CN106480180A - A kind of wheat culm fracture strength molecular marker QWQD4B.4 13 and its application - Google Patents
A kind of wheat culm fracture strength molecular marker QWQD4B.4 13 and its application Download PDFInfo
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Abstract
A kind of wheat culm fracture strength molecular marker, this labelling is located between Semen Tritici aestivi 4B chromosome TDURUM_CONTIG63670_287 and IACX557, is named as QWQD4B.4 13；By the application of this molecular marker, can detect whether there are in wheat breed or strain QWQD4B.4 13 gene locis increasing stalk fracture strength, to accelerate the selection-breeding process of wheat breed resistant to lodging, due to employing special stalk fracture strength molecular marker, not only screen fast accurate, not affected by environment, selection target is clear and definite, and saved production cost, substantially increase efficiency of selection and the quality of High-Yield Wheat Cultivar resistant to lodging or strain, improve effect and the positioning precision of QTL detection, develop effective molecular marker, develop stalk fracture strength marker assisted selection system, breeding practice can be applied to sequencing.
Description
Technical field：
The present invention relates to gene engineering technology field, can be applicable to wheat yield breeding field, specifically provide a kind of little
Wheat stalk fracture strength molecular marker and its application.
Background technology：
Semen Tritici aestivi is the second largest cereal crops of China, and the population in the whole world more than 1/3 is with Semen Tritici aestivi as staple food.Lodging is Semen Tritici aestivi life
Common problem in product, the Semen Tritici aestivi after lodging, not only yield reduction, harvesting inconvenience, and have a strong impact on product quality, make
Seed lack of filling power, unit weight is become to reduce, milling characters is deteriorated, Semen Tritici aestivi commodity and manufacturability can decline, and directly influences high quality wheat
Production and processing.Wheat lodging typically occurs in stalk base portion 10%-30%, i.e. stalk base portion second section and Section three, little
Wheat stalk fracture strength is the aggregative indicator of Semen Tritici aestivi itself lodging resistance, and stalk wall thickness, interstitial content, Stalk structure thick with stem are special
Levy, stalk chemical composition in close relations.The research of morphology, anatomy and physiology to wheat culm for the forefathers is more, but lacks
Weary Study on Genetic Basis.Parse the Genetic Mechanisms of wheat culm fracture strength in gene level, assist choosing to using molecular marker
Select and have great importance with Molecular design breeding.
Emerging molecular marker assisted selection is not affected by environment, can carry out sequencing selection of early generation and predict, can show
Write and improve the accuracy that objective trait selects, quickening wheat yield is improved and ensures that China's grain security has important reality
Meaning and theory value.At present, the gene identification relevant with wheat culm fracture strength seldom, lacks molecular marker assisted selection
Significant notation, main cause is affected by the restriction of following two aspects, on the one hand：The stalk fracture strength that majority is delivered at present
QTLs site is not through breeding process or kind validation verification；On the other hand, due to the gene of single genetical population positioning
Site, poor accuracy, and also because confidence interval is big, QTLs is excessive or there is false positive QTLs, reduces stalk fracture strength
The actual efficiency of molecule assisted Selection, or even cannot effectively be applied in wheat breeding.
A kind of practicality molecular marker being directed to wheat culm fracture strength of offer is therefore provided badly and is beneficial to wheat quality
Breeding.
Content of the invention
The problems referred to above existing for prior art, the invention provides a kind of wheat culm fracture strength molecular marker,
This labelling is located between Semen Tritici aestivi 4B chromosome TDURUM_CONTIG63670_287 and IACX557, is named as QWQD4B.4-13；
By the application of this molecular marker, can detect whether there is in wheat breed or strain increase stalk fracture strength
QWQD4B.4-13 gene locis, to accelerate the selection-breeding process of wheat breed resistant to lodging, the stalk fracture due to employing special is strong
Degree molecular marker, not only screens fast accurate, not affected by environment, selection target clearly, and has saved production cost, significantly
Improve efficiency of selection and the quality of high-quality wheat variety or strain, improve effect and the positioning precision of QTL detection, develop
Effective molecular marker, develops stalk fracture strength marker assisted selection system, can be applied to breeding practice with sequencing.
The present invention employs the following technical solutions：
A kind of wheat culm fracture strength molecular marker QWQD4B.4-13, this labelling is located at Semen Tritici aestivi 4B chromosome TDURUM_
Between CONTIG63670_287 and IACX557；Its nucleotide sequence such as SEQ ID NO:Described in 1.
Inventor finds when containing this molecular marker in Semen Tritici aestivi, and the stalk fracture strength of Semen Tritici aestivi increases.
On the basis of above-mentioned technology, by the application of this molecular marker, whether can detect in wheat breed or strain
Containing increasing stalk fracture strength gene locis QWQD4B.4-13, concretely comprise the following steps：
Using primer amplified target plant leaf DNA, obtain 721bp fragment, its nucleotide sequence is such as
SEQ ID NO:Shown in 1, after utilizing AluI single-minded enzyme enzyme action afterwards, detect respective segments, if using after AluI single-minded enzyme enzyme action
It is still the fragment of 721bp, then proves that this fragment is not switched off, this kind (being) contains stalk fracture strength potentiation gene locis
QWQD4B.4-13, if be containing nucleotide sequence such as SEQ ID NO after detection:Two fragments described in 2 and 3, then proving should
Fragment is cut-off, and this kind (being) does not contain stalk fracture strength potentiation gene locis QWQD4B.4-13.
In order to coordinate this molecular marker, inventor devises its specific primer, its
Forward primer sequence：5 '-GGT GGT TCC TTT CGA TTT TGC GC-3 ' are (as SEQ ID NO:Shown in 4)；
Reverse primer sequences：5 '-ACT AGT GAG TTT GTG TAC CGT AAC A-3 ' are (as SEQ ID NO:5 institutes
Show)；
By the amplification of specific primer, and the enzyme action result of product is judged, can sentence by this method
Stalk fracture strength potentiation gene locis whether are contained in this site fixed although wheat culm fracture strength is subject to multiple gene locis controls
System, but the site being judged by the present invention as major gene loci, it has very for the stalk fracture strength of Semen Tritici aestivi
Important synergistic effect, so would know that the dependency basis whether in wheat plant to be measured with stalk fracture strength synergistic effect
Cause；For stalk fracture strength gene pyramiding so that improve wheat quality provide molecular basises, such that it is able to preferably be applied to product
Plant in selection-breeding and genetic improvement.
In sum, the present inventor provides a kind of wheat culm fracture strength molecular marker, this labelling first
Between Semen Tritici aestivi 4B chromosome TDURUM_CONTIG63670_287 and IACX557；By the application of this molecular marker, permissible
In detection wheat breed or strain, whether there is the QWQD4B.4-13 increasing stalk fracture strength gene locis, to accelerate high yield
The selection-breeding process of wheat breed.Stalk fracture strength molecular marker due to employing special not only screens fast accurate, selects
With clearly defined objective, substantially increase efficiency of selection and the quality of wheat breed resistant to lodging or strain.
Brief description
Fig. 1 is using molecular marker of the present invention and decision method, high stalk fracture strength and low stalk to be ruptured by force
Degree strain pcr amplification product digestion verification result electrophoretogram；
Fig. 2 is using molecular marker of the present invention and decision method, the stalk fracture strength of existing kind to be examined
The result schematic diagram surveyed, in figure kind 1 is mountain agriculture 01-35；2 is mountain agriculture 20；3 is 62008；4 is black wheat 12；5:Weihe River wheat 8；6 are
PH82-2；7 is mountain agriculture 11；8:Glutinous wheat No. 1；9 is Jinan 17；10 is Pseudomonas Jinanensis Cell Wall.
Specific embodiment
In following embodiments in addition to specified otherwise, adopted is state of the art；
The leaf DNA of embodiment 1 Semen Tritici aestivi is extracted
(1) take 0.3-0.5g blade to enter 5mL centrifuge tube, after liquid nitrogen flash freezer, be ground into powder；
(2) the buffer S adding 1600 μ L to be pre-heated to 65 DEG C, is repeatedly inverted and mixes, water-bath 0.5-1h, and jog is several therebetween
Secondary mixed with abundant；
(3) cool to room temperature, wait 10 minutes, plus 37 DEG C of water-bath 30 clocks of 10-15 μ L RNase (10mg/mL), therebetween gently
Shake and mixed with abundant several times, about 1 time/10min；
(4) take out centrifuge tube, add equal-volume 1600 μ L, 4 DEG C of phenol (Tris- balance phenol)：Chloroform：Isoamyl alcohol (volume
Ratio 25：24：1) extract, gently mix 10min, 4 DEG C of refrigerators stand 5min, then 8000rpm centrifugation 10min；
(5) take supernatant in another pipe, about 1300 μ L, add the cold chloroform of equal-volume (4 DEG C of refrigerators are placed), jog 10min
Mix, 8000rpm is centrifuged 10min；
(6) take supernatant in another pipe, plus 100 μ L 3M NaAC (Sodium Acetate Trihydrate), add the cold isopropanol (or 2 of 1000 μ L
The cold ethanol of times volume), fully mix and stand 20min after -20 DEG C of refrigerators；
(7) choose cotton-shaped DNA with pipette tips, with 70% ethanol purge 2-3 time, be centrifuged 5 minutes, remove ethanol, after air dry
(no ethanol abnormal smells from the patient), is dissolved in 200 μ L 1 × TE, gently mixes, -20 DEG C of storages；
(8) with micro visible/ultraviolet spectrophotometer NanoDrop2000, measure DNA concentration, dilution DNA concentration is
200ng/ μ L is as working solution.
Solution employed in it is prepared：
(1)3M NaAc(pH 5.2)
600mL H2408.24g NaAc-3H is added in O2O, uses glacial acetic acid after (or 246.24g anhydrous sodium acetate) dissolving
(glacial acetic acid) adjusts pH value to 5.2, is settled to 1L, and sterilizing is standby.
(2)1×TE(pH 8.0)
800mL H21.211g Tris, 0.3723g Na is added in O2EDTA-2H2O, adjusts pH value to 8.0 with HCL, is settled to
1L, sterilizing, standby.
(3) RNase：If (the subpackage RNase that the 1mL that commercialization configures handles well, can directly use.)
Solid RNase is dissolved in 0.01M NaAc, makes enzyme concentration be 10mg/mL, be heated to 100 DEG C of process 15 20min,
It is slowly cooled to room temperature, be subsequently adding 0.1 times of volume 1M Tris-HCL (pH7.4), subpackage is after -20 DEG C of Refrigerator stores.
(4) buffer S
After preparing according to following formula, it is settled to 1L：
Wherein 10%SDS prepares：
In 900ml water, 100g dissolving electrophoresis level SDS, is heated to 68 degree of hydrotropies, plus several dense HCL adjust solution PH to 7.2,
Add water constant volume to 1L, subpackage is standby.
Embodiment 2 target product expands
Forward primer sequence：5 '-AAC TCG CTC AAC GCC CTC TAC-3 ' are (as SEQ ID NO:Shown in 4)
Reverse primer sequences：5 '-GAT GAT TAG TTA CCA CGG CGT-3 ' are (as SEQ ID NO:Shown in 5)
PCR expands：Its PCR amplification system is 20 μ L
Remarks：Mix can use：Or (Taq enzyme 0.25 μ L, DNK 2.0 μ L, Buffer1.5 μ L, MgCl0.4 μ L configure)
Amplification condition：
The fragment of 721bp can be obtained by above-mentioned amplification, its nucleotide sequence such as SEQ ID NO:Shown in 1.
Embodiment 3 PCR primer specificity enzyme action：
Enzyme action system 10 μ L：
Single-minded enzyme AluI：0.3μL
PCR primer：2μL
10×NE buffer:1.0μL
ddH2O:6μL
Endonuclease reaction condition：Add the single-minded enzyme (market is buied) of 0.3 μ L AluI, 37 DEG C of water-bath 4h. in pcr amplification product
Then by 65 DEG C of inactivation 5min of enzyme action system.
After above-mentioned amplified production is separated by electrophoresis in 8% polyacrylamide gel, amplified production molecular size range is
721bp, detects respective segments, if using after AluI single-minded enzyme enzyme action being still after utilizing AluI single-minded enzyme enzyme action afterwards
The fragment of 721bp, then prove that this fragment is not switched off, and this kind (being) contains stalk fracture strength potentiation gene locis
QWQD4B.4-13, if be containing nucleotide sequence such as SEQ ID NO after detection:Two fragments described in 2 and 3, then proving should
Fragment is cut-off, and this kind (being) does not contain stalk fracture strength potentiation gene locis 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 said method, result is as shown in Figure 2；
Simultaneously through actual measurement, kind (being) the cane fracture strength meansigma methodss containing potentiation gene locis are 15.14N,
Kind (being) cane fracture strength meansigma methodss without potentiation gene locis are 6.74N.And, stalk fracture between the two is strong
Angle value difference reaches pole significant level (P<0.01)：
Two kinds of haplotype QWQD4B.4-13-T/C strength of stem meansigma methodss and significant difference
It can be seen that measured value is corresponding with the testing result in Fig. 2, stalk fracture strength molecular marker provided by the present invention is not
Only screen fast accurate, selection target clearly, substantially increases efficiency of selection and the quality of wheat breed resistant to lodging or strain.
Claims (2)
1. a kind of wheat culm fracture strength molecular marker QWQD4B.4-13 it is characterised in that：This labelling is located at Semen Tritici aestivi 4B and dyes
Between body TDURUM_CONTIG63670_287 and IACX557；Its nucleotide sequence such as SEQ ID NO:Described in 1.
2. wheat culm fracture strength molecular marker QWQD4B.4-13 described in claim 1 application it is characterised in that：For
Whether contain increase stalk fracture strength gene locis QWQD4B.4-13 in detection wheat breed or strain, concretely comprise the following steps：
Using primer amplified target plant leaf DNA, obtain 721bp fragment, its nucleotide sequence such as SEQ
ID NO:Shown in 1, after utilizing AluI single-minded enzyme enzyme action afterwards, detect respective segments, if after utilizing AluI single-minded enzyme enzyme action still
Fragment for 721bp, then prove that this fragment is not switched off, and this kind (being) contains stalk fracture strength potentiation gene locis
QWQD4B.4-13, if be containing nucleotide sequence such as SEQ ID NO after detection:Two fragments described in 2 and 3, then proving should
Fragment is cut-off, and this kind (being) does not contain stalk fracture strength potentiation gene locis QWQD4B.4-13.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109234435A (en) * | 2018-10-23 | 2019-01-18 | 四川省农业科学院作物研究所 | It is a kind of for identifying the method and its primer of wheat culm wall thickness |
| CN111663004A (en) * | 2020-07-28 | 2020-09-15 | 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)
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| CHARLOTTE N. MILLER等: "Elucidation of the genetic basis of variation for stem strength characteristics in bread wheat by Associative Transcriptomics", 《BMC GENOMICS》 * |
| 刘靖: "小麦抗倒伏相关茎秆性状的QTL定位", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109234435A (en) * | 2018-10-23 | 2019-01-18 | 四川省农业科学院作物研究所 | It is a kind of for identifying the method and its primer of wheat culm wall thickness |
| 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 |
| CN111663004A (en) * | 2020-07-28 | 2020-09-15 | 安徽农业大学 | Method for identifying or assisting in identifying strength of wheat stalks and application of method |
| 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 |
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