CN109554498A - A kind of identification single cropping wild rice stem early molecular labeling of late-maturing characteristic and its application, acquisition methods - Google Patents
A kind of identification single cropping wild rice stem early molecular labeling of late-maturing characteristic and its application, acquisition methods Download PDFInfo
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Abstract
A kind of identification single cropping wild rice stem early molecular labeling of late-maturing characteristic and its application, acquisition methods, belongs to technical field of molecular biology.The molecular labeling includes molecular labeling UeSE1 and UeSE2, and the forward primer UeSNPSE1-F nucleotide sequence of the molecular labeling UeSE1 is as shown in SEQ ID NO.1, and reverse primer UeSNPSE1-R nucleotide sequence is as shown in SEQ ID NO.2；The forward primer UeSNPSE2-F nucleotide sequence of the molecular labeling UeSE2 is as shown in SEQ ID NO.3, and reverse primer UeSNPSE2-R nucleotide sequence is as shown in SEQ ID NO.4.Precocious single cropping hay is screened by SNP marker, excellent Zizania latifolia Cultivars can be selected ahead of time, be reduced workload, improve screening efficiency.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of molecule of the early late-maturing characteristic of identification single cropping wild rice stem
Label and its application, acquisition methods.
Background technique
Wild rice stem (Zizania latifolia Turcz.) be grass family perennial herb plant, originate in China and
South east asia is the second largest aquatic vegetable in China.Wild rice stem early has cultivation in China, and before the Western Zhou Dynasty, the seed of wild rice stem is made
It is edible for grain, after the mycoceicidum that is formed because finding that its stem is infected by wild rice smut (Ustilago esculenta) not only may be used
With edible, and meat fertilizer is tender, excellent taste, and is treated as that a kind of vegetables are edible, and the latter stage Tang Dynasty starts by as a kind of vegetables
Plantation extensively.In addition to as vegetables, wild rice stem is also pharmaceutically acceptable, the effect of there is diuresis to quench the thirst, is clearing heat and detoxicating.Single cropping hay is 1 year one
The Zizania latifolia Cultivars that kind one is received harvest season harvesting at the beginning of -10 months generally before and after September part.Single cropping hay is short day property crop, only in the autumn
Ji Zhao just starts pregnant hay after turning short.The general plant of single cropping hay is tall and big, and wild rice stem color, mouthfeel are excellent, and planting range is wider.?
China can be cultivated from Taiwan to Sichuan from Beijing to Guangzhou.The precocious single cropping hay generally pregnant hay time is in Limit of Heat solar term mistake
In afterwards-solar term this period in the Autumnal Equinox, there are also single cropping hays to become kind (such as the beauty of double cropping by many years breeding
No. 1 precious, Jinjiao 2 of hay, water etc.).This period generally before Summer Solstice single cropping hay picking time first time of double cropping, second
Picking time is no different in the 9-10 month and other single cropping hay kinds.This kind of precocity single cropping hay not only has the spy of double season hays " receiving for 1 year two "
The features such as point, there are also wild rice stem head not available for double season hays is big, and color is delicate, therefore the market price is higher than double season hays.With paulownia
For city, township Dong Jia wild rice stem cooperative society, beauty's hay purchasing price that is averaged of mid or late June harvesting is imperial hay 2 harvested at the beginning of 6 months
2-4 times.Therefore the single cropping hay of this kind of precocity of breeding, can be used for double cropping, can not only retain single cropping hay in wild rice stem quality
Advantage relative to double season hays, moreover it is possible to improve crop field utilization rate, increase zizania latifoliai grower income.
The principle of SNP marker is: single nucleotide variations (missing, insertion, frameshit) frequent occurrence, this variation in organism
Result become single nucleotide polymorphism.Although SNPs is located at noncoding region mostly, have with organism phenotype very close
Relationship.With the development of high throughput sequencing technologies, a large amount of SNPs is resolved to be come out, and also genome level has greatly been pushed to comment
Estimate the research of genetic diversity.Since SNP data volume is very huge, the molecular labeling based on SNP exploitation is with higher
Precision.
Summary of the invention
In view of the problems of the existing technology, it is an object of the invention to design, to provide a kind of identification single cropping wild rice stem early late-maturing
The molecular labeling of characteristic and its technical solution of application, acquisition methods.The present invention is by sweeping wild rice smut raw in wild rice stem as SNP
It retouches, multiple early relevant molecular labelings of late-maturing characteristic to single cropping wild rice stem is filtered out, to set up single cropping wild rice stem early late-maturing characteristic
Molecular marker assisted selection system improves the efficiency of selection of single cropping precocity Zizania latifolia Cultivars, lays the foundation for the efficient breeding of wild rice stem.
The molecular labeling of the early late-maturing characteristic of a kind of identification single cropping wild rice stem, it is characterised in that including molecular labeling
The forward primer UeSNPSE1-F nucleotide sequence of UeSE1 and UeSE2, the molecular labeling UeSE1 such as SEQ ID NO.1 institute
Show, reverse primer UeSNPSE1-R nucleotide sequence is as shown in SEQ ID NO.2；The forward primer of the molecular labeling UeSE2
UeSNPSE2-F nucleotide sequence is as shown in SEQ ID NO.3, reverse primer UeSNPSE2-R nucleotide sequence such as SEQ ID
Shown in NO.4.
Application of the molecular labeling in wild rice stem breeding.
The molecular labeling identified in wild rice stem breeding single cropping wild rice stem it is early late-maturing in application.
The preparation method of the molecular labeling, it is characterised in that the following steps are included:
A, the identification of single cropping wild rice stem maturity: the single cropping Zizania latifolia Cultivars of different regions are acquired in batches and are recorded；
B, population analysis
1) the wild rice smut being separately cultured in wild rice stem；
2) CTAB method extracts wild rice smut genomic DNA；
3) in wild rice smut genomic DNA progress two generation of genome of 5 single cropping wild rice stems, is resurveyed into sequence；
4) scanning obtains all SNP；
5) the difference SNP site of single cropping wild rice stem early-maturing variety and late variety wild rice smut is filtered out；
6) 20 acquisitions are chosen to test from the SNP site that the wild rice stem separation wild rice smut of different regions obtains screening
Card；
C, using other with Dan Shuanji characteristic cultivar identification screening SNP site and obtain map；2 pairs of tools are obtained altogether
There are molecular labeling UeSE1, UeSE2 of Dan Shuanji characteristic.
The preparation method, it is characterised in that the identification of the early late-maturing characteristic of single cropping hay in step a specifically: from Zhejiang Province
8-9 month is being collected list in Tongxiang City, Jinhua, Zhejiang Province city, Zhejiang Province Yuyao City, Hangzhou, Zhejiang province city and Suzhou City of Jiangsu Province
Season wild rice stem, record picking time and hay weight；The wild rice stem that hay shape is complete, head is medium, hay leaf is disease-free is chosen when harvesting.
The preparation method, it is characterised in that the 1 of step b) in be separately cultured wild rice smut in wild rice stem specifically:
A, sterile water is added to grind wild rice stem stem；
B, it is applied on the YEPS culture medium containing carbenicillin and kanamycins and cultivates 4-6d.
The preparation method, it is characterised in that the 5 of step b) in filter out in single cropping precocity wild rice stem wild rice smut and list
Season late-maturing wild rice stem wild rice smut difference SNP site specifically:
A, it is two class of A, B by all SNP data markers, represents single cropping precocity, late-maturing two characteristics of single cropping；
B, all SNP of two class of Computer Automatic Recognition A, B and classification；Output format is Genotyping*simple-A/
B.txt.；Data content is SNP_site a/b, and a represents the number with the SNP, and b represents a kind of total number；
C, data are copied to Excel worksheet, using screening function, choose only be in A class be 0 in 1, B class
SNP。
The preparation method, it is characterised in that the 6 of step b) in choose 20 acquisitions and separated from the wild rice stems of different regions
The SNP site that wild rice smut obtains screening carries out verifying and specifically includes:
A, the wild rice stem by 20 acquisitions from different regions carries out grinding separation；
B, whole wild rice smut genomic DNAs are extracted；
C, for the SNP design data AS-PCR primer of acquisition；
D, the wild rice stem wild rice smut DNA for choosing the typical single cropping hay kind with early late maturity carries out AS-PCR verifying, tests
Card obtains 3 SNP sites with polymorphism；Amplimer is detected for this 3 SNP site design HRM；
E, 20 wild rice stem wild rice smut DNA, 3 pairs of HRM primers are expanded, PCR product is detected for HRM；
F, single cropping wild rice stem early late-maturing characteristic is marked with the curve of different colours.
The invention has the following advantages:
1) present invention is swept based on the genome of 5 single cropping wild rice stems with late-maturing characteristic of different morning weight sequencing data
It retouches to obtain SNP data, and screens to obtain the associated site of single cropping wild rice stem maturity by effective means.In conjunction with 20 kinds of different regions
The single cropping wild rice stem wild rice smut of different maturity is the qualification result of object, filters out the early late-maturing relevant molecular labeling of single cropping hay.
2) research cycle is short, using each regional Zizania latifolia Cultivars as material, can exclude environmental factor and do to wild rice stem maturity
It disturbs.
3) SNP marker is based on genome weight sequencing data, has good precision, and site is abundant, contains much information, sieves
The site of choosing has more reliability.
4) wild rice stem breeding cycle is long, and Breeding Process is cumbersome, screens precocious single cropping hay by SNP marker, can be to excellent hay
White kind selects ahead of time, reduces workload, improves screening efficiency.
Detailed description of the invention
Fig. 1 is that 4 typical cases have the single cropping hay wild rice smut 3 of early late maturity to primer AS-PCR polymorphism as a result, screening
Site with polymorphism；
Fig. 2 is LightScanner high-resolution fusion curve in embodiment 3；
Fig. 3 is distribution map of 3 SNP sites in 20 kinds of single cropping wild rice stem wild rice smut.
Specific embodiment
Below with reference to embodiment, the present invention will be further described, and following experimental method kinds are normal unless otherwise specified
Rule method.Embodiment kind in addition to experimental material other can pass through commercial sources purchase obtain.
Embodiment 1: test material and maturity classification
Following tests material is respectively derived from Jinhua, Zhejiang Province city academy of agricultural science, Lishui Jinyun county agriculture
Industry office, Ningbo City, Zhejiang Province Yuyao City Institute of agricultural sciences, Suzhou City of Jiangsu Province academy of agricultural science, Jiaxing City, Zhejiang Province
Tongxiang City Dong Jia wild rice stem cooperative society, Shaoxing, Zhejiang Province city Shengzhou Pu Hui vegetables cooperative society, City of Taizhou Huangyan District vegetables
Research institute, China, Hangzhou, Zhejiang province city measure university life science institute.Sample collecting time was in September, 2016,2017 years 9
The moon, in September, 2018.
1 test sample kind of table, maturity and source
Embodiment 2: population analysis
1) wild rice smut genomic DNA is extracted using CTAB method, measures DNA concentration with nucleic acid microdetermination instrument, and use fine jade
Sepharose electrophoresis detection DNA mass.Use ddH2O is diluted to 100ng/ μ L, is placed in -20 DEG C of preservations.
2) DNA for determining 5 single cropping hay Zizania latifolia Cultivars of maturity phase was sent into for two generations and resurveys sequence.It is that wild rice is black with reference to genome
Powder bacterium MT type genome, WGS INSDC:JTLW00000000.1.
3) sequencing data is compared with GATK, SAMTOOLS, BWA software to genome is referred to, and obtains SNP data file.Altogether
Obtain 21529 SNP sites.
4) it is two class of A, B by the wild rice smut sample number into spectrum of all participation SNP scannings, respectively represents single cropping hay precocity, it is single
Season late-maturing two class of hay.By extracting the Genotype data in SNP scanning result, the SNP data of two class of A, B are exported as SNPc
A/b format, a are represented in such SNP number of c-th of SNP site, and b represents such sample total number.Output result is pasted together
Into Excel worksheet, with Excel screening function, filter out in precocity as b/b, it is late-maturing in be 0/b SNP site.(
In this example, A 10, B 18, C 20, therefore SNPc1=(A arranges=3/3, B column=0/2) is selected, amount to 14 SNP sites.
The identification of the site embodiment 3:SNP
1) obtained SNP site is screened at 14 and selects suitable SNP site verifying: due to reference gene order-checking quality
Problem need to exclude the SNP site in reference genome contig more rearward, by screening, select 3 SNP sites altogether
As verifying site, and design relevant primer.
2) AS-PCR (ApoE gene), AS-PCR are to hold base mispairing that PCR can be made to extend journey using primer 3 '
Sequence interrupts the principle for causing no amplified production, plays an important role in terms of detection SNP, allele genotype.This example devises 3
To AS-PCR primer, all primers subordinate list 2.
2 ApoE gene primer sequence table of table
| Forward primer | Primer sequence (5 ' -3 ') | Reverse primer | Primer sequence (5 ' -3 ') |
| SNPSE 1-F | AGTCACATCACATCACACTGGTCCAT | SNPSE 1-R | TGGAGACTGCTGGTCAAATGAAATTAT |
| SNPSE 2-F | GCGACACTCACACCTCCACGTTT | SNPSE 2-R | GCCCCTGCTCCTTTCCTGAA |
| SNPSE 3-F | CATTTACCCAGTTTTGGCTATCACTCTG | SNPSE 3-R | TCAAGGAAGGGGAATGGTTGC |
3) this example chooses the polymorphism that 4 typical case single cropping hay wild rice smut DNA verify upper table SNP site as template.
4) AS-PCR system: 1 μ L wild rice smut genomic DNA；0.4 μM of forward primer；0.4 μM of reverse primer；5μL2×
Taq Master Plus Mix；3.2μL ddH2O。
5) AS-PCR program is arranged are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s；65 DEG C of annealing 30s；72 DEG C of extensions
15s；Totally 34 circulations；72 DEG C thoroughly extend 7min；12 DEG C of preservations.
6) by 2% agarose gel electrophoresis of all PCR products, test strip whether there is or not.
7) electrophoresis verification result (as shown in Figure 1): 1 representative has band；0 represents band；Band * represents the site in single cropping hay
There is polymorphism in maturity.
8) 3 sites with polymorphism are further detected in this example, detection method is HRM curve (high score
Resolution melting curve).
9) cardinal principle of HRM is according to the length of DNA sequence dna, G/C content and base complementrity sex differernce, using high score
The melting curve of resolution analyzes sample, and high temperature uniformity and temperature resolution can achieve resolving accuracy
Differentiation to single base difference.It is advantageous that low in cost, single detection limit is big, and accuracy is high, and the sample after detection can
Continue on for other experiments, and can be used for distinguishing allele genic homozygote and heterozygote.
10) 3 couples of HRM are designed for above-mentioned 3 SNP sites in this example and detects segment amplimer.All primers subordinate list
3。
3 HRM of table detects segment amplimer
| Forward primer | Primer sequence (5 ' -3 ') | Reverse primer | Primer sequence (5 ' -3 ') |
| HRMSE 1-F | CCAACGGTCCAATCCAACTTCTG | HRMSE 1-R | GCACCTGTCAAGGGCTGTCA |
| HRMSE 2-F | ACTCCAGCGACACTCACACCTC | HRMSE 2-R | GTGGCACACCGCTAGTGATAGTC |
| HRMSE 3-F | AAGCCTATCTGGACACCACACTTG | HRMSE 3-R | GCCCACACTCATCTGACACTAATTG |
11) the 20 kinds of single cropping hays of (Zizania latifolia Cultivars that number is 6-25 in table 1) with different maturity characteristics are used in this example
White wild rice smut DNA is as template verification step 7) in obtained 3 polymorphic SNP sites of screening.
12) HRM-PCR system: 1 μ L wild rice smut genomic DNA；0.6 μM of forward primer；0.6 μM of reverse primer；7.5μL
2×Taq Master Plus Mix；5.3μL ddH2O。
13) HRM-PCR program is arranged are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s；65 DEG C of annealing 30s；72 DEG C of extensions
15s；Totally 40 circulations；72 DEG C thoroughly extend 7min；12 DEG C of preservations.
14) 1 μ L 5 × Eva Green fluorescent dye of Kong Lijia of Xiang Suoyou PCR product, sealing 1000rpm are centrifuged 15s,
Then plus 20 μ L mineral oil are closed.
15) 94 DEG C of denaturation 2min, are integrated to fluorescent dye sufficiently in DNA double chain.
16) LightScanner high-resolution fusion curve system detection product is used, testing result is as shown in Figure 2.
17) the results show that it is HRMSE-1, HRMSE-2, more apparent to single cropping wild rice stem maturity characteristic parting, it therefore, will
HRMSE-1, HRMSE-2 correspond to molecular labeling UeSE1, UeSE2.
18) distribution map of 2 SNP sites in 20 kinds of wild rice stem wild rice smut, as shown in Figure 3.
Sequence table
<110>China's metering university
<120>a kind of identification single cropping wild rice stem early molecular labeling of late-maturing characteristic and its application, acquisition methods
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<170> SIPOSequenceListing 1.0
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<211> 23
<212> DNA
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ccaacggtcc aatccaactt ctg 23
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gcacctgtca agggctgtca 20
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actccagcga cactcacacc tc 22
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gtggcacacc gctagtgata gtc 23
Claims (8)
1. a kind of molecular labeling of the early late-maturing characteristic of identification single cropping hay, it is characterised in that including molecular labeling UeSE1 and UeSE2,
The forward primer UeSNPSE1-F nucleotide sequence of the molecular labeling UeSE1 is as shown in SEQ ID NO.1, reverse primer
UeSNPSE1-R nucleotide sequence is as shown in SEQ ID NO.2；The forward primer UeSNPSE2-F core of the molecular labeling UeSE2
Nucleotide sequence is as shown in SEQ ID NO.3, and reverse primer UeSNPSE2-R nucleotide sequence is as shown in SEQ ID NO.4.
2. application of the molecular labeling as described in claim 1 in wild rice stem breeding.
3. molecular labeling as described in claim 1 identified in wild rice stem breeding single cropping wild rice stem it is early late-maturing in application.
4. the preparation method of molecular labeling as described in claim 1, it is characterised in that the following steps are included:
A, the identification of single cropping hay maturity: the single cropping Zizania latifolia Cultivars of different regions are acquired in batches and are recorded；
B, population analysis
1) the wild rice smut being separately cultured in wild rice stem；
2) CTAB method extracts wild rice smut genomic DNA；
3) in wild rice smut genomic DNA progress two generation of genome of 5 single cropping wild rice stems, is resurveyed into sequence；
4) scanning obtains all SNP；
5) the difference SNP site in single cropping wild rice stem early-maturing variety and late variety wild rice smut is filtered out；
6) 20 acquisitions are chosen to verify from the SNP site that the wild rice stem separation wild rice smut of different regions obtains screening；
C, using other with Dan Shuanji characteristic cultivar identification screening SNP site and obtain map；Obtaining 2 pairs altogether has list
Molecular labeling UeSE1, UeSE2 of double season characteristics.
5. preparation method as claimed in claim 4, it is characterised in that the identification of single cropping wild rice stem maturity in step a specifically: from
Zhejiang Province Tongxiang City, Jinhua, Zhejiang Province city, Zhejiang Province Yuyao City, Hangzhou, Zhejiang province city and Suzhou City of Jiangsu Province are in 8-9 month
Single cropping wild rice stem is collected, picking time and hay weight are recorded；The wild rice stem that hay shape is complete, head is medium, hay leaf is disease-free is chosen when harvesting.
6. preparation method as claimed in claim 4, it is characterised in that the 1 of step b) in be separately cultured wild rice smut in wild rice stem
Specifically:
A, sterile water is added to grind wild rice stem stem；
B, it is applied on the YEPS culture medium containing carbenicillin and kanamycins and cultivates 4-6d.
7. preparation method as claimed in claim 4, it is characterised in that the 5 of step b) in filter out it is black in single cropping precocity wild rice stem wild rice
The difference SNP site of powder bacterium and the late-maturing wild rice stem wild rice smut of single cropping specifically:
A, it is two class of A, B by all SNP data markers, represents single cropping hay precocity, the late-maturing two maturity features of single cropping hay；
B, all SNP of two class of Computer Automatic Recognition A, B and classification；Output format is Genotyping*simple-A/
B.txt.；Data content is SNP_site a/b, and a represents the number with the SNP, and b represents a kind of total number；
C, data are copied to Excel worksheet, using screening function, the SNP that it is 0 that selection, which is in 1, B class in A class,.
8. preparation method as claimed in claim 4, it is characterised in that the 6 of step b) in choose 20 acquisitions from different regions
The SNP site that wild rice stem separation wild rice smut obtains screening carries out verifying and specifically includes:
A, the wild rice stem by 20 acquisitions from different regions carries out grinding separation；
B, whole wild rice smut genomic DNAs are extracted；
C, for the SNP design data AS-PCR primer of acquisition；
D, the wild rice stem wild rice smut DNA for choosing more typical single cropping hay kind carries out AS-PCR verifying, and verifying acquisition 3 has more
The SNP site of state property；Amplimer is detected for this 3 SNP site design HRM；
E, 20 wild rice stem wild rice smut DNA, 3 pairs of HRM primers are expanded, PCR product is detected for HRM；
F, single cropping wild rice stem early late-maturing characteristic is marked with the curve of different colours.
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| CN114703313B (en) * | 2022-03-11 | 2023-11-17 | 中国计量大学 | Wild rice black powder fungus typing identification method and application thereof |
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