CN106434950B - A kind of primer, kit, detection method and its application detecting the low grain protein content of wheat - Google Patents
A kind of primer, kit, detection method and its application detecting the low grain protein content of wheat Download PDFInfo
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- CN106434950B CN106434950B CN201610947212.XA CN201610947212A CN106434950B CN 106434950 B CN106434950 B CN 106434950B CN 201610947212 A CN201610947212 A CN 201610947212A CN 106434950 B CN106434950 B CN 106434950B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses primer, kit, detection method and its applications of a kind of low grain protein content of detection wheat；The upstream primer sequence of the primer is as shown in SEQ ID NO: I, and downstream primer sequence is as shown in SEQ ID NO: II.The method provided by the present invention for detecting the low GPC of wheat is reliable, easy, practical, there is important application prospect in the molecular marker assisted selection of Wheat Germplasm Resources evaluation and breeding, while the wheat breed for the low grain protein content of identification wheat provides reference frame.The present invention can screen low grain protein content in breeding cross 2nd generation, seed lower protein content can only could be screened after hybridization the 4th instead of by overcoming conventional method, therefore reduce breeder to the workload of the character determination, the accuracy and breeding efficiency for improving selection are a kind of efficient, quick new technology of breeding.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of primer of the low grain protein content of detection wheat, examination
Agent box, detection method and its application.
Background technique
With the continuous improvement of China's wheat yield, demand of the people to good quality wheat increasingly increases, foundation wheat quality,
Wheat is divided into strong gluten wheat, middle gluten wheat and weak-gluten wheat by China, and weak-gluten wheat is mainly for the production of biscuit, cake and south
The Flour product such as steamed bun, Protein Content of Wheat Kernel (GPC) are the important indicator of quality, the lower wheat product of grain protein content
Kind can be used as the raw material for producing high-quality weak-gluten wheat product, the high-quality weak-gluten wheat national standard (GB/T17893-1999) in China
Provide that wheat seed crude protein content is no more than 11.5%.
Since grain protein content must just can be carried out attribute test after harvesting, and vulnerable to environmental influence, because
This efficiency of selection is lower, and molecular marker assisted selection can be directed to lower protein content gene or QTL to breeding material in DNA level
The unstability that is selected, thus can be selected in seedling stage, and grain protein content can be overcome to identify reduces
The cost of phenotypic evaluation improves weak-gluten wheat quality breeding efficiency.Blanco etc. (2002) has found 7 control grain proteins
The QTL site of content is located on 4B, 5A, 6A, 6B, 7A and 7B chromosome；Prasad etc. (1999) is control seed protein
The QTL site of matter content is located on 2D chromosome；Huang (2006) etc. utilizes double rate, dyes in 4B and 4D
The QTL of discovery control grain protein content on body；Li et al. (2007) utilizes two recombinations containing 229 and 485 familys
Inbred line population detect respectively 3 and 10 influence grain protein content QTL, be respectively positioned in 2B, 3A, 4A, 4D,
On 5B, 7A, 7B and 1A, 1B, 2A, 2D, 3A, 4B, 5A, 5D, 6B, 7D chromosome, from the point of view of presently disclosed documents and materials, control
The gene or QTL of grain protein content are more, but single effect value is not high, and conventional method can only be after hybridization the 4th instead of
Grain protein content can be checked, therefore increase breeder to the workload of the character determination, accuracy and breeding
Low efficiency.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology, provides a kind of low grain protein content of detection wheat
Primer；
Another object of the present invention is to provide the kits of the low grain protein content of detection wheat；
The third object of the present invention is to provide a kind of detection method of the low grain protein content of detection wheat and its answers
With.
The purpose of the present invention is achieved through the following technical solutions: a kind of detection low grain protein content of wheat draws
Object, the upstream primer sequence of the primer is as shown in SEQ ID NO: I, and downstream primer sequence is as shown in SEQ ID NO: II.
A kind of kit detecting the low grain protein content of wheat, the kit include as described in claim 1
Detect the primer of the low grain protein content of wheat.
Further, it further include: 10 × PCR buffer, dNTP, Taq DNA polymerase, Mg2+It is normal with electrophoresis
Advise reagent.
A method of the low grain protein content of detection wheat, it the following steps are included:
S1.PCR amplification: using the genomic DNA of wheat to be measured as template, PCR amplification is carried out with above-mentioned primer, obtains PCR
Product；
S2. result judges: the size of PCR product obtained by detecting step S1 determines that the wheat to be measured is as follows
No is low grain protein content:
A. the DNA fragmentation for being 2013bp containing size in the PCR product, then the wheat to be measured is low grain protein
The wheat of content；
B. the DNA fragmentation for being 2013 without containing size in the PCR product, then the wheat to be measured is non-low seed protein
The wheat of matter content.
Further, the size is the nucleotide sequence of the DNA fragmentation of 2013bp as shown in SEQ ID NO: III.
Further, the annealing temperature of the PCR amplification is 54 DEG C.
As the application in low content Wheat Grain Protein wheat is being screened and cultivated to above-mentioned method.It specially screens low
The wheat breed of content Wheat Grain Protein；Cultivate the wheat breed of low Wheat Grain Protein.
It in practical applications, can be by the way that PCR product be carried out fine jade when whether judging in PCR product containing target DNA fragment
Sepharose electrophoresis (gel strength concretely 1%), sees whether contain purpose band on electrophorogram, contains on electrophorogram
Purpose band then contains target DNA fragment in PCR product.
The invention has the following advantages that the present invention provides a kind of primers of the low grain protein content of detection wheat, inspection
Survey the kit of the low grain protein content of wheat, the detection method and its application of the low grain protein content of detection wheat.It is logical
Cross to the statistical experiments of 80 parts of wheat breeds the result shows that, 38 parts of wheat breeds of 2013bp purpose band are gone out using primer amplification
GPC luffing is 10.2%-15.2%, and mean value 12.6%, 42 parts of wheat breed GPC luffings for not amplifying 2013bp are
15.0%-19.2%, mean value 16.4%, the results of analysis of variance is shown, the two reaches extremely significant level.As it can be seen that institute of the present invention
The method of the low GPC of detection wheat of offer is reliable, easy, practical, in Wheat Germplasm Resources evaluation and molecular mark
In there is important application prospect, while the wheat breed for the low grain protein content of cultivation wheat provides reference frame.This
Invention can screen low grain protein content in breeding cross 2nd generation, and overcoming conventional breeding can only be in hybridization the 4th
Grain protein content could be checked after instead of, therefore reduce breeder to the workload of the character determination, improved
The accuracy and breeding efficiency of selection are a kind of Gao Yuxiao, quick kind of new technology.
Detailed description of the invention
Fig. 1 is the result that primer of the present invention carries out PCR amplification to the wheat breed of different GPC contents respectively.Wherein, swimming lane
M is DNA molecular amount standard, and each band is descending to be followed successively by 5000bp, 3000bp, 2000bp, 1000bp；The swimming that number is 1
Road is to educate the DNA fragmentation that 2013bp is amplified in 12 in low GPC kind river using primer of the present invention；The swimming lane that number is 2 is to make
The DNA fragmentation of 2013bp is not amplified in non-low GPC kind ZM7186 with primer of the present invention.
Fig. 2 is the result that primer of the present invention carries out PCR amplification to the wheat breed of 16 difference GPC height respectively.Wherein,
Swimming lane M is DNA molecular amount standard, and each band is descending to be followed successively by 5000bp, 3000bp, 2000bp, 1000bp；Number is swimming
The kind of road 1- swimming lane 20 is respectively as follows: ZM6352, ZM5723, ZM12064, ZM6333, hides the spring No. 6, adds Cha Bangda winter wheat, Bainang
Wheat, ZM3489, W5293, river wheat 41, river educate 12, and silk floss 45, silk floss 28 is white, and river educates 20, river wheat 51, silk floss 20.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and embodiments, protection scope of the present invention be not limited to
It is lower described.
Embodiment 1: the measurement of wheat seed GPC
Using 80 parts of wheat breeds in table 1 as experimental material, wheat is measured using method shown in GB 50095-2010
The GPC of seed.The GPC data is that 80 parts of wheat breeds are planted in 2 years mean values between 3 points, and plantation soil fertility is consistent.
Table 1: the measurement result and PCR amplification band of wheat GPC
Note: "+" expression amplifies corresponding purpose band in one column of pcr amplification product in table, and "-" expression does not amplify phase
The purpose band answered.
Embodiment 2: the synthesis and detection method of wheat seed GPC related molecular marker
1, the synthesis of wheat GPC related molecular marker
Synthesize the primer being made of sequence I in sequence table and sequence II, abbreviation GPC-B.
Upstream and downstream primer is as follows in primer GPC-B:
GPC-B-F (sequence I): 5 '-CTACCGTACTCTCTCTGATT-3 ' (1-20 of sequence III)；
GPC-B-R (sequence II): 5 '-TGGATCTCGCAGCAAGTTCT-3 ' (1992-2013 s' of sequence III is anti-
To complementary series).
2, the method for the Markers for Detection wheat GPC content of step 1 is utilized
(1) PCR amplification
Using Wheat volatiles DNA as template, PCR is carried out using primer GPC-B respectively.
Reaction system (20 μ l): 2.0 μ L 10 × PCR buffer, 1.0 μ L of 50ng/ μ L DNA, 2.5mmol/L are total
2.0 μ L of dNTPs, 0.20 μ L of upstream primer (GPC-B-F) that concentration is 10 μm of ol/L, the downstream primer that concentration is 10 μm of ol/L
(GPC-B-R) 0.20 μ L, 0.4 μ L of 5U/ μ L high fidelity enzyme, ddH2O 13.2μL。
The response procedures expanded using primer GPC-B: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 35s；54 DEG C of annealing
35s, 72 DEG C of extension 1min30s are recycled 34 times altogether；Last 72 DEG C of extensions 10min.
After reaction, pcr amplification product is separated with 1% agarose gel electrophoresis, and buffer system is 1 × TAE solution,
Photograph is counted and is saved after 200V electrophoresis 25min, EB dyeing development.
(2) sequencing and analysis of target stripe
It (3) whether is low GPC according to result judgement wheat to be measured
It whether is low GPC with primer pair GPC-B detection wheat: if the DNA for being 2013bp containing size in the PCR product
Segment (sequence III), then the wheat to be measured is low GPC wheat；If the DNA for being 2013bp without containing size in the PCR product
Segment (sequence III), then the wheat to be measured is non-low GPC wheat.
Embodiment 3: primer GPC-B is used to detect the validation verification of the low GPC of wheat
With primer GPC-B to totally 80 parts of wheat breeds have carried out PCR analysis in table 1, operating method is the same as step in embodiment 2
Two.According to the electrophoresis result of pcr amplification product, result is carried out referring to the result judgment criteria in 2 step 22 (3) of embodiment
Judgement.
The results are shown in Table 1, and the Ago-Gel testing result of part wheat breed is as shown in Fig. 2, can be with from result
Find out: in this 80 parts of wheat breeds, the purpose that size is 2013bp has been obtained when carrying out PCR amplification using primer GPC-B
The wheat breed of band (sequence III) has 38 parts, GPC luffing and the mean value mean value between (10.2%-15.2%) as shown in table 1
It is 12.6%；There are 42 parts of wheat breeds not amplify the purpose band that equal size is 2013bp, GPC luffing and mean value such as 1 institute of table
Show that between (15.0%-19.2%), mean value is 16.4%, the results of analysis of variance is shown, the two reach extremely significant water product (P <
0.01)。
SEQUENCE LISTING
<110>Chengdu Inst. of Biology, Chinese Academy of Sciences
<120>primer, kit, detection method and its application of a kind of low grain protein content of detection wheat
<130> 2016
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
ctaccgtact ctctctgatt 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
tggatctcgc agcaagttct 20
<210> 3
<211> 2013
<212> DNA
<213>artificial sequence
<400> 3
ctaccgtact ctctctgatt ctaaatataa gtctttttag agattccaat atggacgttg 60
gcgtcaaggt cagagatatt ctgctatctt ttcagttttg tcatgtcggt acttatgtga 120
cttgtacttt gatctttatg aaagaaataa gacagtatta ccatgcaaaa aaatatgaac 180
tacatacaga gcaaaatgaa tggatctaca ttctaaaatg tgtctatata catctggata 240
tagtttatat taaaatccct aaaatgaatg gagtatttag gaatagagga agtacataag 300
tttgttccat ataattctca taattatgac ttttttaagt aattacggct tttattatta 360
catgaggtac aatctaccgt accgtacaaa agtttacgtg gttaaatgag caatcggttt 420
cccgtttccc ttccgcgatt acgcttcact tatggttttt ccgttctcgg ttcagcgttc 480
caccgtcgtc gtccaaatac gtactagtac caacagcgtc caaatacacc gtacggcacc 540
gccagaagag gagtctgccg gaaatttagc tcggcattca ctcgtttcct tggcgaagct 600
ggcaagcagc cgccacttac catagatgat ctgctttgac tcgcggcgga aatgtttccc 660
cgaaacgatg aaggggacgg ccggattcgt gggattccgc gaggcagagt aattgaccga 720
tcgccgcatt aaaagaatga atgtacagtg gaaggccggt caggtgcaag gcccggcggc 780
ccttatatat acccgccgcc cgcggctacg acagaatcca tcaaatttcc cgagagagag 840
accgacagcg gtgcccgagc ggaggaagag gcggggagag agagggagag agggagagag 900
atggacgtgg agaaggtgac ggaggtggcg ccggaggtgg acgacgacgg ccgcgtaaga 960
acaggtagcg cacacgactc ccccttgtag agagctccgt tgttcgcggt ttcagttcgt 1020
tggttccttc gcttgatgcc ttccgtctga gccgtgatgc gataccggca aacccgctcg 1080
tccttgtggc gaggaggcat ggccggatgc ggtgttttca ggtccgtgcg tgagaaacgg 1140
taggaagcta ggtagacgac agcggtagaa cagttcatgt ctcggttgca gaggttttag 1200
agggcgatgc tggcgatcaa attggcactc aaaatggatt atatcatact cttgccgatt 1260
tgcctctcaa gtctttaggt acttgaagtg gcaaacttct cgttctgcac cggaatgtca 1320
aaaaaagatt ggcacactgc aaactagtac tagcttggta tgggtagtat cacaaattca 1380
caatcatgga gaaaagaaaa agaattatcg catatcctga aactattgtc ttatagaaca 1440
ttaacacaca ttcttttttc cggaaacaaa cgtccatggt taggacttag gacctggaac 1500
tctgctttga cagaggaaca tgttgtagca gagcaattca aaattttgcc catcgcttgt 1560
gatcagcagc gtagcccagg atccaggcag cgctgtcgct tttagatgtt tgcatgcatg 1620
caggccgagc tagtttagta ctccctccgt cccaaaattc ttgtcttaga tttgtttaga 1680
tacggatgta cctaatacta aaacgtgact tggcagtacc tccatatctt ccccatttct 1740
cttgacagtt gacacatcac ttgtccaact ccaatccaga tgacaaccct gacatcggga 1800
cgaaatacat gcaccctgca tctacatgct ctatctgaaa atcagccatc ccctgagaat 1860
gtgccatcca tggtgtggcg cctctgcgcc attcctttta caacgcccca aatgatggga 1920
accgggttgg tggcatggta ttgatgcagg aattaggtcc aaatgccctg tgtccccgat 1980
ccggtagagc aaaagaactt gctgcgagat cca 2013
Claims (7)
1. a kind of primer for detecting the low grain protein content of wheat, which is characterized in that the upstream primer sequence of the primer is such as
Shown in SEQ ID NO:1, downstream primer sequence is as shown in SEQ ID NO:2.
2. a kind of kit for detecting the low grain protein content of wheat, which is characterized in that the kit includes that right such as is wanted
The primer of the low grain protein content of detection wheat described in asking 1.
3. a kind of kit for detecting the low grain protein content of wheat as claimed in claim 2, which is characterized in that it is also wrapped
It includes: 10 × PCR buffer, dNTP, Taq DNA polymerase, Mg2+With electrophoresis conventional reagent.
4. a kind of method of the low grain protein content of detection wheat, which is characterized in that it the following steps are included:
S1. using the genomic DNA of wheat to be measured as template, PCR expansion PCR amplification: is carried out with kit as claimed in claim 2
Increase, obtains PCR product；
S2. result judges: the size of PCR product obtained by detecting step S1, determine as follows the wheat to be measured whether be
Low grain protein content:
A. the DNA fragmentation for being 2013bp containing size in the PCR product, then the wheat to be measured is that low grain protein contains
The wheat of amount；
B. the DNA fragmentation for being 2013bp without containing size in the PCR product, then the wheat to be measured is non-low seed egg
The wheat of white matter content.
5. a kind of method for detecting the low grain protein content of wheat as claimed in claim 4, which is characterized in that the size
For 2013bp DNA fragmentation nucleotide sequence as shown in SEQ ID NO:3.
6. a kind of method for detecting the low grain protein content of wheat as claimed in claim 4, which is characterized in that the PCR
The annealing temperature of amplification is 54 DEG C.
7. application of the method as claimed in claim 4 in the new variety of wheat for screening and cultivating low grain protein content.
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| CN108570517B (en) * | 2018-06-12 | 2021-10-01 | 江苏省农业科学院 | Specific primer related to Ning-Mai No. 9 low protein of weak gluten wheat and application of specific primer |
| CN110373489B (en) * | 2019-07-10 | 2021-01-08 | 江苏省农业科学院 | KASP marker related to wheat grain protein content and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498490A (en) * | 2014-12-24 | 2015-04-08 | 江苏省农业科学院 | Molecular marker tightly linked with low-protein-content QTL of wheat grains and application of molecular marker |
| CN105821052A (en) * | 2016-05-10 | 2016-08-03 | 中国农业大学 | Aegilops speltoides Tausch NAM-S1 gene and molecular marker and application thereof |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498490A (en) * | 2014-12-24 | 2015-04-08 | 江苏省农业科学院 | Molecular marker tightly linked with low-protein-content QTL of wheat grains and application of molecular marker |
| CN105821052A (en) * | 2016-05-10 | 2016-08-03 | 中国农业大学 | Aegilops speltoides Tausch NAM-S1 gene and molecular marker and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| TaAAP6-3B, a regulator of grain protein content selected during wheat improvement;Xiufeng Jin，et al;《BMC Plant Biology》;20180423;第18卷(第71期);1-10 * |
| 小麦籽粒蛋白质组分含量的QTL定位;石培春 等;《麦类作物学报》;20080715;第28卷(第4期);550-554 * |
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