CN103609428A - Method for cultivating corn allopolyploid by using unreduced gamete characteristic of tripsacum dactyloides - Google Patents
Method for cultivating corn allopolyploid by using unreduced gamete characteristic of tripsacum dactyloides Download PDFInfo
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Abstract
The invention discloses a method for cultivating a corn allopolyploid by using an unreduced gamete characteristic of tripsacum dactyloides, belonging to the field of corn distance hybridization. The method comprises the steps of: hybridizing an MTF-1 (metal-responsive transcription factor) as a female parent with corn or tetraploid perennation corn as a male parent, and then selecting a filial generation plant which is capable of overwintering, namely a new corn allopolyploid, wherein the tiller number of the filial generation plant is more than 15 and the chromosome number of the filial generation plant is a sum of the chromosome number of a female parent and the chromosome number of the male parent. According to the method provided by the invention, the dysgenesis of corn affinis species is overcome and a good inheritance basis is provided for breeding ground-breaking corn species by using the bred corn allopolyploid as a bridge material transfer character. In addition, the method provided by the invention provides a model to breed allopolyploids of other species. The corn allopolyploid bred by the method provided by the invention is perennial, adopts vegetative propagation, and provides a material to corn allopolyploid origin and evolution research and allopolyploid breeding. The method provided by the invention is simple, is short in time, high in efficiency and small in workload.
Description
Technical field
The invention belongs to Wide_cross in Plant method field, be specifically related to the method for the unreduced gamete characteristic cultivation corn allopolyploid of utilization friction standing grain.
Background technology
Polyploid is in phalangeal cell, to contain 3 or 3 above genomic organisms, is divided into autopolyploid and the large class of allopolyploid two.Polyploid phenomenon in plant is very general, many important raise crops are as wheat, cotton, peanut, tobacco, sweet potato, potato etc., and many fruit trees, vegetables are polyploid, some other crop is as corn (Gaut, B.S. etc., Proceedings of the National Academy of Sciences.1997.94 (13): 6809-6814), soybean (Shoemaker, R. etc., Genetics, 1996,144 (1): 329-338) etc. its ancestors have also experienced polyploidization process in evolution.In vegetative kingdom, compare the characteristic such as polyploid has the effect that cell volume increases, and often shows stem stalk sturdy, and blade, fruit and seed are larger, the content increase of the nutriments such as carbohydrate and protein with dliploid.In addition, often there is annidation widely in polyploid plant, has stronger plasticity, resistance in specific environment, exactly because the existence of these advantages, polyploid advantage utilization also enjoys the attention of breeding men.Existing polyploid plant is nearly all natural polyploid, and natural polyploid forms and mainly contains two kinds of approach, the one, and biological cell chromosome number doubles, and the 2nd, the generation of unreduced gamete and fusion are that property doubles, and comprise single parent or parents' polyploidization.Synthesized polyploid wheat is mainly to double by chromosome number of somatic, what chromosome doubling was the most generally used is colchicine, this medicine is large to the toxic action of human body, medicine residual serious for environmental pollution, and the required workload of Synthesized polyploid wheat is large, cycle is long, and the polyploid poor stability of acquisition, at present by the artificial synthetic polyploid limited amount obtaining.
Twentieth century thirties, Mangelsdorf etc. have obtained corn-friction standing grain allotriploid hybrid (2n=46) (Mangelsdorf first by dliploid corn (2n=20.Zea mays L.) and the distant hybridization of tetraploid friction standing grain (2n=72), P.C. etc., Journal of Heredity, 1931, 22 (11): 329-343), deep (Brown along with discovery with the research of friction standing grain Apomixis, W.V. etc., American Journal of Botany, 1958:253-263), the people such as Petrov propose the apomictic characteristic of friction standing grain to proceed to the idea of corn, and obtained first corn-friction standing grain allotetraploid hybrid (2n=56) .1979 such as () Petrov by the distant hybridization of tetraploid corn (2n=40) and tetraploid friction standing grain (2n=72), in the research that backcrosses of corn-friction standing grain allotetraploid hybrid and corn, find, corn-friction standing grain allotetraploid and derivative offspring thereof have apomictic characteristic concurrently, can produce a kind of B of uniqueness
iIItype hybrid generation (Kindiger, B. etc., Genetica, 1996a, 97 (1): 103-110, Kindiger, B. etc., Crop science, 1996b, 36 (5): 1108-1113, Sokolov, V. etc., Genetika, 2000,32 (3): 331-353), in parent rubs the hybrid of standing grain, also once had B
iIItype hybrid generation's report (Kindiger, B. etc., Genetica, 1994,92 (3): 197-201), B
iIItype hybrid is by the sperm nucleus of unreduced egg cell (2n gamete) and subtrahend, to be merged the individuality of generation, and the combining form of this 2n+n also has report (Bashaw, E. etc., Crop science, 1990,30 (3): 571-575 in other plant, Bashaw, E. etc., International journal of plant sciences, 1992:466-470, Burson, B.L., Genome, 1992,35 (6): 1002-1006), be 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, take corn-friction standing grain allotetraploid hybrid (2n=56) as maternal, the tetraperennial teosinte (2n=40) of take is male parent, successfully cultivate corn allohexaploid MTF-1(2n=76), MTF-1 comprises complete dliploid maize chromosome group (20), tetraperennial teosinte chromosome set (20) and tetraploid finger-like friction standing grain chromosome set (36), MTF-1 shows as perennial, tiller vigorous, pollen fertility is extremely low, and female fringe part can be educated (Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009).The Sexual polyploid that utilizes apomixis to produce 2n gamete is one of main path of natural polyploid formation, this mechanism also can be used for improving hybrid generation's ploidy level, overcome the obstacle of thing interspecific hybridization, not only can directly the corn allopolyploid of incubation be used as forage grass, the more important thing is that can be used as bridge material transfers to the merit of corn Related species in cultivated maize.
Summary of the invention
Narrow for the hereditary basis existing in current corn breeding, be difficult to produce breakthrough kind, and between corn Related species because there is reproduction isolation, make the merit of corn Related species be difficult to obtain the problem of utilizing in corn breeding, a kind of method that the object of the invention is to provide unreduced gamete characteristic of utilizing friction standing grain to cultivate corn allopolyploid.Utilize the method can synthesize the corn allopolyploid of higher multiple, the polymerization of different corn Related species beneficial genes and transfer are become easily, thereby expand the germ plasm resource basis of corn breeding.
For achieving the above object, technical scheme of the present invention is as follows:
The method of utilizing the unreduced gamete characteristic cultivation corn allopolyploid of friction standing grain, comprises the following steps:
(1), by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76; Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) divide pocket plantation, and maize planting (Zea mays L.) or tetraperennial teosinte (Zea perennis) in batches;
(2), at MTF-1, weave silk the phase, take MTF-1 as maternal, take corn or tetraperennial teosinte to hybridize as male parent, results are the filial generation seed of black or grey; Wherein the female fringe of MTF-1 is at least repeated to pollinate 3 times;
(3), the filial generation seed of step (2) gained is implanted in the nutritive cube of phytotron, in temperature, be to carry out vernalization under 28 ℃, the humidity condition that is 70%, when growth of seedling to 3 leaf 1 heart, transplant to plastic flowerpot or field planting;
(4), in the shooting stage, select tiller number more than 15 plant, and when its plant height 30cm, get the tip of a root and carry out cell chromosome and count evaluation, each individual plant is at least identified 50 cells, and selecting cell chromosome number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum; To selected individual 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 and tetraploid gama grass (Tripsacum dactyloides L., 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 (to see Su Yuegui, Sichuan Agricultural University's Master's thesis, 2009) tip of a root material of the selected individual plant of step (5) is carried out to chromosome sectioning, selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively the chromosome of digoxin and biotin labeling corn and tetraperennial teosinte, identify that its chromosome forms; Selecting root tip cell chromosome number is that maternal chromosome number comprises maize chromosome group (20), tetraperennial teosinte chromosome set (20) and the tetraploid gama grass chromosome set (36) from maternal MTF-1 with male parent andro gamete chromosome number sum and its chromosome formation, and the genomic plant of male parent (corn or tetraperennial teosinte) andro gamete, be new corn allopolyploid (corn allos heptaploid or corn allooctaploid).
Corn allohexaploid MTF-1(Su Yue described in said method step (1) is expensive, Sichuan Agricultural University's Master's thesis, 2009) be tetraploid corn (Zea mays L.4n=40) and tetraploid gama grass (or the title finger-like friction standing grain of the Sichuan Agricultural University Yi Yinzi U.S., Tripsacum dactyloides L., 2n=72) between the F of bigenering
1for female parent, with tetraperennial teosinte (Zea perennis, corn allohexaploid 2n=40) being bred as for paternal hybrid, MTF-1 is one and comprises complete maize chromosome group (20), gama grass chromosome set (36) and tetraperennial teosinte chromosome set (20) be totally 76 chromosomal bigenering, it is a non-existent new species of natural world, Sichuan Agricultural University is that beautiful broomcorn millet standing grain belongs to Yu Qi pouring grass seeds by the corn allohexaploid MTF-1 Classification And Nomenclature being newly bred as, its Latin specific name called after Tripsazea creammaize T. breeding people that wherein T is MTF-1 and classification people Tang are prayed to the abbreviation of woods.
Corn allohexaploid MTF-1(Tripsazea creammaize T.) (see figure 1) combines the feature of corn, tetraperennial teosinte and gama grass, show as perennial, vegetative propagation, plant is healthy and strong, uprightly grow thickly, well developed root system; Taking out male period average plant height is 310cm, and after tasseling stage, plant height reaches as high as 335cm, the thick 9.8-12.9mm of stem, and tasseling stage individual plant is tillered and is reached 50-60, tillers different not obvious with stem stem rough error, and in whole life cycle, constantly generates new root and tiller; Blade is dark green elongated, the long 61-81cm of leaf, and the wide 5.1-5.9cm of leaf, the single stem stalk number of blade is 20-23 sheet; Stem top male flower belongs to panicle, the long 27-30cm of inflorescence, pollen height is sterile, on stem stalk node raw 5-7 branch, branch top is spicate female flower, 9-18 small ear is duplicate rows or four lines to giving birth to arrangement on cob.Plant tassel, leaf sheath and bract are darkviolet.To MTF-1, pollination can obtain a small amount of seed, and seed grain husk shell is hard, is brown or taupe.Cold-resistant, the waterlogging-resistant ability of plant is strong, under 0-5 ℃ and long-term water logging condition, still can continue to grow newly to tiller.
MTF-1 mainly breeds by asexual reproduction methods such as a minute pocket breeding, the cuttage of stem stalk or other vegetative propagation modes.T.2n=76 public Ke Cong Sichuan Agricultural University obtains MTF-1(Tripsazea creammaize) biomaterial.
Corn described in said method step (1) (Zea mays L.) refers to corn inbred line, corn hybrid seed, corn collective species, native maize varieties or corn breeding intermediate materials etc.
The corn inbred lines such as described corn inbred line refers to Mo17,48-2, tuck in 478, Shen 5003 or prosperous 7-2.
Tetraperennial teosinte described in said method step (1) refers to tetraperennial teosinte 9475(Zea perennis9475) or other tetraperennial teosinte materials, public Ke Cong Mexico wheat and corn improvement center freely obtains tetraperennial teosinte 9475(Zea perennis9475).
The method that cell chromosome number described in said method step (4) is identified is: when fine day 10:00～14:00 at noon and temperature get the tip of a root during higher than 25 ℃, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up and is placed on that in fixer, (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) 12-24h with clear water, again by improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing, microscopy, identifies chromosome number.
Minute root and stem of certain plants breeding described in said method step (4) refers to take carries out vegetative method tillering as seedling of plant base portion.
The concrete grammar that the selected plant of step (4) is carried out to aflp analysis described in said method step (5) is: total DNA of the selected plant of extraction step (4), extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass (Tripsacum dactyloides L. simultaneously, total DNA 2n=72), adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L; Then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations; 72 ℃ of 2min, 4 ℃ of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis, and pre-amplimer is:
E00：5’-GACTGCGTACCAATTC-3’（SEQ?ID?No:1），
M00：5’-GATGAGTCCTGAGTAAC-3’；（SEQ?ID?No:2）；
Increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃; 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 ℃ of 2min; Wherein said 16 pairs of selective amplification primers are:
Pair of primers:
1-F：5’-GACTGCGTACCAATTCAGG-3’（SEQ?ID?No:3），
1-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:4）；
Second pair of primer:
2-F：5’-GACTGCGTACCAATTCACC-3’（SEQ?ID?No:5），
2-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:6）；
The 3rd pair of primer:
3-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:7），
3-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:8）；
The 4th pair of primer:
4-F：5’-GACTGCGTACCAATTCAAC-3’（SEQ?ID?No:9），
4-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:10）；
The 5th pair of primer:
5-F：5’-GACTGCGTACCAATTCAGC-3’（SEQ?ID?No:11），
5-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:12）；
The 6th pair of primer:
6-F：5’-GACTGCGTACCAATTCAAG-3’（SEQ?ID?No:13），
6-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:14）；
The 7th pair of primer:
7-F：5’-GACTGCGTACCAATTCACA-3’（SEQ?ID?No:15），
7-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:16）；
The 8th pair of primer:
8-F：5’-GACTGCGTACCAATTCACT-3’（SEQ?ID?No:17），
8-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:18）；
The 9th pair of primer:
9-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:19），
9-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:20）；
The tenth pair of primer:
10-F：5’-GACTGCGTACCAATTCACC-3’（SEQ?ID?No:21），
10-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:22）；
The tenth pair of primers:
11-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:23），
11-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:24）；
The 12 pair of primer:
12-F：5’-GACTGCGTACCAATTCAAC-3’（SEQ?ID?No:25），
12-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:26）；
The 13 pair of primer:
13-F：5’-GACTGCGTACCAATTCAGC-3’（SEQ?ID?No:27），
13-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:28）；
The 14 pair of primer:
14-F：5’-GACTGCGTACCAATTCAAG-3’（SEQ?ID?No:29），
14-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:30）；
The 15 pair of primer:
15-F：5’-GACTGCGTACCAATTCACA-3’（SEQ?ID?No:31），
15-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:32）；
The 16 pair of primer:
16-F：5’-GACTGCGTACCAATTCACT-3’（SEQ?ID?No:33），
16-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:34）。
The method that the unreduced gamete characteristic of above-mentioned utilization friction standing grain is cultivated corn allopolyploid, specifically comprises the following steps:
(1), Sichuan Province, since the last ten-days period in March, minutes 10 batches maize plantings (Zea mays L., 2n=20, as follows), every batch of 70 strains, 10 days, the every batch of plantation interval, corn adopts two strains plantations, and wherein line-spacing is 60cm, and spacing in the rows is 35cm; In the first tenday period of a month in April, according to line-spacing be 2 meters, spacing in the rows be 2 meters distance by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76; Su Yuegui, 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 the female fringe bagging of MTF-1, and after the female fringe of MTF-1 is weaved silk, MTF-1 filigree is cut short to 1～3cm, then be take corn as male parent is to the 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 filial generation seed that step (2) is obtained is implanted in the nutritive cube of climatic cabinate, in temperature, is to carry out vernalization under 28 ℃, the humidity condition that is 70%, transplants to plastic flowerpot or large Tanaka's plantation during plant strain growth to 3 leaf 1 heart;
(4), in the shooting stage, select to have tiller and tiller number more than 15 plant, during plant to be planted plant height 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, and selecting cell chromosome number is the individual plant of maternal chromosome number and male parent andro gamete chromosome number sum (2n=86), and divides 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 (Tripsacum dactyloides L., 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) the residue tip of a root material of the selected individual plant of step (5) is carried out to chromosome sectioning, selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome, identify that its chromosome forms; Selecting root tip cell chromosome number is 86, and its chromosome set is configured to the plant of 36 of 30 of maize chromosomes, 20 of tetraperennial teosinte chromosomes and tetraploid gama grass chromosome numbers, the corn allos heptaploid being bred as.
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: when fine day 10:00～14:00 at noon and temperature get the tip of a root during higher than 25 ℃, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up and is placed on that in fixer, (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) more than 24h with clear water, again by improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing, microscopy, identifies chromosome number.
The method of the aflp analysis described in said method step (5) is: total DNA of the selected plant of extraction step (4), extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass (Tripsacum dactyloides L. simultaneously, total DNA 2n=72), adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L; Then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations; 72 ℃ of 2min, 4 ℃ of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis; Increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃; 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 ℃ of 2min.
The method that the unreduced gamete characteristic of above-mentioned utilization friction standing grain is cultivated corn allopolyploid, specifically comprises the following steps:
(1), Sichuan Province, since the last ten-days period in March, minutes 7 batches plantation tetraperennial teosintes (Zea perennis, 2n=40, as follows), 7 days, the every batch of interval, every batch of 10 strains, tetraperennial teosinte single-strain planting, wherein seeding row spacing is 2m * 2m; In the first tenday period of a month in April, according to line-spacing be 2 meters, spacing in the rows be 2 meters distance by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76; Su Yuegui, 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 the female fringe bagging of MTF-1, and after the female fringe of MTF-1 is weaved silk, MTF-1 filigree is cut short to 1～3cm, then take tetraperennial teosinte as male parent is to the female fringe pollination of MTF-1, each female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, mark, results are the filial generation seed of black or grey;
(3), April next year, the seed that step (2) is obtained is 28 ℃ in temperature in being implanted in the nutritive cube of climatic cabinate, vernalization under the condition that humidity is 70%, transplants to plastic flowerpot or field planting during plant strain growth to 3 leaf 1 heart;
(4), select to have tiller and tiller number more than 15 plant; When plant to be planted plant height is 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, and selecting cell chromosome number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum (2n=96), and divides 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 (Tripsacum dactyloides L., 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) the selected individual plant residue of step (5) tip of a root material is carried out to chromosome sectioning, selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome, identify that chromosome forms; Selecting plant root tip cell chromosome number is that 96 and its chromosome form the complete genomic individual plant that contains 36 of 20 of maize chromosomes, 40 of tetraperennial teosinte chromosomes and gama grass chromosome numbers, 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: when fine day 10:00～14:00 at noon and temperature get the tip of a root during higher than 25 ℃, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up and is placed on that in fixer, (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) more than 24h with clear water, again by improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing, microscopy, identifies chromosome number.
The method of the aflp analysis described in said method step (5) is: total DNA of the selected plant of extraction step (4), extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass (Tripsacum dactyloides L. simultaneously, total DNA 2n=72), adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L; Then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations; 72 ℃ of 2min, 4 ℃ of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis; Increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃; 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 ℃ of 2min.
Advantage of the present invention and good effect: (1), the inventive method have overcome the reproductive disorder between corn Related species, the corn allopolyploid that the inventive method of usining is cultivated is as bridge material, can the merit of corn Related species be transferred in corn at an easy rate, for solving the narrow problem of Germplasm Bases existing in corn breeding, be bred as breakthrough corn variety good genetic germplasm basis is provided.(2) the allopolyploid breeding that the inventive method is other species provides model.(3), the corn allopolyploid cultivated of the inventive method combines the advantage of corn, the perennial teosinte of tetraploid and friction standing grain, output is high, quality better, and perennial, can directly as forage grass kind, apply; (4), the corn allopolyploid good stability that utilizes the inventive method to be bred as, and its perennial, vegetative characteristic having, for providing assurance to its permanently effective utilization.(5) it is perennial utilizing the allopolyploid that the inventive method is bred as, and modes of reproduction is vegetative propagation, and field performance is stable, can keep parent's merit, for allopolyploid origin and Study on Evolution and polyploid breeding provide material.(6) Billy of the present invention is simple by methods such as colchicines, and the time is short, efficiency is high, workload is little.
Accompanying drawing explanation
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 electrophoresis patterns; Wherein 1 is MTFM-1, and 2 is that MTF-1,3 is that tetraploid gama grass, 4 is that tetraperennial teosinte, 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 electrophoresis patterns; Wherein 1 is MTFF-1, and 2 is that MTF-1,3 is that tetraploid gama grass, 4 is that tetraperennial teosinte, 5 is corn.
The plant photo of Fig. 7, corn allooctaploid MTFF-1.
Embodiment
(1) farm, Hui He village, Wenjiang District, Chengdu,, Sichuan Province, since the last ten-days period in March, minutes 10 crowdes two strain maize planting inbred line Mo17 (Zea mays, 2n=20), 10 days, the every batch of interval, every batch of 70 strains, seeding row spacing is 35cm * 60cm; In the first tenday period of a month in April, by MTF-1(Tripsazea creammaize T. Su Yue Gui， Sichuan Agricultural University Master's thesis, 2009.The acquisition of public Ke Cong Sichuan Agricultural University) (see figure 1) divides 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 the female fringe cover of MTF-1 kraft paper bag, after female fringe is weaved silk, filigree is cut short to 1cm, take Mo17 as male parent is to the female fringe pollination of MTF-1, each female fringe at least repeats to pollinate 3 times, after each pollination, on kraft paper bag, makes marks, and results are the filial generation seed of black or grey.
(3), April next year, the seed that step (2) is obtained, in the nutritive cube at climatic cabinate, is 28 ℃ in temperature, humidity is vernalization under 70% condition, when growth of seedling to 3 leaf 1 heart, transplant to plastic flowerpot and plant, the bore of described plastic flowerpot is 40cm, is highly 35cm.
(4), in the shooting stage, selection has tillers, and tiller number is more than 15 plant, during plant to be planted plant height 30cm, when fine day 10:00～14:00 at noon and during 25 ℃ of temperature, get the tip of a root, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up with clear water be placed in fixer (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) 24h, then the tip of a root after fixing is positioned in 70% absolute ethyl alcohol and is kept at 4 ℃; By improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing, identify under the microscope chromosome number, each individual plant is identified 50 cells, selecting cell chromosome number is the plant (see figure 2) of maternal chromosome number and male parent subtrahend andro gamete chromosome number sum (2n=86), and divides 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: total DNA of the young leaflet tablet of the plant that the selected chromosome number of extraction step (4) is 86, and MTF-1(Tripsazea creammaize T.2n=76), dliploid corn (Zea mays L.2n=20), four body gama grass (or claim finger-like friction standing grain, Tripsacum dactyloides L., 2n=72) and tetraperennial teosinte (Zea perennis, total DNA of young leaflet tablet 2n=40), adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L, then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations, 72 ℃ of 2min, 4 ℃ of preservations, pre-amplified production detects with 2% agarose gel electrophoresis, increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant (Fig. 3) consistent with MTF-1 band, genome is not lost, wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃, 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s, 72 ℃ of 2min.
(6), utilize flame seasoning to carry out chromosome sectioning to the individual plant residue tip of a root material of step (5) screening, select division lover's tip of a root combustion sheet to carry out double-colored genomic in situ hybridization, use respectively digoxin and biotin labeling dliploid corn and tetraperennial teosinte chromosome, identify that chromosome forms.Selecting root tip cell chromosome number is 86, and the material that comprises 36 of 30 of maize chromosomes, 20 of tetraperennial teosinte chromosomes and tetraploid gama grass chromosome numbers, qualification result is the perennial corn allos heptaploid (see figure 4) of incubation, names as MTFM-1.
Carry out as follows:
(1) on farm, Hui He village, Wenjiang District, Chengdu, Sichuan Province, since the last ten-days period in March, divide 7 crowdes of plantation tetraperennial teosinte 9475 (Zea perennis9475,2n=40) (Mexico's International Center for Maize and wheat Improvement provides free), 7 days, the every batch of interval, every batch of 10 strains, tetraperennial teosinte single-strain planting, seeding row spacing is 2m * 2m; The corn allohexaploid MTF-1(Tripsazea creammaize T. Su Yue Gui， Sichuan Agricultural University Master's thesis that ，Jiang Sichuan Agricultural University cultivates in the first tenday period of a month in April, 2009.Public Ke Cong Sichuan Agricultural University obtains) 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 the female fringe cover of MTF-1 kraft paper bag, after female fringe is weaved silk, filigree is cut short to 2cm, the tetraperennial teosinte 9475 of take is pollinated to MTF-1 as male parent, each female fringe at least repeats to pollinate 3 times, after each pollination, on kraft paper bag, marks, and results are the filial generation seed of black or grey.
(3), April next year, the filial generation seed that step (2) is obtained is implanted in the nutritive cube of climatic cabinate, in temperature, it is 28 ℃, humidity is to carry out vernalization under 70% condition, during plant strain growth to 3 leaf 1 heart, transplant to plastic flowerpot, the bore of described plastic flowerpot is 40cm, highly for 35cm.
(4), in tillering stage, selection have tiller and tiller number more than 15 offspring plant, when plant to be planted plant height is 30cm, when fine day 10:00～14:00 at noon and during temperature >=25 ℃, get the tip of a root, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, by the pretreated tip of a root with clear water clean up be placed on 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 ℃ standby; The tip of a root that step is fixing is identified to chromosome number with improvement carbolfuchsin dyeing compressing tablet, each individual plant is identified 50 cells, selective staining body number is the plant (see figure 5) of maternal chromosome number and male parent subtrahend andro gamete chromosome number sum (2n=96), and divides 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: the total DNA that adopts the plant young leaflet tablet that the selected chromosome number of 2 * CTAB 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) and tetraperennial teosinte 9475(Mexico wheat and corn improvement center provide free) young leaflet tablet total genomic dna, adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L, then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations, 72 ℃ of 2min, 4 ℃ of preservations, pre-amplified production detects with 2% agarose gel electrophoresis, increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant (Fig. 6) consistent with MTF-1 band, genome is not lost, wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃, 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s, 72 ℃ of 2min.
(6) utilize flame seasoning to carry out chromosome sectioning to the selected individual plant residue of step (5) tip of a root material, select division lover's tip of a root combustion sheet to carry out double-colored genomic in situ hybridization, use respectively digoxin and biotin labeling dliploid corn Mo17 and tetraperennial teosinte chromosome, identify that chromosome forms.Plant root tip cell chromosome number is 96, and its chromosome is configured to the plant that comprises 36 of 20 of maize chromosomes, 40 of tetraperennial teosinte chromosomes and tetraploid gama grass chromosome numbers, be the perennial Hyperploidy corn allooctaploid (see figure 7) of incubation, name as MTFF-1.
Carry out as follows:
(1), expand numerous mode by corn allooctaploid MTFF-1(embodiment 2 by a minute root and stem of certain plants in April, 2012) provide with corn allohexaploid MTF-1(Sichuan Agricultural University) plant in plastic flowerpot (bore 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, choose at random each 3 basins of corn allooctaploid MTFF-1 and corn allohexaploid MTF-1, with ruler and OlympusSZX16 fluorophor formula mirror, measure 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, the long little Hua length of stem, the short and small colored length of stem; The female spike number of statistics plant stem, grain number per spike, male flower branch amount, the 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, and clear water soaks 2-3min after rinsing 3-5 time in distilled water.Put into respectively 10% sucrose solution numbering, take out its vacuum, soak 2-3h, with blade, material is divided into segment, with the embedding of OCT frozen section embedding medium, be placed in the upper section of freezing microtome (LEICA CM1850UC), thickness is 35 μ m, is made into Temporary slide.The pollen flowing out after simultaneously flower pesticide being broken is made into Temporary slide.Section or load are placed under fluorescence microscope (OLYMPUS BX61), respectively with observing and take pictures after green glow, blue light, ultraviolet excitation.With IPP5.1 software (media cybernetics), flower pesticide epidermal thickness, the single colored chamber of flower pesticide area, flower pesticide vascular bundle area, the long little Hua pollen of stem area, the short and small pollen area of stem, filigree cross-sectional area, filigree vascular bundle area, tassel cane vascular bundle area, vascular bundle number, the tassel cane crosscut gross area and 11 indexs of tassel cane skin thickness 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 m 2） | 3015.5 | 3418.86 |
| Filigree length (cm) | 8.93 | 13.7 |
| The long little Hua pollen of stem area (μ m 2） | 3759.68 | 3065.27 |
| The tassel stem stalk crosscut gross area (μ m 2） | 1449177.2 | 975656.36 |
| Short and small pollen area (the μ m of stem 2） | 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 m 2） | 596716.03 | 736324.06 |
Result (in Table 1) is carried out statistical analysis by 23 measured characteristic indexs, wherein, 14 characteristic index there was no significant differences, there is significant difference (table 1) in 9 characteristic indexs.Result shows: on allooctaploid MTFF-1 grain number per spike, produced degeneration, but in 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 length degree, the female spike number of stem, 8 proterties of filigree cross-sectional area, significantly surpass allohexaploid MTF-1, on pollination and ability, significantly surpass allohexaploid MTF-1, illustrate that it shifts Related species gene as bridge crop to corn and has great value.
Claims (9)
1. the method for utilizing the unreduced gamete characteristic cultivation corn allopolyploid of friction standing grain, is characterized in that comprising the following steps:
(1), by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76) divide pocket plantation, and maize planting (Zea mays L.) or tetraperennial teosinte (Zea perennis) in batches;
(2), at MTF-1, weave silk the phase, take MTF-1 as maternal, take corn or tetraperennial teosinte to hybridize as male parent, results are the filial generation seed of black or grey; Wherein the female fringe of MTF-1 is at least repeated to pollinate 3 times;
(3), the filial generation seed of step (2) gained is implanted in the nutritive cube of phytotron, in temperature, be to carry out vernalization under 28 ℃, the humidity condition that is 70%, when growth of seedling to 3 leaf 1 heart, transplant to plastic flowerpot or field planting;
(4), in the shooting stage, select tiller number more than 15 plant, and when its plant height 30cm, get the tip of a root and carry out cell chromosome and count evaluation, each individual plant is at least identified 50 cells, and selecting cell chromosome number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum; To selected individual 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 and tetraploid gama grass (Tripsacum dactyloides L., 2n=72) carry out aflp analysis, select the individual plant consistent with MTF-1 band, genome is not lost;
(6) utilize flame seasoning to carry out chromosome sectioning to the tip of a root material of the selected individual plant of step (5), selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively the chromosome of digoxin and biotin labeling corn and tetraperennial teosinte, identify that its chromosome forms; Selecting root tip cell chromosome number is that maternal chromosome number comprises maize chromosome group, tetraperennial teosinte chromosome set and the tetraploid gama grass chromosome set from maternal MTF-1 with male parent andro gamete chromosome number sum and its chromosome formation, and the genomic plant of male parent andro gamete, be new corn allopolyploid.
2. in accordance with the method for claim 1, it is characterized in that the corn described in step (1) refers to corn inbred line, corn hybrid seed, corn collective species, native maize varieties or corn breeding intermediate materials.
3. in accordance with the method for claim 2, it is characterized in that described corn inbred line refers to Mo17,48-2, tucks in 478, Shen 5003 or prosperous 7-2.
4. in accordance with the method for claim 1, it is characterized in that the tetraperennial teosinte described in step (1) refers to tetraperennial teosinte 9475(Zea perennis9475) or other tetraperennial teosinte materials.
5. in accordance with the method for claim 1, it is characterized in that the method that the cell chromosome number described in step (4) is identified is: when fine day 10:00～14:00 at noon and temperature get the tip of a root during higher than 25 ℃, under dark condition, use α-bromonaphthalene saturated aqueous solution pretreatment 2-3h, the pretreated tip of a root is cleaned up and is placed on that in fixer, (constituent of fixer and ratio thereof are: absolute ethyl alcohol: glacial acetic acid=3:1) 12-24h with clear water, again by improvement carbolfuchsin dyeing compressing tablet for the tip of a root after fixing, microscopy, identifies chromosome number.
6. in accordance with the method for claim 1, it is characterized in that the concrete grammar that the selected plant of step (4) is carried out to aflp analysis described in step (5) is: total DNA of the selected plant of extraction step (4), extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass (Tripsacum dactyloides L. simultaneously, total DNA 2n=72), adopting EcoRI and MseI to carry out enzyme cuts, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L; Then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations; 72 ℃ of 2min, 4 ℃ of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis, and pre-amplimer is:
E00：5’-GACTGCGTACCAATTC-3’（SEQ?ID?No:1），
M00：5’-GATGAGTCCTGAGTAAC-3’；（SEQ?ID?No:2）；
Increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃; 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 ℃ of 2min; Wherein said 16 pairs of selective amplification primers are:
Pair of primers:
1-F：5’-GACTGCGTACCAATTCAGG-3’（SEQ?ID?No:3），
1-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:4）；
Second pair of primer:
2-F：5’-GACTGCGTACCAATTCACC-3’（SEQ?ID?No:5），
2-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:6）；
The 3rd pair of primer:
3-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:7），
3-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:8）；
The 4th pair of primer:
4-F：5’-GACTGCGTACCAATTCAAC-3’（SEQ?ID?No:9），
4-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:10）；
The 5th pair of primer:
5-F：5’-GACTGCGTACCAATTCAGC-3’（SEQ?ID?No:11），
5-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:12）；
The 6th pair of primer:
6-F：5’-GACTGCGTACCAATTCAAG-3’（SEQ?ID?No:13），
6-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:14）；
The 7th pair of primer:
7-F：5’-GACTGCGTACCAATTCACA-3’（SEQ?ID?No:15），
7-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:16）；
The 8th pair of primer:
8-F：5’-GACTGCGTACCAATTCACT-3’（SEQ?ID?No:17），
8-R：5’-GATGAGTCCTGAGTAACCAA-3’（SEQ?ID?No:18）；
The 9th pair of primer:
9-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:19），
9-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:20）；
The tenth pair of primer:
10-F：5’-GACTGCGTACCAATTCACC-3’（SEQ?ID?No:21），
10-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:22）；
The tenth pair of primers:
11-F：5’-GACTGCGTACCAATTCACG-3’（SEQ?ID?No:23），
11-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:24）；
The 12 pair of primer:
12-F：5’-GACTGCGTACCAATTCAAC-3’（SEQ?ID?No:25），
12-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:26）；
The 13 pair of primer:
13-F：5’-GACTGCGTACCAATTCAGC-3’（SEQ?ID?No:27），
13-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:28）；
The 14 pair of primer:
14-F：5’-GACTGCGTACCAATTCAAG-3’（SEQ?ID?No:29），
14-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:30）；
The 15 pair of primer:
15-F：5’-GACTGCGTACCAATTCACA-3’（SEQ?ID?No:31），
15-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:32）；
The 16 pair of primer:
16-F：5’-GACTGCGTACCAATTCACT-3’（SEQ?ID?No:33），
16-R：5’-GATGAGTCCTGAGTAACCAT-3’（SEQ?ID?No:34）。
7. according to method claimed in claim 1, it is characterized in that specifically comprising the following steps:
(1), Sichuan Province, since the last ten-days period in March, minute 10 batches of maize plantings (Zea mays L., 2n=20), every batch of 70 strains, plant interval 10 days for every batch, and corn adopts two strains plantations, and wherein line-spacing is 60cm, and spacing in the rows is 35cm; In the first tenday period of a month in April, according to line-spacing be 2 meters, spacing in the rows be 2 meters distance by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76) divide root and stem of certain plants plantation;
(2), in the phase of weaving silk, before weaving silk, by the female fringe bagging of MTF-1, and after the female fringe of MTF-1 is weaved silk, MTF-1 filigree is cut short to 1～3cm, then be take corn as male parent is to the 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 filial generation seed that step (2) is obtained is implanted in the nutritive cube of climatic cabinate, in temperature, is to carry out vernalization under 28 ℃, the humidity condition that is 70%, transplants to plastic flowerpot or large Tanaka's plantation during plant strain growth to 3 leaf 1 heart;
(4), in the shooting stage, select to have tiller and tiller number more than 15 plant, during plant to be planted plant height 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, and selecting cell chromosome number is the individual plant of maternal chromosome number and male parent andro gamete chromosome number sum (2n=86), and divides 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 (Tripsacum dactyloides L., 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) the residue tip of a root material of the selected individual plant of step (5) is carried out to chromosome sectioning, selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome, identify that its chromosome forms; Selecting root tip cell chromosome number is 86, and its chromosome set is configured to the plant of 36 of 30 of maize chromosomes, 20 of tetraperennial teosinte chromosomes and tetraploid gama grass chromosome numbers, the corn allos heptaploid being bred as.
8. in accordance with the method for claim 7, the method that it is characterized in that the aflp analysis described in step (5) is: total DNA of the selected plant of extraction step (4), extract MTF-1, corn, tetraperennial teosinte and tetraploid gama grass (Tripsacum dactyloides L. simultaneously, total DNA 2n=72), adopt EcoRI and MseI to carry out enzyme and cut, 37 ℃ of enzymes are cut coupled reaction 6h, after taking-up, be placed in 65 ℃ 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), and 10xBuffer2.5 μ L, EcoRI/MseI5.0U, EcoRI joint 5.0Pmol, MseI joint 5.0Pmol, T
4dNA ligase 10.0U, ddH
2o polishing 25.0 μ L; Then the enzyme of take cut with coupled reaction after the DNA of gained be template, take SEQ ID No:1 and SEQ ID No:2 to increase in advance as primer, wherein the PCR response procedures of pre-amplification is: 94 ℃ of sex change 15s, 60 ℃ of renaturation 30s, 72 ℃ are extended 60s, 28 circulations; 72 ℃ of 2min, 4 ℃ of preservations; Pre-amplified production detects with 2% agarose gel electrophoresis; Increase the in advance DNA of gained of take is again template, each primer pair of take respectively in 16 pairs of primers of SEQ ID No:3 to SEQ ID No:34 carries out selective amplification as primer, 6% sex change polyacrylate hydrogel electrophoresis detection for selective amplification product, selects the individual plant consistent with MTF-1 band, genome is not lost; Wherein said selective amplification PCR program is: 94 ℃ of sex change 10s, and 65 ℃ of renaturation 30s, 72 ℃ are extended 60s, 13 circulations, the every circulation of annealing temperature reduces by 0.7 ℃; 94 ℃ of sex change 10s, 56 ℃ of renaturation 30s, 72 ℃ are extended 60s, and 25 circulations are extended every circulation and are increased 1s; 72 ℃ of 2min.
9. in accordance with the method for claim 1, it is characterized in that specifically comprising the following steps:
(1), Sichuan Province, since the last ten-days period in March, minutes 7 batches plantation tetraperennial teosintes (Zea perennis, 2n=40), 7 days, the every batch of interval, every batch of 10 strains, tetraperennial teosinte single-strain planting, wherein seeding row spacing is 2m * 2m; In the first tenday period of a month in April, according to line-spacing be 2 meters, spacing in the rows be 2 meters distance by corn allohexaploid MTF-1(Tripsazea creammaize T.2n=76) divide root and stem of certain plants plantation;
(2), in the phase of weaving silk, before weaving silk by the female fringe bagging of MTF-1, and after the female fringe of MTF-1 is weaved silk, MTF-1 filigree is cut short to 1～3cm, then take tetraperennial teosinte as male parent is to the female fringe pollination of MTF-1, each female fringe at least repeats to pollinate 3 times, after each pollination, on bagging, mark, results are the filial generation seed of black or grey;
(3), April next year, the seed that step (2) is obtained is 28 ℃ in temperature in being implanted in the nutritive cube of climatic cabinate, vernalization under the condition that humidity is 70%, transplants to plastic flowerpot or field planting during plant strain growth to 3 leaf 1 heart;
(4), select to have tiller and tiller number more than 15 plant; When plant to be planted plant height is 30cm, get the tip of a root, identification of cell chromosome number; Each individual plant is at least identified 50 cells, and selecting cell chromosome number is the plant of maternal chromosome number and male parent andro gamete chromosome number sum (2n=96), and divides 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 (Tripsacum dactyloides L., 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 to carry out chromosome sectioning to the selected individual plant residue of step (5) tip of a root material, selective staining body carries out double-colored genomic in situ hybridization in mitosis metaphase and the high tip of a root combustion sheet of dispersity, use respectively digoxin and biotin labeling corn and tetraperennial teosinte chromosome, identify that chromosome forms; Selecting plant root tip cell chromosome number is that 96 and its chromosome form the complete genomic individual plant that contains 36 of 20 of maize chromosomes, 40 of tetraperennial teosinte chromosomes and gama grass chromosome numbers, is bred as corn allooctaploid.
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