2int2/ex4r primer nucleic acid base sequence:
Upstream primer: 5 ' CATGGCCGTAAGTCCCATCGATCGATCATC3 '
Downstream primer: 5 ' TAGCAGTGGAAGAACCTCACCCTCTCGTAC3 '
M5/R7 primer nucleic acid base sequence:
Upstream primer: 5 ' GGACGTCAGCGAGTGGGACC3 '
Downstream primer: 5 ' ACGAGTCCACCGGTGGACGC3 '
M7/R10 primer nucleic acid base sequence:
Upstream primer: 5 ' CACCAGCCGCTTCGAGCCCT3 '
Downstream primer: 5 ' GCCGGCGTGCCGACCACCTT3 '
Wherein, only have the genomic dna amplified production different (other 3 group primer extension products all identical) of ex1/ex2 combination of primers to the glutinous matter Michelia floribunda of millet male parent and maternal stalk matter Yungu No.1, the former amplified fragments is in 5 kb left and right, and the latter's amplified fragments is 828 bp.By confirmation that male parent PCR product is partly checked order, amplified fragments sequence and gi|62318482|dbj|AB210215.1| Setaria italica GBSS1 gene, transposable element TSI-2 in intron 1 mates completely, illustrate that Michelia floribunda belongs to IV type waxy gene, be that the interior unit 1 that is inserted into GBSS1 gene by TSI-2 transposon forms, TSI-2 transposon size is 5251 bp.Utilize first 1 sequence in transposon TSI-2 sequence and GBSS1, design a pair of InDel labeled primer waxy-TSI2/int1, can identify the glutinous matter of IV type isozygoty recessive and heterozygosis dominant gene type; Utilize interior unit 1 sequence at TSI-2 insertion point place, designed again a pair of InDel labeled primer waxy-int1-1F/4R, can differentiate that obstructing matter isozygotys and heterozygous genes type, two pairs of primers are used the interference that can get rid of heterozygous genes type in IV type waxy gene type simultaneously.Two pairs of InDel labeled primer sequences are as follows,
The primer nucleic acid base sequence of waxy-TSI2/int1 mark is:
Upstream primer: 5 ' GAAGTGGAGGATACTGATGGG 3 '
Downstream primer: 5 ' GGATTTGACTGCATGAAAATG 3 '
The primer nucleic acid base sequence of waxy-int1-1F/4R mark is:
Upstream primer: 5 ' CCGTTTCGGTCGGTAGGAAG3 '
Downstream primer: 5 ' AGTGGGTTCGATGTTTGAAC3 '
Primer is synthetic to be completed by Shanghai Sheng Gong company.By synthetic primer, with distilled water dilution, be 10 μ M ,-20 ℃ save backup.
The genomic dna extracting of take is template, by following reaction system, carries out PCR.PCR reaction system (20 μ L) is: 100 ng genomic dna templates, 400 μ M dNTPs, 1 * PCR buffer, each 0.5 μ M upstream and downstream primer, 1.5 U Taq archaeal dna polymerases, mend pure water to 20 μ L.The pcr amplification program of primer 1 is: 95 ℃, and 4 min; 94 ℃ of 30 sec, 60 ℃ of 30 sec, 72 ℃ of 1 min, 35 circulations; 72 ℃, extend 10 min; 10 ℃, cooling 10 min, finish reaction.The pcr amplification program of primer 1 is: 95 ℃, and 4 min; 94 ℃ of 30 sec, 60 ℃ of 30 sec, 72 ℃ of 35 sec, 35 circulations; 72 ℃, extend 10 min; 10 ℃, cooling 10 min, finish reaction.PCR reaction is carried out in ABI VERITI thermal cycler, and amplified production adds indicator (0.25% tetrabromophenol sulfonphthalein, 40% aqueous sucrose solution) rear separated with 1.2% agarose gel electrophoresis, EB dyeing, and gel imaging system is preserved after taking pictures.
Utilize two pairs of labeled primers to " Michelia floribunda/Yungu No.1 " F
2colony's individual plant carries out pcr analysis (Fig. 2), we find marker genetype and phenotype in full accord, i.e. the mark dominant gene type that isozygotys, its phenotype is seed starch and meets iodine and all deepen blueness; Mark heterozygous genes type, its phenotype is that seed starch chance iodine major part deepens blue small part change red-brown; And the glutinous matter allogene of mark type, its phenotype is that starch chance iodine all becomes red-brown, in full accord with Michelia floribunda.
Table 3:F
2segregating population obstructs glutinous marker genetype and phenotype statistics
This mark and waxy gene be divided into from, and can to the glutinous matter proterties of IV type isozygotying and heterozygous genes type is differentiated.
Embodiment 2: utilize the marker gene type analysis of mark to the glutinous material of millet
From Foxtail Millet Germplasm Resources, we have collected 100 parts of Spiked Millet Resources that are recorded as glutinous matter, and country of origin comprises that the regional ，You Ben seminar such as Hebei, Henan, Beijing, ,Jin southeast, Tianjin, Hubei, Gansu, Shanxi preserves.
Every kind of material got 5 seeds and carried out iodine staining, if blue, all occurs with red-brown, selects at random 5 observations of dyeing again.Wherein, the seed endosperm of 59 parts of materials is met iodine and is all become mazarine, illustrates that these materials are all stalk matter; The seed endosperm of 28 parts of materials is met iodine and is all become red-brown, illustrates that these 28 parts of materials are glutinous materials; 13 parts of millet materials, seed endosperm is met iodine and is become indigo plant or red-brown, and illustrative material is impure.
We utilize two pairs of InDel marks to carry out waxy-TSI2/int1 and waxy-int1-1F/4R labeled analysis to 100 parts of millet materials.Two pairs of mark PCR product sepharose part electrophoresis result are shown in Fig. 3.Wherein, 28 parts of iodine stainings are in henna glutinous material, only have 2 parts of marker genetypes with male parent Michelia floribunda, illustrate that they are the glutinous materials that contain IV type waxy gene; The marker genetype that 59 parts of iodine stainings are navy blue stalk material is all with maternal Yungu No.1; 13 parts the marker genetype of pure material is not consistent with its phenotype yet.
The present invention successfully by waxy-TSI2/int1 and waxy-int1-1F/4R tag application in assisted selection, accelerate to utilize the process of the glutinous matter kind that glutinous matter Resource Cultivation contains IV type waxy gene.
>?waxy-TSI2/int1
GAAGTGGAGGATACTGATGGGTATACCAAGTTTTCCAATGCACAAGCAAAGCAAAATTATTGTGGCTTGCATGGCAATTCACAATTTTATCCGAGAGAATAGTGTTGCCGATAGGGAATTTGATTTGTACGATTGTGATGAAAATGGTGTCCCAATGCCCGGAACTTCAAACCGCGGAGGAGGTGAGACAAGTACCCAAGTAGAAGAAGAAGATAGCAACATGAATGCATTTCGAGATGAAATAGCTCATGCCTTGTACAATAGGTCTAGATAAATTGATTTCAATGTTGTAATGATAATGTATTTGTAGTTTAATTTTGGAGTTGTTGGACATTGTAATAGACATAAAATTCCCTTCTTATGCACTGGTGTATGACTGAAAGAGAAAGAGAAGAAGAGAGTGAAAGAGAAAGAGAAGAAGAGAGTGGAGAAGAAACAAGAAGAAAGGCATAGCGCTTTGCATGCATGCAGTGCAAGGAGGAGAACGGAGGGCACGGCACAGCACCGCACCGCTATGCACGCATGCATGCAAGCAGCGCCAGCGAGTGGAGCCAGCGCAGCACGCATGCATAGCAAGGGGCGCCAGCGAGTGGAGCCAGCGCAGCATGCATGCATGCAAGGGGCGCCAGCGAGTGGGGCCAGCGCATGCATGCGCGCGGGCGGGAGGGGCAGCGCGCGCGGGCGGGAGGACAAAAGCAGGGGTATGGTAGTCATTTTCACTATTAGGTGCTAAATTTTAGCCTCCCATCCAAACACCCATGGGTGCTAAATTTTAGCCTCTTTTTTTGAGTGGGCTAAACTTTAGCTCAAGGCTAAATTTTAGCCCCTACTCCCAAACAGGCCAAGTTAGGTTAATTTTGTTTCATGGAAACTGTTTAGTACAACTCATGTTCCTCATTTCGATGCATGCAACCACACACTACTGGAAATATAAAATCTTCAGTTCGTACTCACAAGTCAGAAGCACCCCCGGATAATCCGAAT
>waxy-int1-1F/4R
CCGTTTCGGTCGGTAGGAAGTGCAGGTGCATGCCTCTAGTTTACATCAAGTTTTTTTTTTTTCCTTTTTCGATCCAGTTCGTTTGCCTGTTTCGTATTCTGTCTGAAATCTGAGTCCATGCCGATGTATAGGAAGTGGATGCCTTTAGTTTAGATCAAGGTTTCTAGATCCAGTTTGTTCGTGTGCTTCGTGTTCTGTCTGAATCTAGCTAGGCCTCTGTTGGAGTGGATTAAGTTAGGTTAATTTTGTTTCATGGAAACTGTTTAGTACAACTCATGTTCCTCATTTCGATGCATGCAACCACACACTACTGGAAATATAAAATCTTCAGTTCGTACTCACAAGTCAGAAGCACCCCCGGATAATCCGAATCTGACCTCCAGTGGTAACTGTAGCACAAGTCCACACTTAGATCCGTCCGGAGGGATAATAAATCTGTAATTACTTTTTGACTTGTTGTAAATATATTTGTCATTTTCATGCAGTCAAATCCAAATTTAAATCACCTGAATCACCCATGGTTCAAACATCGAACCCACT
Claims (1)
1. differentiate the molecule marking method of millet waxy gene, it is characterized in that:
With the codominance InDel molecule marker primer of two pairs of specific amplified millet waxy genes, difference called after waxy-TSI2/int1 and waxy-int1-1F/4R:
Waxy-TSI2/int1 upstream primer: 5 ' GAAGTGGAGGATACTGATGGG 3 '
Waxy-TSI2/int1 downstream primer: 5 ' GGATTTGACTGCATGAAAATG 3 '
Waxy-int1-1F/4R upstream primer: 5 ' CCGTTTCGGTCGGTAGGAAG3 '
Waxy-int1-1F/4R downstream primer: 5 ' AGTGGGTTCGATGTTTGAAC3 '
The DNA of amplification millet, if waxy-TSI2/int1 combination of primers can amplify the amplified fragments of 984 bp, and waxy-int1-1F/4R combination of primers can not amplify the amplified fragments of 540 bp, this material is the glutinous matter millet containing IV type waxy gene.
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