CN103275975A - Wheat new few-tillering QTL (quantitative trait locus), primer pair, molecular marker, molecular marking method and application - Google Patents
Wheat new few-tillering QTL (quantitative trait locus), primer pair, molecular marker, molecular marking method and application Download PDFInfo
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Abstract
The invention discloses a wheat new few-tillering QTL (quantitative trait locus), a primer pair, a molecular marker, a molecular marking method and an application. Detection and analysis prove that the molecular marker gpw346 can accurately trace few-tillering major QTL QLtn.sicau-2D.1 of a wheat strain H461 to predicate the tillering characteristics of the wheat so as to further facilitate molecular design breeding. Simultaneously, during laboratory detection employing the molecular marker, the influence of the environment on phenotype can be avoided. The major QTL QLtn.sicau-2D.1 disclosed by the invention and a closely linked molecular marker gpw346 thereof not only provide candidate genes of wheat plant type breeding, but also enhance tillering predication accuracy by utilizing molecular marker assisted selection, the efficiency of plant type breeding is improved, and the purpose of increasing per unit yield of yield can be achieved fast.
Description
Technical field
The invention belongs to the wheat field of molecular breeding, be specifically related to grow wheat a woman who has recently been widowed and tiller main effect QTL QLtn.sicau-2D.1, primer to, molecule marker, molecule marking method and application.
Background technology
Wheat is the second largest food crop that China is only second to paddy rice, and cultivated area accounts for 27% of food crop area more than 2666.67 ten thousand hectares throughout the year; Ultimate production is more than 100,000,000 tons, accounts for 22% of food crop output.
Wheat yield is made of three elements, and namely output=spike number * grain number per spike * grain is heavy.Tiller be influence the wheat spike number what and and then influence one of Main Agronomic Characters of per unit area yield, be again a kind of special branch phenomenon of monocotyledons, have important researching value.Tillering also is the Main Agronomic Characters relevant to planting environment adaptability with wheat.Donald (1968) once proposed, and the desirable wheat genotypes of production potential maximum should have upright blade, big fringe, and the short or only stalk of stalk, tillering is the characteristic that perennial herb is left over, solely the stalk phenotype has formed indirect pushing effect to the tassel seed.Several proterties that the Dofing only bar kinds of discovery such as (1993) provides change phenology to grow comprise showing leaf speed, prematureness and the more characteristic of heading synchronously faster that these characteristics all are the important component parts that promotes productivity under many environment.
The region of high soil fertility condition is suitable for planting the kind of high tiller number, improves the unit surface number of productive ear, in the hope of reaching the largest production potentiality.But in the relatively poor area of soil fertility condition, the moistening live Mai Qu of weather is (for strengthening the wheat growing way; weaken the competitive power of weeds; usually can strengthen application rate; directly cause population density excessive; can't realize high yield) and (it is low that kind should satisfy water loss in the arid area; organism assimilation efficiency height; robust plant; and other poor environments are had higher tolerance) wait these imperfect environment plantations; the stem stalk of the low kind of tillering is sturdy, and lodging tolerance is strong, big fringe; advantages such as many spikelet numbers can be appeared suddenly, and the ultimate guarantee crop yield.
The forming process of tillering can be divided into two key steps, the i.e. formation of tiller bud and elongation.Usually in the axil of every leaf, can both form an axillalry bud, i.e. tiller bud, but only be positioned at the stem culm base do not extend tiller bud on the internode can elongation growth for tillering; And the axillalry bud on the cane upper extended internode does not generally extend and be in dormant state (Jin Wenkui, Liao Pingan. wheat tillering is contained the research of phase birth index. wheat research .2004,25:21-23).
The wheat tillering mutant is less, and the genetic research macro-progress is slow relatively, and present stage work both at home and abroad is main around tillering QTL(quantitative trait locus) molecule marker location and hereditary effect thereof carry out.Up to now, the relevant main effect QTL of having reported of tillering is positioned at 1A, 2A, and 3A, 6A karyomit(e), wherein only the tin gene studies on 1A and the 3A is more deep, has finished Fine Mapping work.
Wheat line H461(derives from cross combination SW94-30921 * allos No. 2) examine kind with respect to kind river farming 16(state) have that the widow is tillered, many grain number per spikes, many spikelet numbers, high thousand seed weight and high fringe grain heavily wait characteristic.Further study the few shooting property of wheat H461, the QTL that setting control is tillered, seek closely linked molecule marker, initiative and the plant type breeding of material of will tillering for wheat is special provides new genetic resources, utilize molecular marker assisted selection simultaneously, with the enhancing prediction accuracy of tillering, improve the plant type breeding efficiency, accelerate to realize increasing the target of yield of wheat.
Molecular marker assisted selection does not rely on phenotype and selects, and namely is not subjected between envrionment conditions, gene influence of various factors such as work, genotype by environment interaction mutually, but directly genotype is selected, thereby can improve breeding efficiency greatly.Simple repeated sequence (simple sequence repeats, be called for short SSR) is that a class extensively is present in the tandem repetitive sequence of being made up of several Nucleotide repeating unit on the genome.Since its distribution a large amount of on genome, the polymorphism height, and operative technique is simple, expense is cheap, is widely used in molecular mark.Therefore, filter out the closely linked molecule marker with the major gene/QTL of tillering, utilize molecule marker that wheat tillering major gene/QTL is selected, effectively regulate and control wheat tillering and take place, moulding reasonably tillers, and colony takes place, and is significant to improving wheat population quality and output.
Summary of the invention
The widow who the purpose of this invention is to provide common wheat strain H461 QTL QLtn.sicau-2D.1 of tillering.
Another object of the present invention provides the compact linkage molecule mark of this QTL.
The primer that the 3rd purpose of the present invention provides above-mentioned molecule marker is right.
The 4th purpose of the present invention provides the tiller application of the closely linked molecule marker of QTL QLtn.sicau-2D.1 of above-mentioned widow.
Purpose of the present invention can be achieved through the following technical solutions:
A woman who has recently been widowed of the present invention is tillered main effect QTL QLtn.sicau-2D.1 from wheat H461, this QTL is positioned at wheat 2D the short arm of a chromosome, in the section apart from galianconism telomere 1.8cM (as shown in Figure 1), significantly reduces wheat tillering, the LOD value is explained about 45% phenotypic variation greater than 3.0.
The present invention is tillered the upstream primer sequence of molecule marker of main effect QTL QLtn.sicau-2D.1 shown in SEQ ID NO.1 for the identification of wheat H461 a woman who has recently been widowed, and the downstream primer sequence is shown in SEQ ID NO.2.
Described molecule marker is that genomic dna with wheat H461 is the amplified fragments of the pcr amplification gained that carries out of substrate, and and QLtn.sicau-2D.1 between genetic distance be 0.8cM.
The molecule marking method that the applicant provides a kind of widow of evaluation to tiller main effect QTL QLtn.sicau-2D.1 comprises: with the DNA of material to be identified as template, with the primer of above-mentioned molecule marker to carrying out pcr amplification; The PCR product carries out native polyacrylamide gel electrophoresis to be separated, and detects with argentation again; Can amplify plant with the H461 same clip and be and contain the tiller plant of main effect QTL QLtn.sicau-2D.1 of widow.
The molecule marking method that the applicant provides a kind of widow of evaluation to tiller main effect QTL QLtn.sicau-2D.1 preferably includes following steps:
1) with the DNA of material to be identified as template, with the primer of the described molecule marker of claim 2 to carrying out pcr amplification: a) pcr amplification reaction system: it is 50 μ l that 5 μ l10xPCR buffer, 1.5U Ex Taq TM archaeal dna polymerase, 2mmol/L MgCl2,0.2mmol/L dNTP, each 150ng of upstream and downstream primer, 100ng template DNA, distilled water add to total amount; B) PCR program: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ of extensions 30s, totally 35 circulations; 72 ℃ are extended 5min; C) the PCR product detects: the PCR product is with 6% denaturing polyacrylamide gel (Acr:Bis=19:1) electrophoretic separation, and electrode buffer is 1xTBE, firm power 80W, 2000 volts of voltages; Gel detects with cma staining at last.
2) labeled primer identifies that the result of QLtn.sicau-2D.1 is: can amplify plant with the H461 same clip and be and contain the tiller plant of main effect QTL QLtn.sicau-2D.1 of widow.
The application of described QTL in the special material initiative of tillering of wheat.
Described molecule marker primer is to the application in the special material initiative of tillering of wheat.
The application of described QTL in the breeding of wheat plant type.
Described molecule marker primer is to the application in the auxiliary wheat plant type breeding of molecule marker.
Tiller QTL and molecule marker thereof of wheat H461 widow of the present invention obtains by the following method:
1) utilizing the wheat widow strain H461 of tillering to be female parent, is paternal hybrid with spike number type wheat river farming 16, obtains hybrid F1, and F1 obtains F2 for individual plant selfing, constitutes F2 genetic mapping colony at 180 individual plants of F2 picked at random.
2) the F2 colony phenotypic evaluation of tillering
The tiller number of F2 colony plant is identified in the wheat aging time field.
3) ssr analysis
A) DNA extraction: extract parent H461, river farming 16 and the plant DNA of F2 colony with the CTAB method.
B) screening of polymorphic molecular marker between the parent: choose GrainGenes(http: // genomic 615 pairs of SSR primers of covering hexaploid wheat A, B, D that wheat.pw.usda.gov/cgi-bin/graingenes) go up to announce, DNA with parent H461 and river farming 16 is template, carry out pcr amplification, obtain 168 pairs of polymorphism SSR molecule markers altogether;
C) ssr analysis of F2 colony: 168 pairs of polymorphism marks that obtain with above-mentioned steps are primer, utilize increase the simultaneously DNA of parent H461, river farming 16 and F2 colony plant of every pair of primer, carry out genotype identification, obtain molecular marker data.The banding pattern of parent H461 is designated as A, and the banding pattern of parent river farming 16 is designated as B.F2 colony strain lace-type derives from the A that is designated as of H461, and derives from the B that is designated as of river farming 16.
D) structure of linkage map: according to the molecular marker data of 168 pairs of primers, utilize mapping software JoinMap4.0 to make up genetic map.Make up linkage group successively with LOD value 3 to 10, seek optimum reference numerals and flag sequence, determine the linkage group of follow-up use.Utilize the interval of software MapQTL6.0 to make graph model (Interval Mapping) and many QTL make graph model (Multiple QTL Model), and in conjunction with the phenotypic data location widow QTL of tillering of tillering of F2 colony, calculate few the tiller position of QTL and the genetic distance between the molecule marker, finding that wheat H461 karyomit(e) 2DS goes up significantly exists a main widow of the effect QTL QLtn.sicau-2D.1 of tillering, interpret table form variation about 45%, its closely linked molecule marker is gpw346, and genetic distance is 0.8cM.
Beneficial effect:
The present invention discloses the main effect QTL QLtn.sicau-2D.1 of tillering from the widow of wheat H461 first, is positioned at wheat 2D the short arm of a chromosome, in the section apart from galianconism telomere 1.8cM (as shown in Figure 1), has significantly reduced wheat tillering, and the LOD value is greater than 3.0; Contribution rate is big, soluble about 45% phenotypic variation.This QTL QLtn.sicau-2D.1 has higher utility value in wheat plant type (regulation and control are tillered) breeding.
The present invention tiller molecule marker gpw346 of main effect QTL QLtn.sicau-2D.1 of disclosed wheat H461 a woman who has recently been widowed first be the codominant marker, and for the identification of whether containing QTL QLtn.sicau-2D.1 in the plant, easy to detect, amplification stablizes, simple and easy to do.
The amplified production of molecule marker gpw346 disclosed by the invention and the genetic distance between the QLtn.sicau-2D.1 only are 0.8cM, chain very tight, utilize the accuracy height of molecular marker assisted selection QLtn.sicau-2D.1, improve the specific breed selection of tillering of the wheat that adapts to varying environment and identify efficient, save cost.
Description of drawings
Fig. 1. wheat H461 widow tiller main effect QTL QLtn.sicau-2D.1 the position on the 2D karyomit(e) and and molecule marker of the present invention between the linkage inheritance collection of illustrative plates.
The electrophoretogram that the F2 plant molecule marker gpw346 of Fig. 2 .H461* river wheat 107 detects; Wherein 1 and 2 be respectively H461 and river farming 16,5,8,9,14,17 and be the widow genotype plant that tillers, 6,11,15,18,20,21,24 are the genotype plant that tillers more, and 3,4,7,10,12,13,16,19,22,23 is heterozygous genes type plant.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1: wheat H461 widow of the present invention the tiller location of main effect QTL and the acquisition of molecule marker
1) utilizing the wheat widow strain H461 of tillering to be female parent, is paternal hybrid with spike number type wheat river farming 16, obtains hybrid F1, and F1 obtains F2 for individual plant selfing, constitutes F2 genetic mapping colony at 180 individual plants of F2 picked at random.
2) the F2 colony phenotypic evaluation of tillering
The tiller number of F2 colony plant is identified in the wheat aging time field.
3) ssr analysis
A) DNA extraction: extract parent H461, river farming 16 and the plant DNA of F2 colony with the CTAB method.
B) screening of polymorphic molecular marker between the parent: choose GrainGenes(http: // genomic 615 pairs of SSR primers of covering hexaploid wheat A, B, D that wheat.pw.usda.gov/cgi-bin/graingenes) go up to announce, DNA with parent H461 and river farming 16 is template, carry out pcr amplification, obtain 168 pairs of polymorphism SSR molecule markers altogether;
C) ssr analysis of F2 colony: 168 pairs of polymorphism marks that obtain with above-mentioned steps are primer, utilize increase the simultaneously DNA of parent H461, river farming 16 and F2 colony plant of every pair of primer, carry out genotype identification, obtain molecular marker data.The banding pattern of parent H461 is designated as A, and the banding pattern of parent river farming 16 is designated as B.F2 colony strain lace-type derives from the A that is designated as of H461, and derives from the B that is designated as of river farming 16.
D) structure of linkage map: according to the molecular marker data of 168 pairs of primers, utilize mapping software JoinMap4.0 to make up genetic map.Make up linkage group successively with LOD value 3 to 10, seek optimum reference numerals and flag sequence, determine the linkage group of follow-up use.Utilize the interval of software MapQTL6.0 to make graph model (Interval Mapping) and many QTL make graph model (Multiple QTL Model), and in conjunction with the phenotypic data location widow QTL of tillering of tillering of F2 colony, calculate few the tiller position of QTL and the genetic distance between the molecule marker, finding that wheat H461 karyomit(e) 2DS goes up significantly exists a main widow of the effect QTL QLtn.sicau-2D.1 of tillering, interpret table form variation about 45%, its closely linked molecule marker is gpw346, and genetic distance is 0.8cM.
Embodiment 2: molecule marker of the present invention is at the application test of selecting on the few main effect QTL QLtn.sicau-2D.1 of tillering
1) utilize the wheat widow strain H461 of tillering to be female parent, be paternal hybrid with the wheat breed river wheat 107 that tillers more, obtains hybrid F1, and F1 obtains F2 for individual plant selfing, at 22 individual plants of F2 picked at random.
2) 22 F2 individual plants that obtain are carried out the gpw346 marker detection, concrete grammar is: the genomic dna that extracts 22 F2 individual plants in seedling stage; Be substrate with the genomic dna, to being that primer carries out pcr amplification, described primer is with the primer of molecule marker gpw346:
Upstream primer: 5' – CGACCTTTCCCAATTCACAC – 3',
Downstream primer: 5' – TGCTTTTATTCCATCGCACA – 3'.
The pcr amplification reaction system: it is 50 μ l that 5 μ l10xPCR buffer, 1.5U Ex Taq TM archaeal dna polymerase, 2mmol/L MgCl2,0.2mmol/L dNTP, each 150ng of upstream and downstream primer, 100ng template DNA, distilled water add to total amount; B) PCR program: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ of extensions 30s, totally 35 circulations; 72 ℃ are extended 5min; With 6% denaturing polyacrylamide gel (Acr:Bis=19:1) electrophoretic separation, electrode buffer is 1xTBE, firm power 80W, 2000 volts of voltages with resulting PCR product; Gel detects with cma staining at last.The electrophoresis result (see figure 2) finds that wherein 5 plant have the gpw346 loci of H461, predict that these 5 plant tiller number after maturation is lower; And 7 plant have the gpw346 loci of river wheat 107, predict that these 7 plant tiller number after maturation is higher.
3) tiller number of 22 F2 plant is identified in the wheat aging time field, and the plant tillering number (1-3) that result's (seeing Table 1) has the gpw346 loci of H461 significantly is lower than the plant tillering number (8-10) of the gpw346 loci with river wheat 107; Actual result is consistent with expected results, illustrates that the widow of the present invention main effect QTL QLtn.sicau-2D.1 of tillering has the effect of remarkable reduction tiller number really; Simultaneously molecule marker gpw346 of the present invention can be used for selecting the widow main effect QTL QLtn.sicau-2D.1 of tillering.
Table 1 is with tiller molecule marker predicted derived offspring's the tillering ability comparing result table (Line represents the row number in field) of main effect QTL QLtn.sicau-2D.1 of wheat H461 widow
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (9)
1. the grow wheat a woman who has recently been widowed main effect QTL of tillering is characterized in that this QTL is positioned at wheat 2D the short arm of a chromosome, in the section apart from galianconism telomere 1.8cM, significantly reduces wheat tillering, and the LOD value is explained about 45% phenotypic variation greater than 3.0.
2. right for the identification of the primer of the molecule marker of the described QTL of claim 1, it is characterized in that the upstream primer sequence of this molecule marker shown in SEQ ID NO.1, the downstream primer sequence is shown in SEQ ID NO.2.
3. the described wheat of the claim 1 a woman who has recently been widowed molecule marker of main effect QTL of tillering, it is characterized in that, described molecule marker is that the genomic dna of wheat H461 is the amplified fragments that substrate carries out the pcr amplification gained, and and QLtn.sicau-2D.1 between genetic distance be 0.8cM.
4. identify the tiller molecule marking method of main effect QTL of the described wheat of claim 1 a woman who has recently been widowed for one kind, it is characterized in that, as template, use primer shown in SEQ ID NO.1, SEQ ID NO.2 to carrying out pcr amplification with the DNA of material to be identified; The PCR product carries out native polyacrylamide gel electrophoresis to be separated, and detects with argentation again; Can amplify plant with the H461 same clip and be and contain the tiller plant of main effect QTL QLtn.sicau-2D.1 of widow.
5. molecule marking method according to claim 4 is characterized in that, comprises the steps:
1) with the DNA of material to be identified as template, use primer shown in SEQ ID NO.1, SEQ ID NO.2 to carrying out pcr amplification: a) pcr amplification reaction system: 5 μ l10xPCR buffer, 1.5U Ex Taq TM archaeal dna polymerase, 2mmol/L MgCl
2, to add to total amount be 50 μ l for 0.2mmol/L dNTP, each 150ng of upstream and downstream primer, 100ng template DNA, distilled water; B) PCR program: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 60 ℃ of annealing 45s, 72 ℃ of extensions 30s, totally 35 circulations; 72 ℃ are extended 5min; C) the PCR product detects: the PCR product is with 6% denaturing polyacrylamide gel (Acr:Bis=19:1) electrophoretic separation, and electrode buffer is 1xTBE, firm power 80W, 2000 volts of voltages; Gel detects with cma staining at last;
2) labeled primer identifies that the result of QLtn.sicau-2D.1 is: can amplify plant with the H461 same clip and be and contain the tiller plant of main effect QTL QLtn.sicau-2D.1 of widow.
6. the application of the described QTL of claim 1 in the special material initiative of tillering of wheat.
7. the described molecule marker primer of claim 2 is to the application in the special material initiative of tillering of wheat.
8. the application of the described QTL of claim 1 in the breeding of wheat plant type.
9. the described molecule marker primer of claim 2 is to the application in the auxiliary wheat plant type breeding of molecule marker.
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| CN104818271A (en) * | 2015-05-04 | 2015-08-05 | 四川农业大学 | Molecular marker HRM5 of wheat few-tillering gene Ltn3 and application thereof |
| CN104818272A (en) * | 2015-05-04 | 2015-08-05 | 四川农业大学 | Molecular marker SSR52 of wheat few-tillering gene Ltn3 and application thereof |
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| CN104818271A (en) * | 2015-05-04 | 2015-08-05 | 四川农业大学 | Molecular marker HRM5 of wheat few-tillering gene Ltn3 and application thereof |
| CN104818272A (en) * | 2015-05-04 | 2015-08-05 | 四川农业大学 | Molecular marker SSR52 of wheat few-tillering gene Ltn3 and application thereof |
| CN104818272B (en) * | 2015-05-04 | 2018-02-23 | 四川农业大学 | The molecular labeling SSR52 of wheat widow Tillering gene Ltn3 a kind of and its application |
| CN104818271B (en) * | 2015-05-04 | 2018-04-27 | 四川农业大学 | The molecular labeling HRM5 of wheat widow Tillering gene Ltn3 a kind of and its application |
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