CN103518615A - Cultivation method and identification method for wheat-leymus mollis multiple alien substitution line containing 3Ns, 6Ns and 7Ns chromosomes - Google Patents
Cultivation method and identification method for wheat-leymus mollis multiple alien substitution line containing 3Ns, 6Ns and 7Ns chromosomes Download PDFInfo
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Abstract
The invention discloses a cultivation method for a wheat-leymus mollis multiple alien substitution line containing 3Ns, 6Ns and 7Ns chromosomes. The cultivation method comprises steps of 1) performing hybridization between tetraploid hard-grain wheat and octoploid tritileymus, performing identification in the F1 generation, the F2 generation, the F3 generation and the F4 generation, and screening individual plants having a chromosome number of 2n=42 and comprising leymus mollis Ns genome chromosomes; and 2) bagging the screened individual plants and selfing, and in the F5 generation, performing breed selection to obtain the wheat-leymus mollis multiple alien substitution line comprising six leymus mollis Ns genome chromosomes (3Ns, 6Ns and 7Ns). The invention discloses an identification method for the wheat-leymus mollis multiple alien substitution line. The wheat-leymus mollis multiple alien substitution line has significant improvements in plant height, ear length, tiller number, and other agronomic characters.
Description
Technical field
The present invention relates to a kind of breeding method of new variety of plant, belong to plant breeding and biogenetics field.
Background technology
Wheat distance edge hybrid is to utilize exogenous genetic material to enrich wheat genetic basis and artificially creating to synthesize one of the important channel of new species and means.Shore wheat (Leymus mollis (Trin.) Hara), has another name called the soft grass that relies, and belongs to grass family, barley subtribe, leymus ruderal, has drought resisting, cold-resistant, Salt And Alkali Tolerance, barren-resistant, anti-multiple fungi, bacteriosis and stem stalk are sturdy, large fringe such as spends more at the characteristic, is the excellent germplasm resource [2,3] of cereal cultivars improvement.The genome of shore wheat is NsNsXmXm, and wherein Ns genome comes from psathyrostachys huashanica (Psathyrostachys huashanica Keng), and the genomic source of Xm is not yet definite at present.
The sixties in last century, Tsitsin etc. cultivate by rataria, have obtained the hybrid of tetraploid, hexaploid wheat and Dalaicao, shore wheat and Sha Shenglai grass.The employing embryo rescues such as Anamthawat-J ó nsson and colchicin double, obtained the hybrid amphidiploid of common wheat, Persian wheat and shore wheat, and reported that 1 comprises whole A and B genome chromosome, 1 pair of D genome chromosome and the 6 couples of chromosomal wheat-shore of shore wheat wheat amphidiploid materials A D99, utilize AD99 and common wheat hybridization, filtered out the homozygous lines of 6 mildew-resistances.Li etc. (2005,2006) generation of Octoploid Tritileymus microspore, gametophytic formation and the chromosomal genetic behavior of shore wheat are analyzed, and by utilizing OCTOPLOID TRITICALE and Octoploid Tritileymus to hybridize, obtained one and comprised 6 chromosomess of rye and 6 the chromosomal wheat-rye-shore of shore wheat wheats three genus hybrids.
Research is saved and colchicin chromosome doubling by embryo, obtained have shore wheat large fringe, spend more, the incomplete amphidiploid M842 of common wheat-shore wheat of large grain, the characteristic such as disease-resistant; Utilize M842 and common wheat and Nullisomic wheat hybridization, cultivated 3 alien addition lines and 2 alien substitutions; The in situ hybridization of applying gene group forms and has carried out molecular cytogenetics analysis the chromosome set of the Octoploid Tritileymus (Octoploid Tritileymus) of 6 types, the chromosome set of Octoploid Tritileymus M842-4, M842-8, M842-12 and M842-16 is formed and is defined as AABBDDNsNs, the chromosome set formation of M842-10 is defined as AABBDDNsNs+2Ta-2Lm (Ns), and the chromosome set formation of M842-13 is defined as AABBDDJJ; Utilize Octoploid Tritileymus and octoploid of wheat-wheatgrass, octoploid tuftlet wheat to hybridize, obtained wheat-shore wheat-couchgrass, wheat-shore wheat-haynaldia villosa three genus hybrids.
Although prior art is cultivated on new wheat breed and obtained huge technological progress at wheat hybridizing, not yet have at present the report of durum wheat and the multiple alien substitution of shore wheat hybridization acquisition.
Summary of the invention
Present inventor is in long-term research and development and breeding process, paying great efforts conducts in-depth research the hybridization of wheat and shore wheat, and the amazing chromosome number that obtained is 2n=42 in the filial generation of durum wheat and shore wheat, contain 6 chromosomal strains of shore wheat, the strain of this acquisition is adopted, morphology, cytogenetics, genomic in situ hybridization and SSR, the technology such as EST-STS molecular labeling conduct in-depth research, not only determined the morphological feature of this strain, genetic behavior, chromosome forms, and the chromosomal Homoeologous groups of contained shore wheat etc., and find that this strain is at plant height, spike length, the economical character aspects such as tiller number have obtained obvious improvement, for wheat breed improvement and chromosome engineering breeding provide new germ plasm resource.
For achieving the above object, the present invention is achieved through the following technical solutions:
Comprise shore wheat 3Ns, the breeding method of the multiple alien substitution of 6Ns and 7Ns chromosomal wheat-shore wheat, comprise the steps: 1) tetraploid durum wheat and Octoploid Tritileymus hybridization, at F1, F2, F3 and F4 all identify from generation to generation, and filtering out chromosome number is 2n=42, and contains the chromosomal individual plant of shore wheat Ns genome; 2) the equal bagging selfing of individual plant filtering out, at F5 from generation to generation, selects and contains shore wheat 3Ns, 6Ns and 7Ns chromosome, totally 6 multiple alien substitutions of the shore chromosomal wheat-shore of wheat Ns wheat.
Wherein, the authentication method that above-mentioned screening process adopts can adopt arbitrarily feasible biological method, preferably adopts cytology chromosome microscopy and genomic in situ hybridization (GISH) to identify, these two kinds of methods can be efficiently, identify accurately.
In the present invention, male parent and female parent used when cultivating beginning can adopt suitable durum wheat kind and Octoploid Tritileymus kind, preferably, described tetraploid durum wheat kind is D4286 (2n=4x=28, AABB) (claim again durum wheat 4286), Octoploid Tritileymus kind is M842-16 (2n=8x=56, AABBDDNsNs).
Applicant has carried out biological analysis evaluation to resulting F5 for wheat-shore multiple alien substitution of wheat, the chromosome configuration of determining the multiple alien substitution of gained wheat-shore wheat is 2n=42=21II, chromosome set becomes by 36 chromosomes of wheat and 6 shore wheat Ns genome chromosomes and forms, 6 Ns genome chromosomes of shore wheat can be paired into 3 bivalent chromosomes completely, and be the 3Ns of shore wheat, 6Ns and 7Ns chromosome.
The morphological analysis experiment that applicant carries out shows, the relative parent of the multiple alien substitution of wheat-shore wheat of breeding method gained of the present invention, and plant height significantly reduces, and tiller number significantly increases, spike length is significantly elongated.
Further, the invention discloses for identifying the authentication method of the multiple alien substitution of wheat-shore wheat, include but not limited to following method:
1. adopt CYTOGENETIC ANALYSIS OF ONE method: by the multiple alien substitution seed of the little shore of F5 offspring wheat indoor germination, clip tip of a root frozen water pretreatment 24h, Kano fixer is fixed, 1% cellulase+1% pectinase enzymatic hydrolysis, aceto-camine dyeing compressing tablet; Pollen mother cell is observed: field is asked and got of the right age young fringe, fixes aceto-camine dyeing compressing tablet, microscopic examination with Kano fixer.
In said method, can determine according to actual conditions the sample time of the tip of a root, frozen water temperature, preferred, when root grows to 1～2cm, the clip tip of a root is in 0～4 ℃ of frozen water pretreatment 24h.
2. adopt genomic in situ hybridization method: adopt CTAB method to extract shore wheat and the psathyrostachys huashanica complete genome DNA row labels of going forward side by side, chromosome sectioning and in situ hybridization, every film-making adds 40 μ l hybridization solutions, and 95 ℃ of sex change 8min are then hybridized in position amenable to process on hybridization instrument and carried out.
Wherein hybridization solution used can adopt in situ hybridization field available composition arbitrarily, the composition and configuration method of this type of hybridization solution is all on the books at common Biochemistry Experiment handbook, preferably, described hybridization solution consists of: 20 * SSC4 μ l, ssDNA (salmon sperm dna, 5ug/uL) 1 μ l, 10% (W/V) SDS is dodecyl sodium sulfate 1 μ l, 50% (W/V) DS is dextran sulfate 8 μ l, deionized formamide 20 μ l, DNA probe 100ng, aseptic deionized water adds to 40 μ l.
This series products that the in situ hybridization instrument that wherein adopted can adopt any producer to provide, its program parameter arranges according to the recommendation setting of instrument, and preferred, in situ hybridization instrument used is HYBrite in situ hybridization instrument, and programming is 75 ℃ of 8min, 37 ℃ of 16h.
3. adopt SSR and EST-STS labeled analysis method: (primer is from Pestsova E, Ganal M and to adopt the chromosomal 14 pairs of SSR primers of wheat D genome 1D～7D
m (2000) .Isolation and mapping of microsatel l ite markers specific for the D genome of bread wheat.Genome.43:689-697. and
mS, Korzun V, Wendehake K, Plaschke J, et a1. (1998) .A microsatellite map of wheat.Genetics.149:2007-2023), the multiple alien substitution of the little shore of F5 offspring wheat and parent thereof are analyzed, determined the chromosomal composition of D genome;
Adopt the chromosomal 10 pairs of EST-STS primers of wheat the 3rd, 6,7 homology groups (primer from: http://wheat.pw.usda.gov/SNP/new/pcr primers.shtml), determine the chromosomal Homoeologous groups of Ns genome; Adopt pcr amplification reaction to increase to DNA, amplified production carries out electrophoresis through 8% non-denaturing polyacrylamide gel, point sample amount 8 μ l, electrophoresis 4～5 h under 150V constant-pressure conditions, cma staining.
The reaction condition that wherein PCR reaction adopts can adopt this area typical conditions, and preferred, the reaction system of described pcr amplification reaction is for comprising 1 * buffer (100mM Tris-HCl pH8.3,1.5mM MgCl2), 0.2M dNTPs, 50ng primer, 1U Taq archaeal dna polymerase, 50～100ng template DNA, reaction condition is 94 ℃ of denaturation 3min, 34 circulations are according to 94 ℃ of sex change 1min, 50,55 or 60 ℃ of annealing 1min, 72 ℃ of extension 2min carry out, the last 10min that fully extends.
The multiple alien substitution of wheat-shore wheat disclosed by the invention provides new germ plasm resource for wheat breed improvement and chromosome engineering breeding, and disclosed authentication method can be identified strain effectively accurately.
Accompanying drawing explanation
Fig. 1 be the multiple alien substitution of gained wheat-shore wheat tip of a root somatic chromosome (A) and and pollen mother cells I in mid-term chromosome (B);
Fig. 2 is that the genomic in situ hybridization of the tip of a root somatic chromosome of the multiple alien substitution 10DM50 of wheat-shore wheat is identified (C) and pollen mother cells I in mid-term chromogene group in situ hybridization evaluation (D);
Fig. 3 is the analysis of the multiple alien substitution of the 3rd homology group EST-STS primer pair little shore wheat;
Fig. 4 is the analysis of the multiple alien substitution of the 6th homology group EST-STS primer pair little shore wheat;
Fig. 5 is the analysis of the multiple alien substitution of the 7th homology group EST-STS primer pair little shore wheat;
Fig. 6 is the multiple alien substitution of wheat-shore wheat and parent's thereof plant (a), tassel (b) and little Hua (c).
Embodiment
Embodiment 1: the cultivation of the multiple alien substitution of wheat-shore wheat
Utilize durum wheat (2n=4x=28, AABB) kind D4286 and Octoploid Tritileymus M842-16 (2n=8x=56, AABBDDNsNs) hybridization, at F1, F2, F3 and F4 all carry out cytology chromosome microscopy and genomic in situ hybridization (GISH) from generation to generation to be identified, screening chromosome number is 2n=42, and contain the chromosomal individual plant of shore wheat Ns genome, to the equal bagging selfing of the individual plant filtering out, at F5 from generation to generation, selecting 1 contains 6 and is respectively shore wheat 3Ns, the multiple alien substitution of the chromosomal shore of 6Ns and 7Ns wheat Ns genome chromosomal wheat-shore wheat, called after 10DM50.
Embodiment 2: the evaluation of the multiple alien substitution of wheat-shore wheat of embodiment 1
The multiple alien substitution 10DM50 of the little shore of F5 offspring wheat of CYTOGENETIC ANALYSIS OF ONE: D4286 * M842-16, seed indoor germination, when root grows to 1～2cm, the clip tip of a root is in 0～4 ℃ of frozen water pretreatment 24h, Kano fixer is fixed, 1% cellulase+1% pectinase enzymatic hydrolysis, aceto-camine dyeing compressing tablet; Pollen mother cell is observed: field is asked and got of the right age young fringe, fixes aceto-camine dyeing compressing tablet, 01ympus BX60 microscopic examination with Kano fixer; Utilize durum wheat kind D4286 (AABB, 2n=28) with M842-16 (AABBDDNsNs, 2n=56) hybridization, offspring is through the bagging selfing of 4 generations, in F5 offspring, by tip of a root somatic chromosome microscopy, screen the plant 10DM50 of 1 strain 2n=42, gather in the crops this individual plant, to its 15 seed indoor germinations, fixedly after the tip of a root, be seeded in land for growing field crops; The tip of a root chromosome number of somatic of 15 seeds is all 2n=42 (Figure 1A).The of the right age young fringe of choosing 10DM50 is asked in field, 40 pollen mother cells I in mid-term chromosome pairing situations are carried out to microscopy observation, result shows, it is 2n=21II that 10DM50 has the chromosome configuration of 38 pollen mother cells, account for 95% of observation of cell number, only having the chromosome configuration of 2 pollen mother cells is 2n=21II+2I, accounts for 5% (Figure 1B) of observation of cell number.
Genomic in situ hybridization: DNA extracts and probe mark: adopt CTAB method to extract shore wheat and the psathyrostachys huashanica complete genome DNA row labels of going forward side by side, chromosome sectioning and in situ hybridization, every film-making adds 40 μ l hybridization solutions and (comprises 20 * SSC4 μ l, ssDNA (salmon sperm dna, 5ug/uL) 1 μ l, 10% (W/V) SDS (dodecyl sodium sulfate), 1 μ l, 50% (W/V) DS (dextran sulfate), 8 μ l, deionized formamide 20 μ l, DNA probe 100ng, aseptic deionized water adds to 40 μ 1).95 ℃ of sex change 8min, hybridization amenable to process on hybrite in situ hybridization instrument: 75 ℃ of 8min, 37 ℃ of 16h carry out;
The tip of a root somatic cell of 10DM50 be take respectively to shore wheat and psathyrostachys huashanica complete genome DNA to be identified as probe carries out genome fluorescence in situ hybridization (GISH), result shows, 10DM50 cell all contains 6 complete yellow green chromosome signals, infers that 10DM50 contains 6 Ns genome chromosomes (Fig. 2 C).
Using psathyrostachys huashanica total genomic dna as probe, its pollen mother cell is carried out to genomic in situ hybridization evaluation, result shows, 10DM50 pollen mother cells I in mid-term cell has 3 complete bivalents to show yellow green signal, shows that 6 Ns genome chromosomes in 10DM50 can be paired into 3 pairs of chromosomes (Fig. 2 D) completely.Explanation thus, 10DM50 is 1 inheritance stability, comprises 36 chromosomes of wheat and 3 pairs of multiple alien substitutions of the shore chromosomal wheat-shore of wheat Ns genome wheat.
SSR and EST-STS labeled analysis: adopt
deng and the chromosomal 14 pairs of SSR primers of wheat D genome 1D～7D announced such as Pestsova, as shown in table 1 below:
Table 1:14 is to SSR primer
10DM50 and parent thereof are analyzed, determine the chromosomal composition of D genome in 10DM50; Adopt the chromosomal 10 pairs of EST-STS primers of wheat the 3rd, 6,7 homology groups, determine the chromosomal Homoeologous groups of Ns genome in 10DM50; Pcr amplification reaction system 20 μ l, comprise 1 * buffer (100mM Tris-HCl pH8.3,1.5mM MgCl2), 0.2M dNTPs, 50ng primer, 1U Taq archaeal dna polymerase, 50～100ng template DNA, 94 ℃ of denaturation 3min, 34 circulations are according to 94 ℃ of sex change 1min, 50,55 or 60 ℃ of annealing 1min, 72 ℃ of extension 2min carry out, the last 10min that fully extends, and amplified production carries out electrophoresis through 8% non-denaturing polyacrylamide gel, point sample amount 8 μ l, electrophoresis 4～5h under 150V constant-pressure conditions, cma staining, digital camera photograph saving result.
SSR interpretation of result shows, the genomic 1D of D, 2D, primer on 4D and 5D chromosome, at 10DM50, in Octoploid Tritileymus M842 and wheat breed 7182, all amplified the set goal band, and 3D, the primer on 6D and 7D chromosome, although amplified the set goal band in Octoploid Tritileymus M842 and wheat breed 7182, but in 10DM50, do not amplify the set goal band, illustrate that 10DM50 may contain the genomic 1D of wheat D, 2D, 4D and 5D chromosome, and disappearance the genomic 3D of wheat D, 6D and 7D chromosome.EST-STS interpretation of result shows, primer (BF291730 on 3 pairs of the 3rd homology group chromosomes, BM134465, CD373475) (Fig. 3), primer (BE426174 on 3 pairs of the 6th homology group chromosomes, BE518349, CD452568) primer (BQ170462 on (Fig. 4) He 4 pairs of the 7th homology group chromosomes, BF293421, BQ161902, CD452422) (Fig. 5) in 10DM50, Octoploid Tritileymus M842-16 and shore wheat (L.mollis), all amplified the special target stripe of Ns genome chromosome.Show: the multiple alien substitution 10DM50 of little shore wheat may contain shore wheat Ns genomic 3Ns, 6Ns and 7Ns chromosome.Illustrate: the genomic 3D of wheat D in 10DM50,6D and 7D chromosome may be replaced by 3Ns, 6Ns and the 7Ns chromosome of shore wheat.
Said process primer used is as shown in table 2 below, and the present invention's this primer used is synthetic by Shanghai Sheng Gong biotechnology Co., Ltd.
Table 2:10 is to ETS primer
Morphological observation: statistics Octoploid Tritileymus M842-16, durum wheat kind D4286; plant height and the tiller number of 10 individual plants of the multiple alien substitution 10DM50 of little shore wheat and Common Wheat Varieties 7182; investigate all spike lengths of tillering of 10 typical individual plants of each material, spikelet number, grain number per spike, thousand kernel weight, ripening rate, utilize SPSS V13.0 software to carry out variance analysis to the significance of difference of each character value.The demonstration of morphology result, 10DM50 has parents' morphological feature, and plant height obviously becomes short (Fig. 5 A); The length of awns is between parents, and its color is black, consistent with parent D4286 (Fig. 5 B); Fringe shape and parent M842-16 are comparatively approaching, are all spindle (Fig. 5 C).The plant height of 10DM50, thousand kernel weight are between parents, and tiller number, spike length are higher than parent, and the proterties such as spikelet number and ripening rate are significantly lower than parents.Variance analysis shows, the plant height of 10DM50 significantly reduces, and tiller number significantly increases, spike length is significantly elongated.
Claims (9)
1. comprise shore wheat 3Ns, the breeding method of the multiple alien substitution of 6Ns and 7Ns chromosomal wheat-shore wheat, it is characterized in that comprising the steps: 1) tetraploid durum wheat and Octoploid Tritileymus hybridization, at F1, F2, F3 and F4 all identify from generation to generation, and filtering out chromosome number is 2n=42, and contains the chromosomal individual plant of shore wheat Ns genome; 2) the equal bagging selfing of individual plant filtering out, at F5 from generation to generation, selects and contains 6 and be respectively shore wheat 3Ns, the multiple alien substitution of wheat-shore wheat that 6Ns and 7Ns are chromosomal.
2. breeding method according to claim 1, is characterized in that described tetraploid durum wheat kind is D4286, and Octoploid Tritileymus kind is M842-16.
3. breeding method according to claim 1, the chromosome configuration that it is characterized in that the multiple alien substitution of gained wheat-shore wheat is 2n=42=21II, chromosome set becomes by 36 chromosomes of wheat and 6 shore wheat Ns genome chromosomes and forms, 6 Ns genome chromosomes of shore wheat can be paired into 3 bivalent chromosomes completely, be respectively the 3Ns of shore wheat, 6Ns and 7Ns chromosome.
4. the authentication method of the multiple alien substitution of wheat-shore wheat, it is characterized in that adopting CYTOGENETIC ANALYSIS OF ONE method: by the multiple alien substitution seed of the little shore of F5 offspring wheat indoor germination, the clip tip of a root is in frozen water pretreatment 24h, Kano fixer is fixed, 1% cellulase+1% pectinase enzymatic hydrolysis, aceto-camine dyeing compressing tablet; Pollen mother cell is observed: field is asked and got of the right age young fringe, fixes aceto-camine dyeing compressing tablet, microscopic examination with Kano fixer.
5. the authentication method of the multiple alien substitution of wheat-shore wheat, it is characterized in that adopting genomic in situ hybridization method: adopt CTAB method to extract shore wheat and the psathyrostachys huashanica complete genome DNA row labels of going forward side by side, chromosome sectioning and in situ hybridization, every film-making adds 40 μ l hybridization solutions, then 95 ℃ of sex change 8min, hybridize in position amenable to process on hybridization instrument and carry out.
6. the authentication method of the multiple alien substitution of wheat-shore wheat according to claim 5, it is characterized in that described hybridization solution consists of: 20 * SSC4 μ l, ssDNA (salmon sperm dna, 5ug/uL) 1 μ l, 10% (W/V) SDS1 μ l, 50% (W/V) DS8 μ l, deionized formamide 20 μ l, DNA probe 100ng, aseptic deionized water adds to 40 μ l.
7. the authentication method of the multiple alien substitution of wheat-shore wheat according to claim 5, is characterized in that in situ hybridization instrument used is HYBrite in situ hybridization instrument, and programming is 75 ℃ of 8min, 37 ℃ of 16h.
8. the authentication method of the multiple alien substitution of wheat-shore wheat, it is characterized in that adopting SSR and EST-STS labeled analysis method: adopt the chromosomal 14 pairs of SSR primers of wheat D genome 1D～7D, the multiple alien substitution of the little shore of F5 offspring wheat and parent thereof are analyzed, determined the chromosomal composition of D genome; Adopt the chromosomal 10 pairs of EST-STS primers of wheat the 3rd, 6,7 homology groups, determine the chromosomal Homoeologous groups of Ns genome; Adopt pcr amplification reaction to increase to DNA, amplified production carries out electrophoresis through 8% non-denaturing polyacrylamide gel, point sample amount 8 μ l, electrophoresis 4～5h under 150V constant-pressure conditions, cma staining.
9. authentication method according to claim 8, is characterized in that the reaction system of described pcr amplification reaction is for comprising 1 * buffer (100mM Tris-HCl pH8.3,1.5mM MgCl
2), 0.2M dNTPs, 50ng primer, 1U Taq archaeal dna polymerase, 50～100ng template DNA, reaction condition is 94 ℃ of denaturation 3min, 34 circulations are according to 94 ℃ of sex change 1min, 50,55 or 60 ℃ of annealing 1min, 72 ℃ of extension 2min carry out, the last 10min that fully extends.
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Non-Patent Citations (2)
| Title |
|---|
| WANLIDU ETAL: "Development and Characterization of a Psathyrostachys huashanica Keng 7Ns Chromosome Addition Line with Leaf Rust Resistance", 《PLOS ONE》 * |
| 无: "Index of /SNP/primers", 《WHEAT.PW.USDA.GOV/SNP/PRIMERS/》 * |
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| CN108866237A (en) * | 2018-09-06 | 2018-11-23 | 西北农林科技大学 | A kind of application of shore wheat genome specific molecular marker |
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Application publication date: 20140122 |