CN103609428B - The method of utilizing the unreduced gamete characteristic of friction standing grain to cultivate corn allopolyploid - Google Patents
The method of utilizing the unreduced gamete characteristic of friction standing grain to cultivate corn allopolyploid Download PDFInfo
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Abstract
The invention discloses the method for utilizing the unreduced gamete characteristic of friction standing grain to cultivate corn allopolyploid, belong to corn distant hybridization field. The present invention is taking MTF-1 as maternal, taking corn or tetraperennial teosinte as paternal hybrid, then select to survive the winter, tiller number more than 15, and chromosome number is the filial generation plant of maternal chromosome number and male parent andro gamete chromosome number sum, is new corn allopolyploid. The inventive method has overcome the dysgenesia of corn Related species, taking the corn allopolyploid that is bred as bridge material transfer proterties, provides good hereditary basis for being bred as breakthrough corn variety. Secondly, the allopolyploid breeding that the inventive method is other species provides model. The allopolyploid that the inventive method is bred as is perennial, and vegetative propagation, for allopolyploid origin and Study on Evolution and polyploid breeding provide material. The inventive method is simple, and the time is short, efficiency is high, workload is little.
Description
Technical field
The invention belongs to Wide_cross in Plant method field, be specifically related to utilize the unreduced gamete of friction standing grainCharacteristic is cultivated the method for corn allopolyploid.
Background technology
Polyploid is in phalangeal cell, to contain 3 or 3 above genomic organisms, is divided into homologyPolyploid and the large class of allopolyploid two. Polyploid phenomenon in plant is very general, many important cultivationsCrop is as wheat, cotton, peanut, tobacco, sweet potato, potato etc., and many fruit trees, vegetables are equalFor polyploid, some other crop is as corn (Gaut, B.S. etc., ProceedingsoftheNationalAcademyofSciences.1997.94 (13): 6809-6814), soybean (Shoemaker,R. etc., Genetics, 1996,144 (1): 329-338) etc. its ancestors in evolution, also experienced many timesChange process. In plant kingdom, compared with dliploid, polyploid has the effect that cell volume increases, pastToward to show stem stalk sturdy, blade, fruit and seed are larger, the containing of the nutriments such as carbohydrate and proteinThe characteristics such as amount increase. In addition, often there is annidation widely in polyploid plant, at specific ringIn border, there is stronger plasticity, resistance, exactly because the existence of these advantages, polyploid advantage profitWith the attention that also enjoys breeding men. Existing polyploid plant is nearly all natural polyploid, natural manyTimes body forms and mainly contains two kinds of approach, and the one, biological cell chromosome number doubles, and the 2nd, subtrahend is not joinedGeneration and the fusion of son are that property doubles, and comprise single parent or parents' polyploidization. Synthesized polyploid wheat is mainBe to double by chromosome number of somatic, what chromosome doubling was the most generally used is colchicine, this medicineProduct are large to the toxic action of human body, medicine residual serious for environmental pollution, and manually synthetic many timesThe required workload of body is large, and the cycle is long, and the polyploid poor stability of acquisition, at present by artificial synthetic obtainingThe polyploid limited amount obtaining.
Twentieth century thirties, Mangelsdorf etc. are by dliploid corn (2n=20.ZeamaysL.)Obtain first corn-friction standing grain allotriploid hybrid with the distant hybridization of tetraploid friction standing grain (2n=72)(2n=46) (Mangelsdorf, P.C. etc., JournalofHeredity, 1931,22 (11):329-343), along with the discovery of friction standing grain Apomixis and research deep (Brown, W.V. etc.,AmericanJournalofBotany, 1958:253-263), the people such as Petrov propose to rub standing grainApomictic characteristic proceeds to the idea of corn, and rubs by tetraploid corn (2n=40) and tetraploidWipe standing grain (2n=72) distant hybridization and obtained first corn-friction standing grain allotetraploid hybrid (2n=56).1979 such as () Petrov sends out in the research that backcrosses of corn-friction standing grain allotetraploid hybrid and cornExisting, corn-friction standing grain allotetraploid and derivative offspring thereof have apomictic characteristic concurrently, can produce onePlant unique BⅢType hybrid generation (Kindiger, B. etc., Genetica, 1996a, 97 (1): 103-110;Kindiger, B. etc., Cropscience, 1996b, 36 (5): 1108-1113; Sokolov, V.Deng, Genetika, 2000,32 (3): 331-353), in rubbing the hybrid of standing grain, parent also once had BⅢType hybrid generation reports (Kindiger, B. etc., Genetica, 1994,92 (3): 197-201), BⅢType hybrid is to merge by the sperm nucleus of unreduced egg cell (2n gamete) and subtrahend the individuality producing, thisThe combining form of kind of 2n+n in other plant, also have report (Bashaw, E. etc., Cropscience,1990,30 (3): 571-575; Bashaw, E. etc., Internationaljournalofplantsciences,1992:466-470;Burson,B.L.,Genome,1992,35(6):1002-1006)，It is a kind of special mode of reproduction.
This apomictic characteristics of utilization friction standing grain such as woods are prayed by Tang of Sichuan Agricultural University, with corn-frictionStanding grain allotetraploid hybrid (2n=56) is maternal, taking tetraperennial teosinte (2n=40) as fatherThis, successfully cultivate corn allohexaploid MTF-1(2n=76), MTF-1 comprises complete dliploid jadeRice genome (20), tetraperennial teosinte genome (20) and tetraploid finger-likeFriction standing grain genome (36), that MTF-1 shows as is perennial, it is vigorous to tiller, and pollen fertility is extremely low,And female fringe part can be educated (Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009). Utilize apomixiaThe Sexual polyploid that produces 2n gamete is one of main path of natural polyploid formation, and this mechanism also canFor improving hybrid generation's ploidy level, overcome the obstacle of thing interspecific hybridization, not only can be directly byThe corn allopolyploid being bred as is used as forage grass, the more important thing is and can be used as bridge material by jadeThe merit of rice Related species is transferred in cultivated maize.
Summary of the invention
Narrow for the hereditary basis existing in current corn breeding, be difficult to produce breakthrough kind, and beautifulBetween rice Related species, because there being reproduction isolation, make the merit of corn Related species be difficult to educate at cornIn kind, obtain the problem of utilizing, the object of the invention is to provide a kind of unreduced gamete spy who utilizes friction standing grainProperty cultivate corn allopolyploid method. Utilize the method can synthesize the corn allos of higher multiple manyTimes body, becomes easily the polymerization of different corn Related species beneficial genes and transfer, thereby expands cornThe germ plasm resource basis of breeding.
For achieving the above object, technical scheme of the present invention is as follows:
The method of utilizing the unreduced gamete characteristic of friction standing grain to cultivate corn allopolyploid, comprises following stepRapid:
(1), by corn allohexaploid MTF-1(TripsazeacreammaizeT.2n=76; Su YueExpensive, Sichuan Agricultural University's Master's thesis, 2009) divide pocket plantation, and maize planting (Zeamays in batchesOr tetraperennial teosinte (Zeaperennis) L.);
(2), weave silk the phase at MTF-1, taking MTF-1 as maternal, with corn or the perennial class of tetraploidCorn is that male parent is hybridized, and results are the filial generation seed of black or grey; Wherein female to MTF-1Fringe at least repeats to pollinate 3 times;
(3), the filial generation seed of step (2) gained is implanted in the nutritive cube of phjytotron,Be to carry out vernalization under 28 DEG C, the humidity condition that is 70% in temperature, treat 1 heart time shift of growth of seedling to 3 leafPlant to plastic flowerpot or field planting;
(4), in the jointing stage, select the plant of tiller number more than 15, and in the time of its plant height 30cm,Get the tip of a root and carry out cell chromosome and count qualification, each individual plant is at least identified 50 cells, selects cell dyeingBody number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum; Selected individual plant is carried outDivide root and stem of certain plants breeding;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte and tetraploid gama grass (TripsacumdactyloidesL., 2n=72)Carry out aflp analysis, select the individual plant consistent with MTF-1 band (swimming band), genome is not lost;
(6) utilize flame seasoning (seeing Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009)Tip of a root material to the selected individual plant of step (5) carries out chromosome sectioning, and selective staining body is in there being silk pointSplit the tip of a root combustion sheet that mid-term and decentralization are high and carry out double-colored genomic in situ hybridization, use respectively digoxin and lifeThe chromosome of thing element mark corn and tetraperennial teosinte, identifies its chromosome composition; Select rootTip Cells chromosome number is maternal chromosome number and male parent andro gamete chromosome number sum and its chromosome structureBecome to comprise maize chromosome group (20), tetraperennial teosinte dyeing from maternal MTF-1Body group (20) and tetraploid gama grass genome (36), and male parent (corn orTetraperennial teosinte) the genomic plant of andro gamete, be new corn allopolyploid (jadeMeter Yi Yuan heptaploid or corn allooctaploid).
Corn allohexaploid MTF-1(Su Yue described in said method step (1) is expensive, sichuan agricultureUniversity's Master's thesis, 2009) be that Sichuan Agricultural University is to draw the tetraploid corn (Zeamays from the U.S.L.4n=40) and tetraploid gama grass (or claim finger-like friction standing grain, TripsacumdactyloidesL.,The F of bigenering 2n=72)1For female parent, with tetraperennial teosinte (Zeaperennis,Corn allohexaploid 2n=40) being bred as for paternal hybrid, MTF-1 is one and comprises complete corn dyeingBody group (20), gama grass genome (36) and tetraperennial teosinte dyeingBody group (20) totally 76 chromosomal bigenering, are non-existent new species of nature,Sichuan Agricultural University is that beautiful broomcorn millet standing grain belongs to Yu Qi pouring by the corn allohexaploid MTF-1 Classification And Nomenclature being newly bred asGrass seeds, by its Latin specific name called after TripsazeacreammaizeT. wherein T be MTF-1Breeding people and classification people Tang pray the abbreviation of woods.
Corn allohexaploid MTF-1(TripsazeacreammaizeT.) (see figure 1) combine corn,The feature of tetraperennial teosinte and gama grass, shows as perennially, and vegetative propagation, plantsStrain is healthy and strong, uprightly grows thickly, and well developed root system; Taking out male period average plant height is 310cm, plant height after tasseling stageReach as high as 335cm, the thick 9.8-12.9mm of stem, tasseling stage individual plant is tillered and is reached 50-60, tiller withStem stem rough error is different not obvious, and in whole life cycle, the new root of continuous generation is tillered; Blade is dark green thinLong, the long 61-81cm of leaf, the wide 5.1-5.9cm of leaf, the single stem stalk number of blade is 20-23 sheet; Stem topMale flower belongs to panicle, the long 27-30cm of inflorescence, pollen height is sterile, on stem stalk node raw 5-7 pointBranch, branch top is spicate female flower, 9-18 small ear is duplicate rows or four lines to raw row on cobRow. Plant tassel, leaf sheath and bract are darkviolet. To MTF-1, pollination can obtain a small amount of seed, plantsThe clever shell of son is hard, is brown or taupe. Cold-resistant, the waterlogging-resistant ability of plant is strong, 0-5 DEG C and long-term water loggingUnder condition, still can continue to grow newly to tiller.
MTF-1 is mainly by vegetative propagation sides such as a point pocket breeding, the cuttage of stem stalk or other vegetative propagation modesMethod is bred. The public can obtain MTF-1(Tripsazeacreammaize from Sichuan Agricultural UniversityT.2n=76) biomaterial.
Corn (ZeamaysL.) described in said method step (1) refers to corn inbred line, cornCenospecies, corn collective species, native maize varieties or corn breeding intermediate materials etc.
The corns such as described corn inbred line refers to Mo17,48-2, tuck in 478, Shen 5003 or prosperous 7-2 fromHand over system.
Tetraperennial teosinte described in said method step (1) refers to the perennial class jade of tetraploidRice 9475(Zeaperennis9475) or other tetraperennial teosinte materials, the public can be fromMexico's wheat and corn improvement center freely obtains tetraperennial teosinte 9475(Zeaperennis9475）。
The method that cell chromosome number described in said method step (4) is identified is: at fine day noonWhen 10:00～14:00 and temperature get the tip of a root during higher than 25 DEG C, saturated water-soluble with α-bromonaphthalene under dark conditionLiquid pretreatment 2-3h, cleans up the pretreated tip of a root to be placed in fixer (fixer with clear waterConstituent and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) 12-24h, then the tip of a root after fixing is usedImprovement carbolfuchsin dyeing compressing tablet, microscopy, qualification chromosome number.
Point root and stem of certain plants breeding described in said method step (4) referred to taking tillering as seedling of plant base portion to be carried outVegetative method.
Described in said method step (5), step (4) selected plant is carried out to aflp analysisConcrete grammar is: total DNA of the selected plant of extraction step (4), extract simultaneously MTF-1, corn,Tetraperennial teosinte and tetraploid gama grass (TripsacumdactyloidesL., 2n=72)Total DNA, adopt EcoRI and MseI to carry out enzyme and cut, 37 DEG C of enzymes are cut coupled reaction 6h, after taking-up,Be placed in 65 DEG C of water-bath 10min and make enzyme-deactivating, be cooled to room temperature; Wherein enzyme is cut with enzyme coupled reaction system and is:Total DNA5.0 μ L(100ng/ μ L), 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI connects5.0Pmol, MseI joint 5.0Pmol, T4DNA ligase 10.0U, ddH2O polishing 25.0 μ L;Then taking enzyme cut with coupled reaction after the DNA of gained as template, with SEQIDNo:1 and SEQIDNo:2For primer increases in advance, wherein the PCR response procedures of pre-amplification is: 94 DEG C of sex change 15s, 60 DEG C multipleProperty 30s, 72 DEG C extend 60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations; Pre-amplified production is used2% agarose gel electrophoresis detects, and pre-amplimer is:
E00：5’-GACTGCGTACCAATTC-3’（SEQIDNo:1），
M00：5’-GATGAGTCCTGAGTAAC-3’；（SEQIDNo:2）；
Again taking the DNA of the gained that increases in advance as template, respectively with SEQIDNo:3 to SEQIDNo:3416 pairs of primers in each primer pair be that primer carries out selective amplification, selective amplification product is with 6%Sex change polyacrylate hydrogel electrophoresis detection, select the individual plant consistent with MTF-1 band, genome is not lost;Wherein said selective amplification PCR program is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s, 72 DEG C are prolongedStretch 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation30s, 72 DEG C are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 DEG C of 2min; Wherein16 pairs of described selective amplification primers are:
Pair of primers:
1-F：5’-GACTGCGTACCAATTCAGG-3’（SEQIDNo:3），
1-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:4）；
Second pair of primer:
2-F：5’-GACTGCGTACCAATTCACC-3’（SEQIDNo:5），
2-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:6）；
The 3rd pair of primer:
3-F：5’-GACTGCGTACCAATTCACG-3’（SEQIDNo:7），
3-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:8）；
The 4th pair of primer:
4-F：5’-GACTGCGTACCAATTCAAC-3’（SEQIDNo:9），
4-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:10）；
The 5th pair of primer:
5-F：5’-GACTGCGTACCAATTCAGC-3’（SEQIDNo:11），
5-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:12）；
The 6th pair of primer:
6-F：5’-GACTGCGTACCAATTCAAG-3’（SEQIDNo:13），
6-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:14）；
The 7th pair of primer:
7-F：5’-GACTGCGTACCAATTCACA-3’（SEQIDNo:15），
7-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:16）；
The 8th pair of primer:
8-F：5’-GACTGCGTACCAATTCACT-3’（SEQIDNo:17），
8-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQIDNo:18）；
The 9th pair of primer:
9-F：5’-GACTGCGTACCAATTCACG-3’（SEQIDNo:19），
9-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:20）；
The tenth pair of primer:
10-F：5’-GACTGCGTACCAATTCACC-3’（SEQIDNo:21），
10-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:22）；
The tenth pair of primers:
11-F：5’-GACTGCGTACCAATTCACG-3’（SEQIDNo:23），
11-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:24）；
The 12 pair of primer:
12-F：5’-GACTGCGTACCAATTCAAC-3’（SEQIDNo:25），
12-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:26）；
The 13 pair of primer:
13-F：5’-GACTGCGTACCAATTCAGC-3’（SEQIDNo:27），
13-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:28）；
The 14 pair of primer:
14-F：5’-GACTGCGTACCAATTCAAG-3’（SEQIDNo:29），
14-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:30）；
The 15 pair of primer:
15-F：5’-GACTGCGTACCAATTCACA-3’（SEQIDNo:31），
15-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:32）；
The 16 pair of primer:
16-F：5’-GACTGCGTACCAATTCACT-3’（SEQIDNo:33），
16-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQIDNo:34）。
The method that the unreduced gamete characteristic of above-mentioned utilization friction standing grain is cultivated corn allopolyploid, specifically bagDraw together following steps:
(1), in Sichuan Province, since the last ten-days period in March, points of 10 batches maize plantings (ZeamaysL., 2n=20,As follows), every batch of 70 strains, plant interval 10 days for every batch, and corn adopts two strains plantations, and wherein line-spacing is60cm, spacing in the rows is 35cm; In the first tenday period of a month in April, be that 2 meters, spacing in the rows are that the distance of 2 meters will according to line-spacingCorn allohexaploid MTF-1(TripsazeacreammaizeT.2n=76; Su Yuegui, sichuan agricultureUniversity's Master's thesis, 2009) divide the root and stem of certain plants to plant;
(2), in the phase of weaving silk, before weaving silk, by female MTF-1 fringe bagging, and weave silk at the female fringe of MTF-1After MTF-1 filigree is cut short to 1～3cm, then taking corn as male parent to the pollination of the female fringe of MTF-1, everyIndividual female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, marks; Results are black or greyFilial generation seed;
(3), April next year, filial generation seed that step (2) is obtained is implanted in growth cabinetIn nutritive cube, be to carry out vernalization, plant strain growth to 3 leaf under 28 DEG C, the humidity condition that is 70% in temperatureWhen 1 heart, transplant to plastic flowerpot or large Tanaka's plantation;
(4), in the jointing stage, select to have and tillers and the plant of tiller number more than 15, plant to be plantedWhen plant height 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells,Selecting cell chromosome number is the list of maternal chromosome number and male parent andro gamete chromosome number sum (2n=86)Strain, and divide root and stem of certain plants breeding to selected individual plant;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte, tetraploid gama grass (TripsacumdactyloidesL., 2n=72)Carry out aflp analysis, select the individual plant consistent with MTF-1 band (swimming band), genome is not lost;
(6), utilize flame seasoning (Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) to stepSuddenly the residue tip of a root material of (5) selected individual plant carries out chromosome sectioning, and selective staining body is in there being silk pointSplit the tip of a root combustion sheet that mid-term and decentralization are high and carry out double-colored genomic in situ hybridization, use respectively digoxin and lifeThing element mark corn and tetraperennial teosinte chromosome, identify its chromosome composition; Select the tip of a rootCell chromosome number is 86, and its genome is configured to 30 of maize chromosomes, tetraploid is manyThe plant that 20 of year life class maize chromosomes and tetraploid gama grass chromosome number are 36, educatesThe corn allos heptaploid becoming.
The bore of the plastic flowerpot described in said method step (3) is 40cm, is highly 35cm.
Identification of cell chromosome number object method described in said method step (4) is: at fine day noonWhen 10:00～14:00 and temperature get the tip of a root during higher than 25 DEG C, under dark condition with α-bromonaphthalene saturation waterSolution pretreatment 2-3h, cleans up the pretreated tip of a root to be placed on (fixer in fixer with clear waterConstituent and ratio thereof be: absolute ethyl alcohol: glacial acetic acid=3:1) more than 24h, then by the root after fixingImprovement carbolfuchsin dyeing compressing tablet for point, microscopy, qualification chromosome number.
The method of the aflp analysis described in said method step (5) is: extraction step (4) is selectedTotal DNA of plant, extract MTF-1, corn, tetraperennial teosinte and tetraploid duck simultaneouslyTotal DNA of thatch shape friction standing grain (TripsacumdactyloidesL., 2n=72), adopt EcoRI andMseI carries out enzyme and cuts, and 37 DEG C of enzymes are cut coupled reaction 6h, after taking-up, is placed in 65 DEG C of water-bath 10min and makes enzymeDeactivation, is cooled to room temperature; Wherein enzyme is cut with enzyme coupled reaction system and is: total DNA5.0 μ L(100ng/ μ L),10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol，T4DNA ligase 10.0U, ddH2O polishing 25.0 μ L; Then cut and coupled reaction with enzymeThe DNA of rear gained is template, increases in advance taking SEQIDNo:1 and SEQIDNo:2 as primer,Wherein the PCR response procedures of pre-amplification is: 94 DEG C of sex change 15s, 60 DEG C of renaturation 30s, 72 DEG C of extensions60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations; 2% Ago-Gel electricity for pre-amplified productionSwimming detects; Again taking the DNA of the gained that increases in advance as template, respectively with SEQIDNo:3 to SEQIDNo:3416 pairs of primers in each primer pair be that primer carries out selective amplification, selective amplification product is with 6%Sex change polyacrylate hydrogel electrophoresis detection, select the individual plant consistent with MTF-1 band, genome is not lost;Wherein said selective amplification PCR program is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s, 72 DEG C are prolongedStretch 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation30s, 72 DEG C are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 DEG C of 2min.
The method that the unreduced gamete characteristic of above-mentioned utilization friction standing grain is cultivated corn allopolyploid, specifically bagDraw together following steps:
(1), in Sichuan Province, since the last ten-days period in March, points of 7 crowdes plantation tetraperennial teosinte (ZeaPerennis, 2n=40, as follows), 7 days, the every batch of interval, every batch of 10 strains, the perennial class jade of tetraploidRice single-strain planting, wherein seeding row spacing is 2m × 2m; In the first tenday period of a month in April, be 2 meters, spacing in the rows according to line-spacingBe that the distance of 2 meters is by corn allohexaploid MTF-1(TripsazeacreammaizeT.2n=76; Soviet UnionThe moon is expensive, Sichuan Agricultural University's Master's thesis, 2009) divide the root and stem of certain plants to plant;
(2), in the phase of weaving silk, before weaving silk, by female MTF-1 fringe bagging, and weave silk at the female fringe of MTF-1After MTF-1 filigree is cut short to 1～3cm, then give taking tetraperennial teosinte as male parentThe female fringe pollination of MTF-1, each female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, marks,Results are the filial generation seed of black or grey;
(3), April next year, the seed that step (2) is obtained is implanted in the nutritive cube of growth cabinetIn, be 28 DEG C in temperature, vernalization under the condition that humidity is 70%, transplants when plant strain growth to 3 leaf 1 heartTo plastic flowerpot or field planting;
(4), select to have to tiller and the plant of tiller number more than 15; Plant to be planted plant height is 30cmTime, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, selects cell to dyeColour solid number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum (2n=96), and to selectedIndividual plant divide root and stem of certain plants breeding;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte, tetraploid gama grass (TripsacumdactyloidesL., 2n=72)Carry out aflp analysis, select the individual plant consistent with MTF-1 band (swimming band), genome is not lost;
(6), utilize flame seasoning (Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) to stepSuddenly (5) selected individual plant residue tip of a root material carries out chromosome sectioning, and selective staining body is in mitosisThe tip of a root combustion sheet that mid-term and decentralization are high carries out double-colored genomic in situ hybridization, uses respectively digoxin and biologyElement mark corn and tetraperennial teosinte chromosome, qualification chromosome composition; Select the plant tip of a rootCell chromosome number be 96 and its chromosome form contain 20 of maize chromosomes, tetraploid is manyThe complete genomic list that 40 of year life class maize chromosomes and gama grass chromosome number are 36Strain, is bred as corn allooctaploid.
The bore of the plastic flowerpot described in said method step (3) is 40cm, is highly 35cm.
Identification of cell chromosome number object method described in said method step (4) is: at fine day noonWhen 10:00～14:00 and temperature get the tip of a root during higher than 25 DEG C, under dark condition with α-bromonaphthalene saturation waterSolution pretreatment 2-3h, cleans up the pretreated tip of a root to be placed on (fixer in fixer with clear waterConstituent and ratio thereof be: absolute ethyl alcohol: glacial acetic acid=3:1) more than 24h, then by the root after fixingImprovement carbolfuchsin dyeing compressing tablet for point, microscopy, qualification chromosome number.
The method of the aflp analysis described in said method step (5) is: extraction step (4) is selectedTotal DNA of plant, extract MTF-1, corn, tetraperennial teosinte and tetraploid duck simultaneouslyTotal DNA of thatch shape friction standing grain (TripsacumdactyloidesL., 2n=72), adopt EcoRI andMseI carries out enzyme and cuts, and 37 DEG C of enzymes are cut coupled reaction 6h, after taking-up, is placed in 65 DEG C of water-bath 10min and makes enzymeDeactivation, is cooled to room temperature; Wherein enzyme is cut with enzyme coupled reaction system and is: total DNA5.0 μ L(100ng/ μ L),10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol，T4DNA ligase 10.0U, ddH2O polishing 25.0 μ L; Then cut and coupled reaction with enzymeThe DNA of rear gained is template, increases in advance taking SEQIDNo:1 and SEQIDNo:2 as primer,Wherein the PCR response procedures of pre-amplification is: 94 DEG C of sex change 15s, 60 DEG C of renaturation 30s, 72 DEG C of extensions60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations; 2% Ago-Gel electricity for pre-amplified productionSwimming detects; Again taking the DNA of the gained that increases in advance as template, respectively with SEQIDNo:3 to SEQIDNo:3416 pairs of primers in each primer pair be that primer carries out selective amplification, selective amplification product is with 6%Sex change polyacrylate hydrogel electrophoresis detection, select the individual plant consistent with MTF-1 band, genome is not lost;Wherein said selective amplification PCR program is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s, 72 DEG C are prolongedStretch 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation30s, 72 DEG C are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 DEG C of 2min.
Advantage of the present invention and good effect: (1), the inventive method have overcome between corn Related speciesDysgenesia, the corn allopolyploid of cultivating using the inventive method, can be near by corn as bridge materialEdge belongs to the merit of planting to be transferred in corn easily, is to solve the germplasm base existing in corn breedingThe narrow problem of plinth, is bred as breakthrough corn variety good genetic germplasm basis is provided. (2) thisThe allopolyploid breeding that bright method is other species provides model. (3) jade that, the inventive method is cultivatedRice allopolyploid combines the advantage of corn, the perennial teosinte of tetraploid and friction standing grain, output is high,Quality better, and perennial, can directly apply as forage grass kind; (4), utilize the inventive method to educateThe corn allopolyploid good stability becoming, and its perennial, vegetative characteristic having, forProvide guarantee to its permanently effective utilization. (5) allopolyploid that utilizes the inventive method to be bred as is manyNian Sheng, modes of reproduction is vegetative propagation, field performance is stable, can keep parent's merit, forAllopolyploid origin and Study on Evolution and polyploid breeding provide material. (6) Billy of the present invention uses the autumnThe methods such as narcissus element are simple, and the time is short, efficiency is high, workload is little.
Brief description of the drawings
Fig. 1, corn allohexaploid MTF-1 plant photo.
Fig. 2, corn allos heptaploid MTFM-1 cell chromosome in situ hybridization microphoto.
Fig. 3, corn allos heptaploid MTFM-1 and MTF-1 and three parent's aflp analysis electrophoretogramsSpectrum; Wherein 1 is MTFM-1, and 2 is that MTF-1,3 is that tetraploid gama grass, 4 is tetraploidPerennial class corn, 5 is corn.
The plant photo of Fig. 4, corn allos heptaploid MTFM-1.
Fig. 5, corn allooctaploid MTFF-1 cell chromosome in situ hybridization microphoto.
Fig. 6, corn allooctaploid MTFF-1 and MTF-1 and three parent's aflp analysis electrophoretogramsSpectrum; Wherein 1 is MTFF-1, and 2 is that MTF-1,3 is that tetraploid gama grass, 4 is tetraploidPerennial class corn, 5 is corn.
The plant photo of Fig. 7, corn allooctaploid MTFF-1.
Detailed description of the invention
Embodiment 1, utilize the unreduced gamete characteristic of MTF-1 and friction standing grain to cultivate corn allos heptaploid
(1), on farm, Hui He village, Wenjiang District, Chengdu, Sichuan Province, since the last ten-days period in March, points of 10 batches two strainsMaize planting self-mating system Mo17 (Zeamays, 2n=20), 10 days, the every batch of interval, every batch of 70 strains, strainLine-spacing is 35cm × 60cm; In the first tenday period of a month in April, by MTF-1(TripsazeacreammaizeT. Su YueExpensive, Sichuan Agricultural University's Master's thesis, 2009. The public can obtain from Sichuan Agricultural University) (see figure 1)Divide the root and stem of certain plants to plant in land for growing field crops, seeding row spacing 2m × 2m, totally 50 strains;
(2), in the phase of weaving silk, before the female fringe of MTF-1 is weaved silk, by female MTF-1 fringe cover kraft paper bag,After female fringe is weaved silk, filigree is cut short to 1cm, to the female fringe pollination of MTF-1, each taking Mo17 as male parentFemale fringe at least repeats to pollinate 3 times, after each pollination, on kraft paper bag, makes marks, and results are black or ashThe filial generation seed of look.
(3), April next year, the seed that step (2) is obtained in the nutritive cube at growth cabinet,Be 28 DEG C in temperature, vernalization under the condition that humidity is 70% is transplanted extremely in the time of growth of seedling to 3 leaf 1 heartIn plastic flowerpot, plant, the bore of described plastic flowerpot is 40cm, is highly 35cm.
(4), in the jointing stage, select to have and tillers, and the plant of tiller number more than 15 plant to be plantedWhen plant height 30cm, in the time of fine day 10:00～14:00 at noon and when 25 DEG C of temperature, get the tip of a root, at dark barUnder part, with α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up to postposition with clear water(constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) 24h in fixer,Again the tip of a root after fixing is positioned in 70% absolute ethyl alcohol and is kept at 4 DEG C; By the tip of a root after fixing with changingGood carbolfuchsin dyeing compressing tablet, identifies chromosome number under the microscope, 50 cells of each individual plant qualification,Selecting cell chromosome number is maternal chromosome number and male parent subtrahend andro gamete chromosome number sum (2n=86)Plant (see figure 2), and divide root and stem of certain plants breeding to selected individual plant.
(5), the selected plant of step (4) is carried out to aflp analysis, concrete grammar is: extraction step(4) total DNA of the young leaflet tablet of the plant that selected chromosome number is 86, and MTF-1(TripsazeacreammaizeT.2n=76), dliploid corn (ZeamaysL.2n=20), four bodiesGama grass (or claiming finger-like friction standing grain, TripsacumdactyloidesL., 2n=72) and tetraploidTotal DNA of the young leaflet tablet of perennial class corn (Zeaperennis, 2n=40), adopt EcoRI andMseI carries out enzyme and cuts, and 37 DEG C of enzymes are cut coupled reaction 6h, after taking-up, is placed in 65 DEG C of water-bath 10min and makes enzymeDeactivation, is cooled to room temperature; Wherein enzyme is cut with enzyme coupled reaction system and is: total DNA5.0 μ L(100ng/ μ L),10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol，T4DNA ligase 10.0U, ddH2O polishing 25.0 μ L; Then cut and coupled reaction with enzymeThe DNA of rear gained is template, increases in advance taking SEQIDNo:1 and SEQIDNo:2 as primer,Wherein the PCR response procedures of pre-amplification is: 94 DEG C of sex change 15s, 60 DEG C of renaturation 30s, 72 DEG C of extensions60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations; 2% Ago-Gel electricity for pre-amplified productionSwimming detects; Again taking the DNA of the gained that increases in advance as template, respectively with SEQIDNo:3 to SEQIDNo:3416 pairs of primers in each primer pair be that primer carries out selective amplification, selective amplification product is with 6%Sex change polyacrylate hydrogel electrophoresis detection, select the individual plant consistent with MTF-1 band, genome is not lost(Fig. 3); Wherein said selective amplification PCR program is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s,72 DEG C are extended 60s, 13 circulations, and the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG CRenaturation 30s, 72 DEG C are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 DEG C of 2min.
(6), utilize flame seasoning to dye to the individual plant residue tip of a root material of step (5) screeningSystem sheet, selects division lover's tip of a root combustion sheet to carry out double-colored genomic in situ hybridization, uses respectively digoxinWith biotin labeling dliploid corn and tetraperennial teosinte chromosome, qualification chromosome composition.Selecting root tip cell chromosome number is 86, and comprise 30 of maize chromosomes, tetraploid is perennialThe material that 20 of class maize chromosomes and tetraploid gama grass chromosome number are 36, qualification resultBe the perennial corn allos heptaploid (see figure 4) of incubation, name as MTFM-1.
Embodiment 2, utilize the unreduced gamete characteristic of MTF-1 and friction standing grain to cultivate corn allooctaploid
Carry out as follows:
(1) on farm, Hui He village, Wenjiang District, Chengdu, Sichuan Province, since the last ten-days period in March, point 7 batches of plantations fourTimes perennial class corn 9475 of body (Zeaperennis9475,2n=40) (the international corn of Mexico and wheatImprovement center provides free), 7 days, the every batch of interval, every batch of 10 strains, tetraperennial teosinte individual plantPlantation, seeding row spacing is 2m × 2m; In the first tenday period of a month in April, the corn allos six that Sichuan Agricultural University is cultivatedTimes body MTF-1(TripsazeacreammaizeT. Su Yue is expensive, Sichuan Agricultural University's Master's thesis, 2009.The public can obtain from Sichuan Agricultural University) divide a root and stem of certain plants to plant in land for growing field crops, seeding row spacing is 2m × 2m, totally 50 strains;
(2), in the phase of weaving silk, before the female fringe of MTF-1 is weaved silk, by female MTF-1 fringe cover kraft paper bag,After female fringe is weaved silk, filigree is cut short to 2cm, given taking tetraperennial teosinte 9475 as male parentMTF-1 pollination, each female fringe at least repeats to pollinate 3 times, after each pollination, on kraft paper bag, marks,Results are the filial generation seed of black or grey.
(3), April next year, filial generation seed that step (2) is obtained is implanted in growth cabinetIn nutritive cube, be 28 DEG C in temperature, under the condition that humidity is 70%, carry out vernalization, plant strain growth to 3 leafWhen 1 heart, transplant to plastic flowerpot, the bore of described plastic flowerpot is 40cm, highly for 35cm.
(4), in tillering stage, select to have and tillers and the offspring plant of tiller number more than 15, treatWhen plant plant height is 30cm, in the time of fine day 10:00～14:00 at noon and when temperature >=25 DEG C, get the tip of a root,Under dark condition, with α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned dry with clear waterOnly be placed in fixer (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1)24 hours, then by the tip of a root after fixing be positioned in 70% absolute ethyl alcohol, be kept at 4 DEG C for subsequent use; By stepImprovement carbolfuchsin dyeing compressing tablet qualification chromosome number for the fixing tip of a root, 50 cells of each individual plant qualification,Selective staining body number is planting of maternal chromosome number and male parent subtrahend andro gamete chromosome number sum (2n=96)Strain (see figure 5), and divide root and stem of certain plants breeding to selected individual plant.
(5), the selected plant of step (4) is carried out to aflp analysis, concrete grammar is: adopt 2 × CTABTotal DNA of the plant young leaflet tablet that the selected chromosome number of method extraction step (4) is 96, and MTF-1,Corn inbred line Mo17, tetraploid gama grass (Mexico's wheat and corn improvement center provides free)Provide free with tetraperennial teosinte 9475(Mexico wheat and corn improvement center) young tender leafSheet total genomic dna, adopts EcoRI and MseI to carry out enzyme and cuts, and 37 DEG C of enzymes are cut coupled reaction 6h, getAfter going out, be placed in 65 DEG C of water-bath 10min and make enzyme-deactivating, be cooled to room temperature; Wherein enzyme is cut and enzyme coupled reactionSystem is: total DNA5.0 μ L(100ng/ μ L), 10xBuffer2.5 μ L, EcoRI/MseI5.0U,EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T4DNA ligase 10.0U, ddH2OPolishing 25.0 μ L; Then taking enzyme cut with coupled reaction after the DNA of gained as template, with SEQIDNo:1With SEQIDNo:2 be that primer increases in advance, wherein the PCR response procedures of pre-amplification is: 94 DEG C of changesProperty 15s, 60 DEG C of renaturation 30s, 72 DEG C extend 60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations;Pre-amplified production detects with 2% agarose gel electrophoresis; Again taking the DNA of the gained that increases in advance as template,Enter taking the each primer pair in 16 pairs of primers of SEQIDNo:3 to SEQIDNo:34 as primer respectivelyRow selective amplification, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, select withThe individual plant (Fig. 6) that MTF-1 band is consistent, genome is not lost; Wherein said selective amplification PCRProgram is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s, 72 DEG C are extended 60s, 13 circulations, annealingThe every circulation of temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation 30s, 72 DEG C are extended 60s, 25Individual circulation, extends every circulation and increases 1s; 72 DEG C of 2min.
(6) utilize flame seasoning to carry out chromosome to the selected individual plant residue of step (5) tip of a root materialFilm-making, selects division lover's tip of a root combustion sheet to carry out double-colored genomic in situ hybridization, use respectively digoxin andBiotin labeling dliploid corn Mo17 and tetraperennial teosinte chromosome, qualification genomeBecome. Plant root tip cell chromosome number is 96, and its chromosome is configured to and comprises maize chromosomeArticle 20,, 40 of tetraperennial teosinte chromosomes and tetraploid gama grass chromosome number 36The plant of bar, is the perennial Hyperploidy corn allooctaploid (see figure 7) of incubation, names as MTFF-1.
Embodiment 3, corn allooctaploid MTFF-1 and the female fringe of corn allohexaploid MTF-1 and heroFringe property determination contrast test
Carry out as follows:
(1), expand numerous mode by corn allooctaploid MTFF-1(in fact by a point root and stem of certain plants in April, 2012Execute example 2) provide with corn allohexaploid MTF-1(Sichuan Agricultural University) plant in plastic flowerpot (mouthFootpath 40cm × high 33cm), each material is planted 10 basins.
(2), mensuration period and the morphological characters surveyed thereof
Plant enters takes out the hero after date of weaving silk, and chooses at random corn allooctaploid MTFF-1 and corn allosEach 3 basins of hexaploid MTF-1, use ruler and OlympusSZX16 fluorophor formula mirror measurement filigree length,Female spike length degree, inflorescence length, the long little Hua anther length of stem, the short and small colored anther length of stem, stem are grown littleFlower length, the short and small colored length of stem; The female spike number of statistics plant stem, grain number per spike, male flower branch amount, masterThe long little Hua weight of stem, the short and small colored weight of stem, totally 12 proterties.
(3), assay method: material is taken out from fixer, clear water rinse 3-5 all over after in distilled waterSoak 2-3min. Put into respectively 10% sucrose solution numbering, take out its vacuum, soak 2-3h bladeMaterial is divided into segment, with the embedding of OCT frozen section embedding medium, is placed in freezing microtome (LEICACM1850UC) upper section, thickness is 35 μ m, is made into Temporary slide. After flower pesticide being broken simultaneouslyThe pollen flowing out is made into Temporary slide. Section or load are placed in to fluorescence microscope (OLYMPUSBX61) under, respectively with observing and take pictures after green glow, blue light, ultraviolet excitation. With IPP5.1 software (mediaCybernetics) long to flower pesticide epidermal thickness, the single colored chamber of flower pesticide area, flower pesticide vascular bundle area, stemLittle Hua pollen area, the short and small pollen area of stem, filigree cross-sectional area, filigree vascular bundle area, heroEar stem bar vascular bundle area, vascular bundle number, the tassel cane crosscut gross area and tassel cane skin thickness11 indexs are measured.
Table 1MTFF-1 and MTF-1 characteristic index are measured comparative test result (mean value)
| Testing index | MTF-1 | MTFF-1 |
| Grain number per spike (grain) | 17.3 | 10.33 |
| Flower pesticide vascular bundle area (μ m2） | 3015.5 | 3418.86 |
| Filigree length (cm) | 8.93 | 13.7 |
| The long little Hua pollen of stem area (μ m2） | 3759.68 | 3065.27 |
| The tassel stem stalk crosscut gross area (μ m2） | 1449177.2 | 975656.36 |
| Short and small pollen area (the μ m of stem2） | 3593.29 | 3200.62 |
| Female spike length degree (cm) | 7.37 | 8.02 |
| The female spike number of stem (individual) | 5.33 | 8.33 |
| Filigree cross-sectional area (μ m2） | 596716.03 | 736324.06 |
23 measured characteristic indexs are carried out statistical analysis by result (in table 1), wherein, and 14 individual characteiesShape index there was no significant difference, there is significant difference (table 1) in 9 characteristic indexs. Result shows: allosOn octoploid MTFF-1 grain number per spike, produced degeneration, but flower pesticide vascular bundle area, filigree length,The long little Hua pollen of stem area, the tassel cane crosscut gross area, the short and small pollen area of stem, female spike lengthIn degree, the female spike number of stem, 8 proterties of filigree cross-sectional area, significantly exceed allohexaploid MTF-1,Pollination and ability on significantly exceed allohexaploid MTF-1, illustrate its as bridge crop to cornShift Related species gene and there is great value.
Claims (7)
1. the method for utilizing the unreduced gamete characteristic of friction standing grain to cultivate corn allopolyploid, its feature existsIn comprising the following steps:
(1), divide pocket kind by corn allohexaploid MTF-1 (TripsazeacreammaizeT.)Plant, and maize planting (ZeamaysL.) or tetraperennial teosinte (Zeaperennis) in batches;
(2), weave silk the phase at MTF-1, taking MTF-1 as maternal, with corn or the perennial class of tetraploidCorn is that male parent is hybridized, and results are the filial generation seed of black or grey; Wherein female to MTF-1Fringe at least repeats to pollinate 3 times;
(3), the filial generation seed of step (2) gained is implanted in the nutritive cube of phjytotron,Be to carry out vernalization under 28 DEG C, the humidity condition that is 70% in temperature, treat 1 heart time shift of growth of seedling to 3 leafPlant to plastic flowerpot or field planting;
(4), in the jointing stage, select the plant of tiller number more than 15, and in the time of its plant height 30cm,Get the tip of a root and carry out cell chromosome and count qualification, each individual plant is at least identified 50 cells, selects cell dyeingBody number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum; Selected individual plant is carried outDivide root and stem of certain plants breeding;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte and tetraploid gama grass (TripsacumdactyloidesL.) carry outAflp analysis, selects the individual plant consistent with MTF-1 band, genome is not lost;
The concrete grammar that wherein the selected plant of step (4) is carried out to aflp analysis is: extraction step (4)Total DNA of selected plant extracts MTF-1, corn, tetraperennial teosinte and four times simultaneouslyTotal DNA of body gama grass (TripsacumdactyloidesL.), adopts EcoRI and MseICarry out enzyme and cut, 37 DEG C of enzymes are cut coupled reaction 6h, and after taking-up, be placed in 65 DEG C of water-bath 10min and make enzyme-deactivating,Be cooled to room temperature; Wherein enzyme is cut with enzyme coupled reaction system and is: concentration is total DNA of 100ng/ μ L5.0 μ L, 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0pmol, MseI connects5.0pmol, T4DNA ligase 10.0U, ddH2O polishing 25.0 μ L; Then cut with enzyme and connectMeeting the DNA of gained after reaction is template, carries out in advance taking SEQIDNo:1 and SEQIDNo:2 as primerAmplification, wherein the PCR response procedures of pre-amplification is: 94 DEG C of sex change 15s, 60 DEG C of renaturation 30s, 72 DEG CExtend 60s, 28 circulations; 72 DEG C of 2min, 4 DEG C of preservations; Pre-amplified production is solidifying with 2% agaroseGel electrophoresis detects, and pre-amplimer is:
E00：5’-GACTGCGTACCAATTC-3’(SEQIDNo:1)，
M00：5’-GATGAGTCCTGAGTAAC-3’(SEQIDNo:2)；
Again taking the DNA of the gained that increases in advance as template, respectively with SEQIDNo:3 to SEQIDNo:3416 pairs of primers in each primer pair be that primer carries out selective amplification, selective amplification product is with 6%Sex change polyacrylate hydrogel electrophoresis detection, select the individual plant consistent with MTF-1 band, genome is not lost;Wherein said selective amplification PCR program is: 94 DEG C of sex change 10s, and 65 DEG C of renaturation 30s, 72 DEG C are prolongedStretch 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation30s, 72 DEG C are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 DEG C of 2min; Wherein16 pairs of described selective amplification primers are:
Pair of primers:
1-F：5’-GACTGCGTACCAATTCAGG-3’(SEQIDNo:3)，
1-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:4)；
Second pair of primer:
2-F：5’-GACTGCGTACCAATTCACC-3’(SEQIDNo:5)，
2-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:6)；
The 3rd pair of primer:
3-F：5’-GACTGCGTACCAATTCACG-3’(SEQIDNo:7)，
3-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:8)；
The 4th pair of primer:
4-F：5’-GACTGCGTACCAATTCAAC-3’(SEQIDNo:9)，
4-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:10)；
The 5th pair of primer:
5-F：5’-GACTGCGTACCAATTCAGC-3’(SEQIDNo:11)，
5-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:12)；
The 6th pair of primer:
6-F：5’-GACTGCGTACCAATTCAAG-3’(SEQIDNo:13)，
6-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:14)；
The 7th pair of primer:
7-F：5’-GACTGCGTACCAATTCACA-3’(SEQIDNo:15)，
7-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:16)；
The 8th pair of primer:
8-F：5’-GACTGCGTACCAATTCACT-3’(SEQIDNo:17)，
8-R：5’-GATGAGTCCTGAGTAACCAA-3’(SEQIDNo:18)；
The 9th pair of primer:
9-F：5’-GACTGCGTACCAATTCACG-3’(SEQIDNo:19)，
9-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:20)；
The tenth pair of primer:
10-F：5’-GACTGCGTACCAATTCACC-3’(SEQIDNo:21)，
10-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:22)；
The tenth pair of primers:
11-F：5’-GACTGCGTACCAATTCACG-3’(SEQIDNo:23)，
11-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:24)；
The 12 pair of primer:
12-F：5’-GACTGCGTACCAATTCAAC-3’(SEQIDNo:25)，
12-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:26)；
The 13 pair of primer:
13-F：5’-GACTGCGTACCAATTCAGC-3’(SEQIDNo:27)，
13-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:28)；
The 14 pair of primer:
14-F：5’-GACTGCGTACCAATTCAAG-3’(SEQIDNo:29)，
14-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:30)；
The 15 pair of primer:
15-F：5’-GACTGCGTACCAATTCACA-3’(SEQIDNo:31)，
15-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:32)；
The 16 pair of primer:
16-F：5’-GACTGCGTACCAATTCACT-3’(SEQIDNo:33)，
16-R：5’-GATGAGTCCTGAGTAACCAT-3’(SEQIDNo:34)；
(6) utilize flame seasoning to the tip of a root material of the selected individual plant of step (5) system that dyesSheet, it is former that selective staining body carries out double-colored genome in mitosis metaphase and the high tip of a root of decentralization combustion sheetHybridize position, uses respectively the chromosome of digoxin and biotin labeling corn and tetraperennial teosinte,Identify its chromosome composition; Selecting root tip cell chromosome number is that maternal chromosome number and male parent andro gamete dyeColour solid counts sum and its chromosome forms the maize chromosome group, the tetraploid that comprise from maternal MTF-1Perennial class maize chromosome group and tetraploid gama grass genome, and male parent andro gamete dyesThe plant of colour solid group, is new corn allopolyploid.
2. method according to claim 1, is characterized in that the corn described in step (1) isRefer to corn inbred line, corn hybrid seed, corn collective species, native maize varieties or corn breeding intermediate woodMaterial.
3. method according to claim 2, it is characterized in that described corn inbred line refer to Mo17,48-2, tuck in 478, Shen 5003 or prosperous 7-2.
4. method according to claim 1, is characterized in that the tetraploid described in step (1)Perennial class corn refers to tetraperennial teosinte 9475.
5. method according to claim 1, is characterized in that the cell described in step (4) dyesThe method that colour solid number is identified is: in the time of fine day 10:00～14:00 at noon and temperature get the tip of a root during higher than 25 DEG C,Under dark condition, with α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned with clear waterTotally be placed on 12-24h in fixer, then by improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing,Microscopy, qualification chromosome number; The constituent of described fixer and ratio thereof are: absolute ethyl alcohol: ice vinegarAcid=3:1.
6. method according to claim 1, is characterized in that specifically comprising the following steps:
(1), in Sichuan Province, since the last ten-days period in March, point 10 batches of maize plantings (ZeamaysL.), everyCriticize 70 strains, plant interval 10 days for every batch, corn adopts two strains plantations, and wherein line-spacing is 60cm, strainDistance is 35cm; In the first tenday period of a month in April, be that 2 meters, spacing in the rows are that the distance of 2 meters is by corn allos according to line-spacingHexaploid MTF-1 (TripsazeacreammaizeT.) divides root and stem of certain plants plantation;
(2), in the phase of weaving silk, before weaving silk, by female MTF-1 fringe bagging, and weave silk at the female fringe of MTF-1After MTF-1 filigree is cut short to 1～3cm, then taking corn as male parent to the pollination of the female fringe of MTF-1, everyIndividual female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, marks; Results are black or greyFilial generation seed;
(3), April next year, filial generation seed that step (2) is obtained is implanted in growth cabinetIn nutritive cube, be to carry out vernalization, plant strain growth to 3 leaf under 28 DEG C, the humidity condition that is 70% in temperatureWhen 1 heart, transplant to plastic flowerpot or large Tanaka's plantation;
(4), in the jointing stage, select to have and tillers and the plant of tiller number more than 15, plant to be plantedWhen plant height 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells,Selecting cell chromosome number is the individual plant that maternal chromosome number and male parent andro gamete chromosome number sum are 86,And divide root and stem of certain plants breeding to selected individual plant;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte, tetraploid gama grass (TripsacumdactyloidesL.) carry outAflp analysis, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein to step (4)The method that selected plant carries out aflp analysis is: total DNA of the selected plant of extraction step (4),Extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass simultaneously(TripsacumdactyloidesL.) total DNA, adopts EcoRI and MseI to carry out enzyme and cuts, 37 DEG CEnzyme is cut coupled reaction 6h, after taking-up, is placed in 65 DEG C of water-bath 10min and makes enzyme-deactivating, is cooled to room temperature;Wherein enzyme is cut with enzyme coupled reaction system and is: concentration is total DNA5.0 μ L of 100ng/ μ L, 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0pmol, MseI joint 5.0pmol, T4DNALigase 10.0U, ddH2O polishing 25.0 μ L; Then with enzyme cut with coupled reaction after the DNA of gainedFor template, increase in advance taking SEQIDNo:1 and SEQIDNo:2 as primer, wherein pre-amplificationPCR response procedures is: 94 DEG C of sex change 15s, and 60 DEG C of renaturation 30s, 72 DEG C are extended 60s, and 28 are followedRing; 72 DEG C of 2min, 4 DEG C of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis; Again withThe DNA of pre-amplification gained is template, draws respectively with 16 couple of SEQIDNo:3 to SEQIDNo:34Each primer pair in thing is that primer carries out selective amplification, and selective amplification product gathers third with 6% sex changeAlkene detected through gel electrophoresis, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein saidSelective amplification PCR program be: 94 DEG C of sex change 10s, 65 DEG C of renaturation 30s, 72 DEG C extend 60s,13 circulations, the every circulation of annealing temperature reduces by 0.7 DEG C; 94 DEG C of sex change 10s, 56 DEG C of renaturation 30s, 72 DEG CExtend 60s, 25 circulations, extend every circulation and increase 1s; 72 DEG C of 2min;
(6), utilize flame seasoning to dye to the residue tip of a root material of the selected individual plant of step (5)System sheet, selective staining body carries out double-colored gene in mitosis metaphase and the high tip of a root combustion sheet of decentralizationGroup in situ hybridization, uses respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome,Identify its chromosome composition; Selecting root tip cell chromosome number is 86, and its genome is configured to30 of maize chromosomes, 20 of tetraperennial teosinte chromosomes and tetraploid gama grassThe plant that chromosome number is 36, the corn allos heptaploid being bred as.
7. method according to claim 1, is characterized in that specifically comprising the following steps:
(1), in Sichuan Province, since the last ten-days period in March, points of 7 crowdes plantation tetraperennial teosinte (ZeaPerennis), 7 days, the every batch of interval, every batch of 10 strains, tetraperennial teosinte single-strain planting, whereinSeeding row spacing is 2m × 2m; In the first tenday period of a month in April, be that 2 meters, spacing in the rows are that the distance of 2 meters is by jade according to line-spacingRice allohexaploid MTF-1 (TripsazeacreammaizeT.) divides root and stem of certain plants plantation;
(2), in the phase of weaving silk, before weaving silk, by female MTF-1 fringe bagging, and weave silk at the female fringe of MTF-1After MTF-1 filigree is cut short to 1～3cm, then give taking tetraperennial teosinte as male parentThe female fringe pollination of MTF-1, each female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, marks,Results are the filial generation seed of black or grey;
(3), April next year, the seed that step (2) is obtained is implanted in the nutritive cube of growth cabinet,Be 28 DEG C in temperature, vernalization under the condition that humidity is 70%, transplants to moulding when plant strain growth to 3 leaf 1 heartMaterial flowerpot or field planting;
(4), select to have to tiller and the plant of tiller number more than 15; Plant to be planted plant height is 30cmTime, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, selects cell to dyeColour solid number is the plant that maternal chromosome number and male parent andro gamete chromosome number sum are 96, and to selectedIndividual plant divides root and stem of certain plants breeding;
(5), the selected plant of step (4) is carried out to aflp analysis, simultaneously to MTF-1, corn,Tetraperennial teosinte, tetraploid gama grass (TripsacumdactyloidesL.) carry outAflp analysis, selects the individual plant consistent with MTF-1 band, genome is not lost;
(6), utilize flame seasoning to carry out chromosome to the selected individual plant residue of step (5) tip of a root materialFilm-making, selective staining body carries out double-colored genome in mitosis metaphase and the high tip of a root combustion sheet of decentralizationIn situ hybridization, uses respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome,Qualification chromosome composition; Selecting plant root tip cell chromosome number is that 96 and its chromosome form and containsThere are 20 of maize chromosomes, 40 of tetraperennial teosinte chromosomes and gama grass dyeingThe complete genomic individual plant that body number is 36, is bred as corn allooctaploid.
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| CN106818458B (en) * | 2017-01-24 | 2019-01-29 | 四川农业大学 | Corn-friction standing grain monosomic addition line method is cultivated using corn allopolyploid |
| CN106818459B (en) * | 2017-01-24 | 2019-01-25 | 四川农业大学 | Corn-teosinte monosomic addition line method is cultivated using corn allopolyploid |
| CN106857228B (en) * | 2017-01-24 | 2019-03-01 | 四川农业大学 | A method of cold-resistant corn variety is cultivated using corn allopolyploid |
| CN107155866A (en) * | 2017-04-10 | 2017-09-15 | 四川农业大学 | The method that perennial forage maize is cultivated using non-multiple corn allopolyploid |
| CN107148910B (en) * | 2017-07-18 | 2020-10-16 | 四川农业大学 | Method for cultivating near-allotetraploid corn material |
| CN110972930B (en) * | 2019-11-22 | 2022-04-22 | 四川农业大学 | Method for cultivating high-fertility heterotetraploid corn |
| CN114467744B (en) * | 2022-03-21 | 2023-05-16 | 玉林师范学院 | Method for creating star-oil vine polyploid resource |
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