CN103898109A - Molecular marker closely interlocked with wheat sharp eyespot resistance QTL (Quantitative Trait Loci) and application thereof - Google Patents
Molecular marker closely interlocked with wheat sharp eyespot resistance QTL (Quantitative Trait Loci) and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker closely interlocked with wheat sharp eyespot resistance QTL (Quantitative Trait Loci) and an application of the molecular marker. The molecular marker Xgwm484-145 with the size of 145bp is obtained by the steps of performing PCR (Polymerase Chain Reaction) amplification on DNA (Deoxyribonucleic Acid) of a wheat variety CI12633 by using primer pairs SEQNo.1 and SEQNo.2, and performing electrophoresis separation on an amplification product on 6% polyacrylamide gel; the molecular marker is used for detecting the wheat variety CI12633 and derivative varietes or strain genetypes of the wheat variety CI12633, so as to judge whether the varieties or the strains have the wheat sharp eyespot resistance. By adopting the molecular marker, the defects that wheat sharp eyespot resistance identification is easily affected by the environment in conventionality breeding and the wheat sharp eyespot resistance can only be identified and screened in the grain milk-ripe stage are overcome, the sharp eyespot resistance of wheat plants can be predicted and screened by detecting the molecular marker at the seedling stage, infected plants can be eliminated, and the breeding efficiency is improved.
Description
Technical field
The present invention relates to wheat breeding and biology field, particularly a kind of and the closely linked molecule marker of wheat sharp eyespot resistance QTL and application thereof.
Background technology
Wheat hypochnus is the important disease of wheat of China middle and lower reach of Yangtze River Mai Qu and Yellow River-Huai River region, mainly infect and cause by Rhizoctonia cerealis (RhizoctoniacerealisVanderHoeven) etc., this disease causes wheat yield loss serious, only 2005～2009, China suffers approximately 670～8,000,000 hectares of the wheatland areas that banded sclerotial blight causes harm every year, and the financial loss causing is thus more than billions of units.The control of this disease is taking chemical prevention as main at present, because disease happening part is at wheat stalk culm base, control not only needs dose greatly and prevention effect is subject to the such environmental effects such as weather, chemical prevention has also increased agriculture originally, the extensive chemical agent influence ecological environment that is bound to that uses, cultivates and uses disease-resistant variety to prevent and treat beyond doubt the most economical and effective means of wheat hypochnus.
The cultivation of disease-resistant variety need to have the anti-source that resistance is stable to combine for preparing hybrid.In the last thirty years, China researchist has carried out sharp eyespot resistance qualification to more than 3000 parts of wheat idioplasms, though screen some anti-sources, the genetics of resistance rule in these anti-sources is known little about it, most of anti-source agronomy is poor, and the anti-source of banded sclerotial blight that can use in breeding still lacks.
The research report of carrying out wheat sharp eyespot resistance is abroad less, report to wheat hypochnus disease-resistant gene (QTL) location mainly concentrates on domestic, utilize toothpick to embed and trench sowing (the wheat crops journals such as disease wheat Resistance Identification method Tang Ting that carry disease germs, 2004,24 (4): 11-16) adopt recombinant inbred lines ARz/ Yangmai No.158, on 2D, 3B, 3D and 7D karyomit(e), sharp eyespot resistance QTL detected, explain respectively the 8%-14% of sharp eyespot resistance; Open (the plant genetic resources journals such as hamlet, 2005,6 (3): 276-279) adopt potted plant seed dressing inoculation method to identify the sharp eyespot resistance of recombinant inbred lines river 35050/ mountain agriculture 483, QTL positioning result shows that 1A karyomit(e) exists anti-banded sclerotial blight QTL, the sharp eyespot resistance of this QTL soluble 21.57%, detect that 4 pairs are made QTL mutually, total contribution rate is 52.2% simultaneously; Adopt Shandong wheat 21/ mountain agriculture 0431 recombinant inbred lines, the anti-banded sclerotial blight QTL chain with mark Xgwm526 detected at 2B karyomit(e).(the wheat crops journal such as Ren Lijuan, 2007,27 (3): 416-420) utilize toothpick to embed and the method such as table soil inoculation is identified recombinant inbred lines Sumai 3/exempt from the in vain sharp eyespot resistance of No. 3, in conjunction with genotype data, on karyomit(e) 2B, 3B, 5A, 6A and 6B, find anti-banded sclerotial blight QTL, phenotype explanation rate is respectively 9%-13%; Chen etc. (TheoreticalandAppliedGenetics, 2013,126 (11): 2865-2878) also find to exist sharp eyespot resistance QTL at 1A, 2B, 3B, 4A, 5D, 6B and 7B karyomit(e).
CI12633 is the anti-source of Powdery Mildew containing Pm2 and Pm6 that China introduces, and this anti-source is double Resistant Gene To Rust and smut simultaneously, and Resistance Identification shows for many years, and this anti-source stem stalk is tough and tensile, has more stable sharp eyespot resistance, can be used for varietal resistance improvement.Genetic analysis shows, the sharp eyespot resistance of CI12633 is the quantitative character by major gene and minor gene co-controlling, be conducive to molecule location and the molecular marker assisted selection (Ren Lijuan etc. of major gene, Jiangsu agricultural journal, 2010, 26 (6): 1156-1161), we adopt CI12633/ Yangmai No.158 recombinant inbred lines to locate its sharp eyespot resistance main effect QTL, at 6B, on 5A karyomit(e), find that phenotype explanation rate exceedes 10% anti-banded sclerotial blight main effect QTL, GWM608 with 6B and 5A karyomit(e) resistance main effect QTL compact linkage, GWM88, WMC727 and GWM410.2 molecule marker are applied for a patent (patent No.: ZL200710019279.8), but have not yet to see report with the molecule marker of sharp eyespot resistance main effect QTL compact linkage in CI12633 kind 2D karyomit(e).
Summary of the invention
The object of the invention is to provide a kind of molecule marker whether existing in wheat 2D karyomit(e) with the sharp eyespot resistance main effect QTL compact linkage of wheat breed CI12633 that detects, judge whether wheat plant contains sharp eyespot resistance main effect QTL, thereby the sharp eyespot resistance of prediction wheat plant, the selection progress of accelerating anti-banded sclerotial blight wheat, concrete steps are as follows:
One and the closely linked molecule marker of wheat sharp eyespot resistance QTL, with the DNA of wheat breed CI12633, carry out pcr amplification with primer pair SEQNo:1 and SEQNo:2, amplified production is on 6% polyacrylamide gel after electrophoretic separation, and obtaining size is the molecule marker Xgwm484-145 of 145bp.
In the present invention, the DNA of wheat breed CI12633 refers to the blade of CI12633 wheat plant separated to the DNA obtaining, and described 6% polyacrylamide gel refers to and in 100ml polyacrylamide sol solution, contains 5.7g acrylamide and 0.3g methylene diacrylamide.
A kind of application of above-mentioned and the closely linked molecule marker of wheat sharp eyespot resistance QTL, molecule marker Xgwm484-145 is used for to wheat breed CI12633 and derived varieties or the genotypic detection of strain, to judge whether this kind or strain have sharp eyespot resistance.
In application of the present invention, the described method that judges whether this kind or strain have a sharp eyespot resistance is: be male parent or maternal and other wheat hybridizings by CI12633 and derived varieties thereof or product and multiply to F
2more than generation, extract wheat plant leaf DNA, carry out pcr amplification with primer pair SEQNo:1 and SEQNo:2, amplified production is on 6% polyacrylamide gel after electrophoretic separation, check whether contain size for 145bp with the closely linked molecule marker Xgwm484-145 of wheat sharp eyespot resistance QTL, there is this molecule marker, " in the anti-" level of predicting that the resistance of the banded sclerotial blight of this wheat at least reaches; Described " in anti-" level refers to that wheat hypochnus disease index is 20.1-30.0%.
In application of the present invention, described wheat breed CI12633 and derived varieties thereof or strain refer to, taking CI12633 as parent, by conventional hybridization or adopt corn and wheat hybridizing to induce monoploid, then double to obtain wheat breed or the strain of double haploid by colchicine.
The present invention can overcome wheat sharp eyespot resistance in conventional breeding and identify easily affected by environment, and can only be in the shortcoming of seed milk stage evaluation and screening, just can the sharp eyespot resistance of wheat plant be predicted and be screened by detection molecules mark in seedling stage, eliminate disease plant, reduce the waste of manpower and materials, improve breeding efficiency.
Embodiment
Embodiment 1 verifies in seed materials with the molecule marker of wheat sharp eyespot resistance main effect QTL compact linkage:
The primer pair that the present embodiment relates to:
SEQNO:15’ACATCGCTCTTCACAAACCC3’；
SEQNO:25’AGTTCCGGTCATGGCTAGG3’
1. the strain that is customized for W001～W018 is taking CI12633 as male parent, taking Yangmai No.158 as maternal, hybridizes and multiplies to F
6the seed materials in generation; The strain that is customized for W019～W048 is taking CI12633 as male parent, taking peaceful wheat No. 9 as maternal, hybridizes and multiplies to F
6the seed materials in generation; The strain that is customized for W049～W068 is taking CI12633 as male parent, and taking Yangmai No.158 as maternal, hybridization F1, adopts Pollen Maydis induction monoploid, and double haploid strain after doubling by colchicine.
2. pair each seed materials adopts respectively the method for CTAB to extract leaf DNA.
3. the DNA that pair step (2) obtains carries out pcr amplification with PCR primer pair SEQNO:1 and SEQNO:2 respectively, and PCR reaction system is: cumulative volume 20 μ l, 1.5mmol/LMgCl
2, 0.2mmol/LdNTP
s, 0.4mmol/L primer SEQNo:1 and primer SEQNo:2,50ng wheat template DNA, 1uTag enzyme, surplus is 1 × PCR damping fluid; PCR reaction conditions: 95 DEG C of sex change 3min, then 94 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 30s totally 35 circulations, last 72 DEG C are extended 5min;
Amplified production is on 6% polyacrylamide gel after electrophoretic separation, check and whether contain the Xgwm484-145 molecule marker of size for 145bp, described 6% polyacrylamide gel is in 100ml polyacrylamide sol solution, to contain 5.7g acrylamide and 0.3g methylene diacrylamide.
4. as contain Xgwm484-145 molecule marker, the resistance of the banded sclerotial blight of measurable this wheat at least reaches " in anti-" level.
5. the sick garden in field, carries out wheat sharp eyespot resistance qualification to W001～W068 seed materials, in wheat during jointing stage inoculation banded sclerotial blight germ, and the milk stage investigation banded sclerotial blight state of an illness, severity criterion is as follows: 0 grade: without illness; 1 grade: leaf sheath has typical banded sclerotial blight scab, but do not invade stem; 2 grades: germ is invaded people's stem stalk, scab width ring stem is no more than 1/2 of stem stalk girth; 3 grades: germ is invaded people's stem stalk, on stem stalk, scab ring stem width is between 1/2-3/4 stem stalk girth; 4 grades: germ is invaded people's stem stalk, on stem stalk scab around stem stalk more than 3/4, or the soft corruption of stem stalk; 5 grades: withered booting or withered dead ears.
Disease index calculates as follows:
Disease index=[(Σ diseased plant numbers at different levels × typical values at different levels)/(total strain number × highest typical value)] × 100
Resistance criterion: wheat hypochnus disease index is " resisting " below 20.0%, 20.1-30.0% is " in anti-", 30.1-40.0% is " middle sense ", more than 40.1% is " sense ".
6. Xgwm484-145 Molecular Prediction result and Disease garden identification actual result are compared, the results are shown in subordinate list 1, predict the outcome very identical with measured result, as long as there is Xgwm484-145 molecule marker to exist, this strain banded sclerotial blight disease index is all lower than 30%, wherein 3 parts of material disease indexs, lower than 20.0%, reach the level of " resisting ".
Table 1 Molecular Prediction result and actual result comparison
Note :+expressive notation exists, and-expressive notation does not exist.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
Mono-kind of <120> and the closely linked molecule marker of wheat sharp eyespot resistance QTL and application thereof
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
acatcgctct tcacaaaccc 20
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
agttccggtc atggctagg 19
Claims (5)
1. one kind and the closely linked molecule marker of wheat sharp eyespot resistance QTL, it is characterized in that, size is the molecule marker Xgwm484-145 of 145bp, the DNA with wheat breed CI12633, carry out pcr amplification with primer pair SEQ No:1 and SEQ No:2, amplified production obtains after electrophoretic separation on 6% polyacrylamide gel.
2. described in requiring 1 according to power with the closely linked molecule marker of wheat sharp eyespot resistance QTL, it is characterized in that, the DNA of described wheat breed CI12633 refers to the blade of CI12633 wheat plant separated to the DNA obtaining, and described 6% polyacrylamide gel refers to and in 100ml polyacrylamide sol solution, contains 5.7g acrylamide and 0.3g methylene diacrylamide.
3. the application of molecule marker as claimed in claim 1 or 2, is characterized in that, molecule marker Xgwm484-145 is used for to wheat breed CI12633 and derived varieties or the genotypic detection of strain, to judge whether this kind or strain have sharp eyespot resistance.
4. the application of molecule marker according to claim 3, is characterized in that, the described method that judges whether this kind or strain have a sharp eyespot resistance is: be male parent or maternal and other wheat hybridizings by CI12633 and derived varieties thereof or product and multiply to F
2more than generation, extract wheat plant leaf DNA, carry out pcr amplification with primer pair SEQ No:1 and SEQ No:2, amplified production is on 6% polyacrylamide gel after electrophoretic separation, check whether contain size for 145bp with the closely linked molecule marker Xgwm484-145 of wheat sharp eyespot resistance QTL, there is this molecule marker, " in the anti-" level of predicting that the resistance of the banded sclerotial blight of this wheat at least reaches; Described " in anti-" level refers to that wheat hypochnus disease index is 20.1-30.0%.
5. the application of molecule marker according to claim 4, it is characterized in that, described wheat breed CI12633 and derived varieties thereof or strain refer to, taking CI12633 as parent, by conventional hybridization or adopt corn and wheat hybridizing to induce monoploid, then double to obtain wheat breed or the strain of double haploid by colchicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104498503A (en) * | 2014-11-28 | 2015-04-08 | 安徽拜森生物科技有限公司 | Corn sheath blight anti-disease gene GRMZM2G127328 and application thereof |
| CN106834527A (en) * | 2017-04-01 | 2017-06-13 | 江苏省农业科学院 | Molecular labeling and its application with wheat seedling sharp eyespot resistance QTL close linkages |
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| CN101003839A (en) * | 2007-01-11 | 2007-07-25 | 江苏省农业科学院 | Molecule tag close linked to resistance QTL of wheat sharp eyespot, and application |
| CN103688846A (en) * | 2013-12-02 | 2014-04-02 | 西北农林科技大学 | Wheat-tritileymus 3D (3Ns) alien substitution line cultivation method and identification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003839A (en) * | 2007-01-11 | 2007-07-25 | 江苏省农业科学院 | Molecule tag close linked to resistance QTL of wheat sharp eyespot, and application |
| CN103688846A (en) * | 2013-12-02 | 2014-04-02 | 西北农林科技大学 | Wheat-tritileymus 3D (3Ns) alien substitution line cultivation method and identification method |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498503A (en) * | 2014-11-28 | 2015-04-08 | 安徽拜森生物科技有限公司 | Corn sheath blight anti-disease gene GRMZM2G127328 and application thereof |
| CN106834527A (en) * | 2017-04-01 | 2017-06-13 | 江苏省农业科学院 | Molecular labeling and its application with wheat seedling sharp eyespot resistance QTL close linkages |
| CN106834527B (en) * | 2017-04-01 | 2020-09-04 | 江苏省农业科学院 | Molecular marker closely linked with wheat seedling sheath blight resistance QTL and application thereof |
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