CN106480180B - A kind of wheat culm breaking strength molecular labeling QWQD4B.4-13 and its application - Google Patents
A kind of wheat culm breaking strength molecular labeling QWQD4B.4-13 and its application Download PDFInfo
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- CN106480180B CN106480180B CN201610871079.4A CN201610871079A CN106480180B CN 106480180 B CN106480180 B CN 106480180B CN 201610871079 A CN201610871079 A CN 201610871079A CN 106480180 B CN106480180 B CN 106480180B
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Abstract
A kind of wheat culm breaking strength molecular labeling, the label are located between wheat 4B chromosome TDURUM_CONTIG63670_287 and IACX557, are named as QWQD4B.4-13；Pass through the application of the molecular labeling, whether can detecte in wheat breed or strain has the QWQD4B.4-13 gene loci for increasing stalk breaking strength, to accelerate the breeding process of wheat breed resistant to lodging, due to using dedicated stalk breaking strength molecular labeling, not only screen fast accurate, it is not affected by environment, selection target is clear, and production cost is saved, substantially increase the efficiency of selection and quality of High-Yield Wheat Cultivar resistant to lodging or strain, improve the effect and positioning accuracy of QTL detection, develop effective molecular labeling, develop stalk breaking strength marker assisted selection system, it can be applied to breeding practice with sequencing.
Description
Technical field:
The present invention relates to gene engineering technology fields, can be applied to wheat yield breeding field, specifically provide a kind of small
Wheat stalk breaking strength molecular labeling and its application.
Background technique:
Wheat is the second largest cereal crops in China, and the population in 1/3 or more the whole world is using wheat as staple food.Lodging is that wheat is raw
Common problem in production, the wheat after lodging, not only yield reduces, harvesting is inconvenient, but also seriously affects product quality, makes
At seed lack of filling power, bulk density reduction, milling characters are deteriorated, wheat commodity and manufacturability can decline, high quality wheat is directly influenced
Production and processing.Wheat lodging typically occurs in stalk base portion 10%-30%, i.e. the second section of stalk base portion and third section, small
Wheat stalk breaking strength is the overall target of wheat itself lodging resistance, special with stem thickness and stalk wall thickness, interstitial content, Stalk structure
Sign, stalk chemical component are in close relations.Forefathers are more to the research of the morphology of wheat culm, anatomy and physiology, but lack
Weary Study on Genetic Basis.In the Genetic Mechanisms of gene level parsing wheat culm breaking strength, select using molecular labeling auxiliary
It selects and has great importance with Molecular design breeding.
Emerging molecular marker assisted selection is not affected by environment, can carry out the early generation selection of sequencing and prediction, can show
The accuracy for improving objective trait selection is write, improves and ensure that China's grain security has important reality to wheat yield is accelerated
Meaning and theoretical value.Currently, identified for genes related with wheat culm breaking strength is seldom, lack molecular marker assisted selection
Significant notation, main cause be by following two in terms of limitation, on the one hand: the stalk breaking strength that majority is delivered at present
Breeding process or kind validation verification are not passed through in the site QTLs；On the other hand, due to the gene of single genetic group positioning
Site, poor accuracy, and since confidence interval is big, QTLs is excessive or there are false positive QTLs, reduces stalk breaking strength
The actual efficiency of molecule assisted Selection, or even can not effectively be applied in wheat breeding.
Therefore it is urgent to provide a kind of practicability molecular labelings for being directed to wheat culm breaking strength in favor of wheat quality
Breeding.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, the present invention provides a kind of wheat culm breaking strength molecular labeling,
The label is located between wheat 4B chromosome TDURUM_CONTIG63670_287 and IACX557, is named as QWQD4B.4-13；
Whether by the application of the molecular labeling, can detecte has increase stalk breaking strength in wheat breed or strain
QWQD4B.4-13 gene loci is strong due to using the fracture of dedicated stalk to accelerate the breeding process of wheat breed resistant to lodging
Molecular labeling is spent, not only screens fast accurate, not affected by environment, selection target is clear, and has saved production cost, significantly
The efficiency of selection and quality for improving high-quality wheat variety or strain improve the effect and positioning accuracy of QTL detection, develop
Effective molecular labeling develops stalk breaking strength marker assisted selection system, can be applied to breeding practice with sequencing.
The invention adopts the following technical scheme:
A kind of wheat culm breaking strength molecular labeling QWQD4B.4-13, the label are located at wheat 4B chromosome TDURUM_
Between CONTIG63670_287 and IACX557；Its nucleotide sequence is as described in SEQ ID NO:1.
When containing the molecular labeling in wheat, the stalk breaking strength of wheat increases inventor's discovery.
On the basis of above-mentioned technology, by the application of the molecular labeling, can detecte in wheat breed or strain whether
Contain increase stalk breaking strength gene loci QWQD4B.4-13, specific steps are as follows:
Using the leaf DNA of primer amplified target plant, the segment of 721bp is obtained, nucleotide sequence is such as
Shown in SEQ ID NO:1, later using respective segments are detected after the single-minded enzyme digestion of AluI, if after using the single-minded enzyme digestion of AluI
Still it is the segment of 721bp, then proves that the segment is not switched off, which contains stalk breaking strength synergy gene loci
QWQD4B.4-13, if being two segments containing nucleotide sequence as described in SEQ ID NO:2 and 3 after detection, proving should
Segment has been cut off, which does not contain stalk breaking strength synergy gene loci QWQD4B.4-13.
In order to cooperate the molecular labeling, inventor devises its specific primer,
Forward primer sequence: ggtggttcct ttcgattttg cgc (as shown in SEQ ID NO:4)；
Reverse primer sequences: tgttacggta cacaaactca ctagt (as shown in SEQ ID NO:5)；
Determine by the amplification of specific primer, and to the digestion result of product, can sentence by this method
Whether the fixed site contains stalk breaking strength synergy gene loci, although wheat culm breaking strength is by multiple gene loci controls
System, but since the site that the present invention is determined is major gene loci, have very for the stalk breaking strength of wheat
Important synergistic effect, so you can learn that whether there is the dependency basis of stalk breaking strength synergistic effect in wheat plant to be measured
Cause；For stalk breaking strength gene pyramiding and then improvement wheat quality provides molecular basis, so as to preferably be applied to product
In kind breeding and genetic improvement.
In conclusion the present inventor provides a kind of wheat culm breaking strength molecular labeling, the label for the first time
Between wheat 4B chromosome TDURUM_CONTIG63670_287 and IACX557；It, can be with by the application of the molecular labeling
Whether have the QWQD4B.4-13 that increases stalk breaking strength gene loci, to accelerate high yield if detecting in wheat breed or strain
The breeding process of wheat breed.Fast accurate is not only screened due to using dedicated stalk breaking strength molecular labeling, is selected
It is with clearly defined objective, substantially increase the efficiency of selection and quality of wheat breed resistant to lodging or strain.
Detailed description of the invention
Fig. 1 is to be broken by force using molecular labeling of the present invention and determination method to high stalk breaking strength and low stalk
Spend strain pcr amplification product digestion verification result electrophoretogram；
Fig. 2 is to be examined using molecular labeling of the present invention and determination method to the stalk breaking strength of existing kind
The result schematic diagram of survey, kind 1 is mountain agriculture 01-35 in figure；2 be mountain agriculture 20；3 be 62008；4 be black wheat 12；5: Weihe River wheat 8；6 are
PH82-2；7 be mountain agriculture 11；8: glutinous wheat No. 1；9 be Jinan 17；10 be Pseudomonas Jinanensis Cell Wall.
Specific embodiment
In following embodiments in addition to specified otherwise, used is state of the art；
The leaf DNA of 1 wheat of embodiment is extracted
(1) it takes 0.3-0.5g blade to enter 5mL centrifuge tube, is ground into a powder after liquid nitrogen flash freezer；
(2) the buffer S that 1600 μ L are pre-heated to 65 DEG C is added, is repeatedly inverted and mixes, water-bath 0.5-1h, jog is several therebetween
It is secondary to mix well；
(3) room temperature is cooled to, waits 10 minutes, adds 37 DEG C of 30 clocks of water-bath of 10-15 μ L RNA enzyme (10mg/mL), therebetween gently
It shakes several times to mix well, about 1 time/10min；
(4) centrifuge tube is taken out, isometric 1600 μ L, 4 DEG C of phenol (Tris- balance phenol): chloroform: isoamyl alcohol (volume are added
Than 25:24:1) extracting, 10min is mixed gently, 4 DEG C of refrigerators stand 5min, and then 8000rpm is centrifuged 10min；
(5) take supernatant in another pipe, isometric cold chloroform (4 DEG C of refrigerators are placed), jog 10min is added in about 1300 μ L
It mixes, 8000rpm is centrifuged 10min；
(6) supernatant is taken in another pipe, adds 100 μ L 3M NaAC (sodium acetate), and the cold isopropanol (or 2 of 1000 μ L is added
The cold ethyl alcohol of times volume), 20min are stood in -20 DEG C of refrigerators after mixing well；
(7) choose cotton-shaped DNA with pipette tips, cleaned 2-3 times with 70% ethyl alcohol, is centrifuged 5 minutes, remove ethyl alcohol, after gas is dry
(no ethyl alcohol smell) is dissolved in 200 μ 1 × TE of L, mixes gently, -20 DEG C of storages；
(8) with micro visible/ultraviolet specrophotometer NanoDrop2000, DNA concentration is measured, dilution DNA concentration is
200ng/ μ L is as working solution.
Wherein used solution is prepared:
(1)3M NaAc(pH 5.2)
600mL H2Add 408.24g NaAc-3H in O2O uses glacial acetic acid after (or 246.24g anhydrous sodium acetate) dissolution
(glacial acetic acid) adjusts pH value to 5.2, is settled to 1L, sterilizing is spare.
(2)1×TE(pH 8.0)
800mL H2Add 1.211g Tris, 0.3723g Na in O2EDTA-2H2O is settled to HCL tune pH value to 8.0
1L, sterilizing are spare.
(3) it RNA enzyme: (if being commercialized the packing RNA enzyme that configured 1mL is handled well, can directly use.)
Solid RNA enzyme is dissolved in 0.01M NaAc, makes enzyme concentration 10mg/mL, is heated to 100 DEG C of 15 20min of processing,
It is slowly cooled to room temperature, 0.1 times of volume 1M Tr is-HCL (pH 7.4) is then added, is saved after packing in -20 DEG C of refrigerators.
(4) buffer S
After preparing as following formula, it is settled to 1L:
Wherein 10%SDS is prepared:
100g dissolves electrophoresis grade SDS in 900ml water, is heated to 68 degree of hydrotropies, and the dense HCL of a few drops is added to adjust solution PH to 7.2,
Add water constant volume to 1L, dispenses spare.
The amplification of 2 target product of embodiment
Forward primer sequence: ggtggttcct ttcgattttg cgc (as shown in SEQ ID NO:4)
Reverse primer sequences: tgttacggta cacaaactca ctagt (as shown in SEQ ID NO:5)
PCR amplification: its PCR amplification system is 20 μ L
Remarks: Mix is available: or (Taq enzyme 0.25 μ L, DNK 2.0 μ L, Buffer1.5 μ L, MgCl0.4 μ L configuration)
Amplification condition:
It can be obtained the segment of 721bp by above-mentioned amplification, nucleotide sequence is as shown in SEQ ID NO:1.
3 PCR product specificity digestion of embodiment:
10 μ L of digestion system:
Single-minded enzyme AluI:0.3 μ L
PCR product: 2 μ L
10×NE buffer:1.0μL
ddH2O:6μL
Endonuclease reaction condition: the 0.3 single-minded enzyme of μ L AluI (market is bought), 37 DEG C of water-bath 4h. are added in pcr amplification product
Then by 65 DEG C of inactivation 5min of digestion system.
After above-mentioned amplified production is separated by electrophoresis on 8% polyacrylamide gel, amplified production molecular size range is
721bp, later using respective segments are detected after the single-minded enzyme digestion of AluI, if using being still after the single-minded enzyme digestion of AluI
The segment of 721bp then proves that the segment is not switched off, which contains stalk breaking strength synergy gene loci
QWQD4B.4-13, if being two segments containing nucleotide sequence as described in SEQ ID NO:2 and 3 after detection, proving should
Segment has been cut off, which does not contain stalk breaking strength synergy gene loci QWQD4B.4-13.
Experimental example
To known wheat breed mountain agriculture 01-35, mountain agriculture 20,62008, black wheat 12, Weihe River wheat 8, PH82-2, mountain agriculture 11, glutinous wheat 1
Number, Jinan 17 and Pseudomonas Jinanensis Cell Wall are detected using the above method, as a result as shown in Figure 2；
Pass through actual measurement simultaneously, the kind of the gene loci containing synergy (is) that stem breaking strength average value is 15.14N,
Kind without synergy gene loci (is) that stem breaking strength average value is 6.74N.Also, stalk fracture between the two is strong
Angle value difference reaches extremely significant horizontal (P < 0.01):
Two kinds of haplotype QWQD4B.4-13-T/C strength of stem average values and significant difference
It can be seen that measured value is corresponding with the testing result in Fig. 2, stalk breaking strength molecular labeling provided by the present invention is not
Fast accurate is only screened, selection target is clear, substantially increases the efficiency of selection and quality of wheat breed resistant to lodging or strain.
<110>Shandong Agricultural University
<120>a kind of wheat culm breaking strength molecular labeling QWQD4B.4-13 and its application
<160>5
<210>1
<211>721
<212>DNA
<213>artificial sequence
<400>1
ggtggttcct ttcgattttg cgctcggggt acagaaggag gggaattaac acctgcgttg 60
cagttgtagc atatatccaa tcttagtttt ggttagtacc aaagaggata tcaatgtact 120
tatgtgatta ctcctgttat tgtcctgcat attacacctt tgtggaatgt cttccatttt 180
gctctaaact cattcgagtg cattctaaat ccgcctctaa cacttcttgg tgtcattatg 240
tttaacctct ccaccatttt gtttctttgc aaagatgtca gaacttttct gtgtgcatgt 300
tatctgtagt agtggcaaga actaacttgt gattgaacat gtttatttgc ttatttctat 360
cgtgagatga accacattta tgcatcttac atctctatgt tgaatactca gggggatagt 420
ctgatgagga ttctaagcgt tgcagcattt gctgtttctg gttatgcctc tacggatgga 480
aggagttgta agcccttcaa ccctcttctt ggagagacct atgaagcaga ttatccagac 540
aaaggtcttc gttttttctc agaaaaggta tgtttatgaa cctttagaat ccagtaatat 600
gaataagagg ttcttactgt tggttagcct agtccaatgt tcattctcgc aacttatttc 660
ttacattcta ctaatgagtt tggggtcccg cacacaacta gtgagtttgt gtaccgtaac 720
a 721
<210>2
<211>485
<212>DNA
<213>artificial sequence
<400>2
ggtggttcct ttcgattttg cgctcggggt acagaaggag gggaattaac acctgcgttg 60
cagttgtagc atatatccaa tcttagtttt ggttagtacc aaagaggata tcaatgtact 120
tatgtgatta ctcctgttat tgtcctgcat attacacctt tgtggaatgt cttccatttt 180
gctctaaact cattcgagtg cattctaaat ccgcctctaa cacttcttgg tgtcattatg 240
tttaacctct ccaccatttt gtttctttgc aaagatgtca gaacttttct gtgtgcatgt 300
tatctgtagt agtggcaaga actaacttgt gattgaacat gtttatttgc ttatttctat 360
cgtgagatga accacattta tgcatcttac atctctatgt tgaatactca gggggatagt 420
ctgatgagga ttctaagcgt tgcagcattt gctgtttctg gttatgcctc tacggatgga 480
aggag 485
<210>3
<211>236
<212>DNA
<213>artificial sequence
<400>3
ctgtaagccc ttcaaccctc ttcttggaga gacctatgaa gcagattatc cagacaaagg 60
tcttcgtttt ttctcagaaa aggtatgttt atgaaccttt agaatccagt aatatgaata 120
agaggttctt actgttggtt agcctagtcc aatgttcatt ctcgcaactt atttcttaca 180
ttctactaat gagtttgggg tcccgcacac aactagtgag tttgtgtacc gtaaca 236
<210>4
<211>23
<212>DNA
<213>artificial sequence
<400>4
ggtggttcct ttcgattttg cgc 23
<210>5
<211>25
<212>DNA
<213>artificial sequence
<400>5
tgttacggta cacaaactca ctagt 25
Claims (1)
1. a kind of wheat culm breaking strength molecular labeling QWQD4B.4-13, it is characterised in that: the label is located at wheat 4B dyeing
Between body TDURUM_CONTIG63670_287 and IACX557；Its nucleotide sequence is as described in SEQ ID NO:1.
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| CN106119391A (en) * | 2016-08-24 | 2016-11-16 | 山东农业大学 | Semen Tritici aestivi precipitation number molecular marker QSed1B 26 and application thereof |
| CN109234435B (en) * | 2018-10-23 | 2021-03-23 | 四川省农业科学院作物研究所 | Method for identifying wheat straw wall thickness and primer thereof |
| CN111663004B (en) * | 2020-07-28 | 2022-03-22 | 安徽农业大学 | Method for identifying or assisting in identifying strength of wheat stalks and application of method |
Citations (2)
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| CN103993018A (en) * | 2014-03-13 | 2014-08-20 | 中国科学院遗传与发育生物学研究所 | Gene for controlling Oryza sativa plant height, enhancing lodging resistance, increasing effective tiller number and yield and its application |
| CN104164428A (en) * | 2014-07-25 | 2014-11-26 | 山东农业大学 | Wheat grain weight molecular marker and its use in breeding |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993018A (en) * | 2014-03-13 | 2014-08-20 | 中国科学院遗传与发育生物学研究所 | Gene for controlling Oryza sativa plant height, enhancing lodging resistance, increasing effective tiller number and yield and its application |
| CN104164428A (en) * | 2014-07-25 | 2014-11-26 | 山东农业大学 | Wheat grain weight molecular marker and its use in breeding |
Non-Patent Citations (2)
| Title |
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| Elucidation of the genetic basis of variation for stem strength characteristics in bread wheat by Associative Transcriptomics;Charlotte N. Miller等;《BMC Genomics》;20160716;第17卷;500 * |
| 小麦抗倒伏相关茎秆性状的QTL定位;刘靖;《中国优秀硕士学位论文全文数据库 农业科技辑》;20160515(第5期);D047-99 * |
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