CN105256044A - Wheat molecular barcode based on single nucleotide polymorphism - Google Patents

Wheat molecular barcode based on single nucleotide polymorphism Download PDF

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CN105256044A
CN105256044A CN201510736738.9A CN201510736738A CN105256044A CN 105256044 A CN105256044 A CN 105256044A CN 201510736738 A CN201510736738 A CN 201510736738A CN 105256044 A CN105256044 A CN 105256044A
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snp site
nucleotide
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wheat
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高丽锋
孔秀英
贾继增
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses a wheat molecular barcode based on single nucleotide polymorphism. The invention provides the application of a substance for detecting the polymorphisms or genotypes of 43 SNP (single nucleotide polymorphism) sites in identification or help to identify wheat varieties; the research plans to utilize present high-throughput SNP chips, a set of SNP marks in the smallest number, which has the highest solution, are screened out to serve as the barcode, so that the utilization efficiency of wheat resources in wheat breeding and scientific research can be increased.

Description

A kind of wheat molecular barcode based on single nucleotide polymorphism
Technical field
The present invention relates to biological technical field, particularly relate to a kind of wheat molecular barcode based on single nucleotide polymorphism.
Background technology
DNA fingerprinting technology has been widely used in the qualification of crop material, has analyzed and kind protection (Wang2006). and the molecule marker as DNA fingerprinting experienced by three phases: first-generation molecule marker is restriction fragment length polymorphism (RFLP), but its complicated operation, polymorphism are low and spend high, utilize the time shorter (Grover & Sharma2015); S-generation molecule marker is simple sequence repeats (SSR), and its operation is relatively simple, polymorphism is higher and cost is lower, is therefore widely used in crop genetic research (Wu & Tanksley1993; Brunel1994; al.1998); SNP is third generation molecule marker, along with the development of sequencing technologies of future generation and genome sequencing cost progressively reduce, many important crops genome sequencings complete, and create a large amount of SNP data and develop SNP chip (Cavanaghetal.2013 accordingly; Chenetal.2014; Mondenetal.201; Unterseeretal.2014; Wangetal.2014; Hulse-Kempetal.2015; Leeetal.2015; ).The stdn of SNP chip operating process, the data produced between different experiments room have comparability.Theoretically, on chip, the position of SNP point on karyomit(e) or genome of Boulez is unique, thus largely avoid the somatotype puzzlement that multiple copied number gene SNP brings.This is even more important for the polyploid species that common wheat is such.In recent years, there is a few money wheat SNP chip product successively, 35K, 817K and 660KSNP chip that 9K, 90KSNP chip released as illumina company and Affymetrix company release.These wheat SNP chip are for wheat population structure, heritable variation, selection evolution and whole-genome association (Cavanaghetal.2013; Farisetal.2014; Wangetal.2014; Zankeetal., 2015).
Summary of the invention
The present invention's object is to provide and detects the polymorphism of 43 SNP site or the purposes of genotypic material in Wheat volatiles.
The invention provides the polymorphism or the application of genotypic material in qualification or assistant identification wheat breed that detect 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or the genotypic material application in the test kit of characterization or assistant identification wheat breed in detection Wheat volatiles;
Described 43 SNP site are as follows: IWA530, IWA2783, IWA5405, IWA5186, IWA4153, IWA4154, IWA4155, IWA4031, IWA695, IWA4525, IWA4884, IWA2938, IWA2442, IWA4323, IWA2961, IWA5285, IWA5106, IWA2037, IWA8303, IWA662, IWA5618, IWA6279, IWA3710, IWA2124, IWA3835, IWA3011, IWA1691, IWA4854, IWA5375, IWA4805, IWA5326, IWA1755, IWA2698, IWA6526, IWA7177, IWA224, IWA8380, IWA3312, IWA2476, IWA2506, IWA4187, IWA1437 and IWA304.
In above-mentioned application, identify or assistant identification wheat breed for identify or whether assistant identification wheat to be measured is certain wheat breed shown in table 2 and/or table 3, concrete grammar comprises the steps: to detect 43 SNP site genotype described in wheat breed genome to be measured, 43 SNP site genotype of each wheat breed shown in described wheat breed to be measured 43 SNP site genotype and table 2 and/or table 3 are compared, determines whether wheat to be measured is table 2 and/or table 3 certain wheat breed shown.
Another object of the present invention is to provide and detects the polymorphism of 43 SNP site or the purposes of genotypic material in Wheat volatiles.
The invention provides the polymorphism or the application of genotypic material in differentiation or supplementary globe wheat breed that detect 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or genotypic material are distinguished or application in supplementary globe wheat breed product in preparation in detection Wheat volatiles.
In above-mentioned application, differentiation or supplementary globe wheat breed are distinguish at least two kinds shown in table 2 and/or table 3, concrete grammar comprises the steps: 43 SNP site genotype described at least two kind genomes shown in detection table 2 and/or table 3, two kinds, 43 SNP site genotype described in comparison, distinguish at least two kinds shown in table 2 and/or table 3.
The present invention's the 3rd object is to provide and detects the polymorphism of 43 SNP site or the purposes of genotypic material in Wheat volatiles.
The invention provides the polymorphism or the application of genotypic material in wheat breed classification that detect 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or genotypic material are preparing the application in wheat breed sort product in detection Wheat volatiles.
In above-mentioned application,
Described SNP site IWA530 is sequence 1 the 51st Nucleotide;
Described SNP site IWA2783 is sequence 2 the 101st Nucleotide;
Described SNP site IWA5405 is sequence 3 the 101st Nucleotide;
Described SNP site IWA5186 is sequence 4 the 101st Nucleotide;
Described SNP site IWA4153 is sequence 5 the 101st Nucleotide;
Described SNP site IWA4154 is sequence 6 the 101st Nucleotide;
Described SNP site IWA4155 is sequence 7 the 101st Nucleotide;
Described SNP site IWA4031 is sequence 8 the 43rd Nucleotide;
Described SNP site IWA695 is sequence 9 the 101st Nucleotide;
Described SNP site IWA4525 is sequence 10 the 101st Nucleotide;
Described SNP site IWA4884 is sequence 11 the 101st Nucleotide;
Described SNP site IWA2938 is sequence 12 the 101st Nucleotide;
Described SNP site IWA2442 is sequence 13 the 101st Nucleotide;
Described SNP site IWA4323 is sequence 14 the 101st Nucleotide;
Described SNP site IWA2961 is sequence 15 the 101st Nucleotide;
Described SNP site IWA5285 is sequence 16 the 101st Nucleotide;
Described SNP site IWA5106 is sequence 17 the 101st Nucleotide;
Described SNP site IWA2037 is sequence 18 the 101st Nucleotide;
Described SNP site IWA8303 is sequence 19 the 26th Nucleotide;
Described SNP site IWA662 is sequence 20 the 101st Nucleotide;
Described SNP site IWA5618 is sequence 21 the 101st Nucleotide;
Described SNP site IWA6279 is sequence 22 the 101st Nucleotide;
Described SNP site IWA3710 is sequence 23 the 101st Nucleotide;
Described SNP site IWA2124 is sequence 24 the 101st Nucleotide;
Described SNP site IWA3835 is sequence 25 the 101st Nucleotide;
Described SNP site IWA3011 is sequence 26 the 101st Nucleotide;
Described SNP site IWA1691 is sequence 27 the 101st Nucleotide;
Described SNP site IWA4854 is sequence 28 the 101st Nucleotide;
Described SNP site IWA5375 is sequence 29 the 101st Nucleotide;
Described SNP site IWA4805 is sequence 30 the 101st Nucleotide;
Described SNP site IWA5326 is sequence 31 the 101st Nucleotide;
Described SNP site IWA1755 is sequence 32 the 101st Nucleotide;
Described SNP site IWA2698 is sequence 33 the 101st Nucleotide;
Described SNP site IWA6526 is sequence 34 the 101st Nucleotide;
Described SNP site IWA7177 is sequence 35 the 101st Nucleotide;
Described SNP site IWA224 is sequence 36 the 61st Nucleotide;
Described SNP site IWA8380 is sequence 37 the 51st Nucleotide;
Described SNP site IWA3312 is sequence 38 the 101st Nucleotide;
Described SNP site IWA2476 is sequence 39 the 101st Nucleotide;
Described SNP site IWA2506 is sequence 40 the 101st Nucleotide;
Described SNP site IWA4187 is sequence 41 the 101st Nucleotide;
Described SNP site IWA1437 is sequence 42 the 101st Nucleotide;
Described SNP site IWA304 is sequence 43 the 61st Nucleotide.
The present invention's the 4th object is to provide a kind of method of multiple wheat to be measured being carried out to assortment.
Method provided by the invention, comprise the steps: to detect respectively 43 SNP site genotype described in multiple Wheat volatiles to be measured, whether identical according to described multiple wheats to be measured 43 SNP site genotype, determine that in described multiple wheat to be measured, which belongs to a class.
Above-mentioned multiple wheat to be measured is at least 2 wheats to be measured;
Above-mentioned wheat to be measured in the kind shown in the kind shown in table 2 or table 3 any one.
The present invention's the 5th object is to provide the polymorphism or genotypic material that detect 43 SNP site in Wheat volatiles.
Material provided by the invention is a)-c) in any one product:
A) product of qualification or assistant identification wheat breed;
B) differentiation or supplementary globe wheat breed;
C) wheat breed sort product.
In above-mentioned detection Wheat volatiles, the polymorphism of 43 SNP site or genotypic material comprise InfiniumBeadchip platform and scans I lluminawheat9KInfiniumAssay chip.
Above-mentioned wheat in the kind shown in the kind shown in table 2 or table 3 any one.
Experiment of the present invention proves, present invention finds 43 SNP site, and it can be used for qualification, differentiation or sorted table 2 and/or the kind shown in table 3, and raising wheat breeding and scientific research are to the utilising efficiency of wheat resource.
Accompanying drawing explanation
Fig. 1 is wheat SNP barcode screening process.
Fig. 2 is 43 SNP barcode distributions on chromosome.
Fig. 3 is 43 SNP bar code label linkage disequilibrium (LD) collection of illustrative plates.
Fig. 4 is that the UPGMA dendrogram of table 2 shows 43 SNP barcode high resolving power.
Fig. 5 is the Quick Response Code that 43 SNP barcodes differentiate common wheat.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The screening of embodiment 1, SNP site
Wheat 429 parts of common wheat compositions as shown in Table 2 of the present embodiment, these wheats comprise local variety and modern improved variety, extensively plant in China's wheat cultivation region from the thirties in last century.These materials carry Main Agronomic Characters gene, such as stripe rust resisting, banded sclerotial blight or head blight gene, the excellent proterties of drought resisting, moisture-proof saline-resisting and alkaline-resisting gene, high-quality, large grain, large fringe, dwarf gene etc.In addition, some materials, as backbone parent, have been bred as a collection of wheat breed.First set material is for screening SNP barcode.
1, SNP marker screening
1) DNA is extracted
Supplying shown in all tables 2 tries material selection plump seed, germinates in room temperature, until growth of seedling after one week, with DNA extraction kit (CN.DP321 Beijing Tian Gen biotech company), according to operation instructions, extracts seedling DNA.
2) genotype detection
DNA sample is (1.8<OD260/280<2.0) after quality examination is qualified, utilize InfiniumBeadchip platform, scans I lluminawheat9KInfiniumiSelectSNPAssay ((Illumina, SanDiego, CA, Cavanaghetal.2013) chip, detects these material genotype.
When analyzing 9KSNP chip, in order to verify the exactness of genotyping result, repetition is artificially designed 1 time to wheat breed exhibition No. 1 material of laying down.When genotype data arranges, the SNP that in first exhibition No. 1 two of laying down being repeated, genotype is inconsistent is all deleted.Then, have the SNP site of typical AA, AB, BB tri-kinds of genetic pattern from those that (wherein AA, BB are homozygous genotype, AB is heterozygous genotypes), only retain typical two condition SNP, because the two condition SNP most common type that is single nucleotide polymorphism correct raw data according to the method for (2013) such as Cavanagh.After above-mentioned initial filtration, in first set material 429 parts of wheat breeds, remain 3489 effective SNP site, these SNP will be used to the screening of SNP barcode.
SNP barcode screening process based on 429 parts of materials is shown in Fig. 1.First, utilize PowerMarker software, analyze the feature of 3489 SNP, by singlet or missing data, the SNP site more than 10% is deleted, and then calculates the PIC value of residue SNP site, retains the SNP site that 50 PIC values are the highest.Then, utilize the genotype of this 50 SNP site 429 parts of materials, build dendrogram, to detect the resolving power of these SNP marker to material by UPGMA method.Result shows, although select the SNP marker that PIC value is the highest, some materials still cannot distinguish.Wherein, 364 parts of materials (84.8%) can obviously distinguish mutually.All the other materials of 15% due to sibship near and cannot distinguish.Such as, raising wheat No. 5 is choosing from Ah husband, and good star 66 and good star 99 have common parent's donor Ji 91102.
Afterwards, according to the material that cannot distinguish that above-mentioned UPGMA dendrogram provides, separately these materials are compared between two, screening can distinguish the SNP marker of the very near material of sibship, before adding in 50 SNP site, progressively delete that specific mark screened by non-material simultaneously, after deleting SNP site, all use UPGMA dendrogram to check the resolving power retaining SNP site at every turn.Finally, filter out 43 SNP site (table 1), and these 43 SNP site can distinguish (table 2 and Fig. 4) by portion common wheat of 429 shown in table 2 completely.Fully show the validity of 43 SNP marker, the finger printing specially that every part of material has it unique, thus be different from other materials (Fig. 3).In 429 parts of wheat lines, recently (0.023), agricultural university 311 and No. 4, Mount Taishan genetic distance are farthest (1.6818) for continuous wheat 1403 and continuous wheat 23 genetic distance.UPGMA dendrogram shows 43 SNP barcode high resolving power, and the finger printing of 429 parts of materials shows the abundant genetic diversity that these kind of matter contains.Every part of material 43 SNP barcode finger printings change into Quick Response Code, are convenient to mobile telephone scanning (Fig. 5).
Table 1 is 43SNP bar code information and the polymorphism in first set material thereof
asNP numbers, chromosome position information, and SNP type and I type and the change of II type are from (2013) and Wang etc. (2014) such as document Cavanagh;
bpIC, polymorphisminformationcontent polymorphism information content; The minimum gene frequency of MAF, minorallelefrequency
Two, SNP characteristics of bar code
A SNP barcode site, shown in the table 1 filtered out 43 is distributed in (Fig. 2 on common wheat 21 karyomit(e)s, every bar line represents a SNP site, according to minimum gene frequency is different, color mark is different, chromosome length (CentiMorgan, cM) scale is in figure left side), karyomit(e) 1B and 3B marks relatively many (table 1).Analyze and find, relatively independent between these SNP site, the lower (R of average l/d value 2=0.1), but on karyomit(e) 1B and 3B, each 2 SNP site show as height linkage inheritance (R 2=0.9, Fig. 3).In 43 SNP site, 34 SNP are translation type (A/G or T/C), and 9 is transversion (A/C or T/G), and wherein 18 SNP site are same sense mutation, the amino acid mutation (table 1) that 13 SNP cause encoding.
43 SNP barcodes are in 429 parts of materials, and minimum gene frequency (MAF) is 8% to 50%, average out to 37%; Polymorphism level (PIC) is between 0.14 to 0.38, average out to 0.34, compared with original 3489 average polymorphisms of SNP site (PIC=0.29), the polymorphism of 43 SNP barcodes does not reduce, and increases 17% (table 1).Therefore, can think according to MAF and PIC value, 43 the SNP barcodes filtered out represent abundant variation.
Therefore, can find out, the SNP site of 43 shown in table 2 can be used for identifying or portion wheat of 429 shown in differentiation table 2, and 43 SNP site of each wheat are all incomplete same, can as the SNP barcode of qualification wheat.
Embodiment 2,43 SNP site are distinguishing the application in wheat breed
Second cover material is by the hybrid dna sample composition (table 3) of 193 portions of common wheats of Stochastic choice in first set material and 96 to wild two (Triticumdicoccoides) and aegilops tauschii (Aegilopstauschii).
1, DNA is extracted
All for examination material selection plump seed, germinate in room temperature, after growth of seedling one week (common wheat and wild emmer) and two weeks (aegilops tauschii), with DNA extraction kit (CN.DP321 Beijing Tian Gen biotech company,), according to operation instructions, extract seedling DNA.
2, genotype detection
DNA sample is (1.8<OD260/280<2.0) after quality examination is qualified, utilize InfiniumBeadchip platform, scans I lluminawheat90KInfiniumAssay (Cavanaghetal.2013) chip, detects these material genotype.The data produced are analyzed with GenomeStudiov2011.1 software (Illumina).As previously mentioned, although in theory each SNP site on chromosome position should be unique, but common wheat is allohexaploid, have A, B, D tri-genomes, in addition because some gene is gene family, sequence similarity is very high, therefore, inevitably, portion homologous gene or orthologous gene can cause SNP site position is unique on chromosome, thus affect the accuracy of material genotyping result.Therefore, on the basis of GenomeStudiov2011.1 software automatic parting direction, need the somatotype pattern of each SNP site of artificial adjustment, to guarantee result accuracy.
With Powermarkerv3.25 (Liu & Muse2002) software analysis each SNP site gene frequency (minimum gene frequency, minorallelefrequency, and polymorphism index (PIC, polymorphisminformationcontent) MAF).In order to understand the mutual relation of SNP barcode filtered out, utilize linkage relationship between Powermarker and Tassel3.0 (Bradburyetal.2007) software estimation mark, calculate linkage disequilibrium value. utilize the SNP bar code label filtered out, based on NeiShi (1972) genetic distance method, employing UPGMA method, dendrogram is built to institute's analysis of material, dendrogram Powermarker Software Create, Mega5 (Tamuraetal.2011) software is browsed.
Result is as shown in Fig. 4 and table 3, and 43 SNP site can not only distinguish common wheat, and can differentiate the wheat lines of Different Ploidy.Common wheat is hexaploid, and wild emmer Triticumdicoccoides is tetraploid, and aegilops tauschii Aegilopstauschii is diploid, and the latter two are common wheat ancestors kinds.As can be seen from Fig. 4 and table 3, can plant with its ancestors completely for 190 parts in 193 portions of common wheats and distinguish, all the other 3 parts is U.S.'s wheat triumph wheat (Triumph, 212), Gansu 96 (129) and XJ1 (Xinjiang Wheat, 263).Further demonstrate that 43 SNP barcodes not only can be used as common wheat New variety protection and discriminating, and may be used for novel material classification.

Claims (5)

1. detect polymorphism or the application of genotypic material in qualification or assistant identification wheat breed of 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or the genotypic material application in the test kit of characterization or assistant identification wheat breed in detection Wheat volatiles;
Described 43 SNP site are as follows: IWA530, IWA2783, IWA5405, IWA5186, IWA4153, IWA4154, IWA4155, IWA4031, IWA695, IWA4525, IWA4884, IWA2938, IWA2442, IWA4323, IWA2961, IWA5285, IWA5106, IWA2037, IWA8303, IWA662, IWA5618, IWA6279, IWA3710, IWA2124, IWA3835, IWA3011, IWA1691, IWA4854, IWA5375, IWA4805, IWA5326, IWA1755, IWA2698, IWA6526, IWA7177, IWA224, IWA8380, IWA3312, IWA2476, IWA2506, IWA4187, IWA1437 and IWA304.
2. detect polymorphism or the application of genotypic material in differentiation or supplementary globe wheat breed of 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or genotypic material are distinguished or application in supplementary globe wheat breed product in preparation in detection Wheat volatiles.
3. detect polymorphism or the application of genotypic material in wheat breed classification of 43 SNP site in Wheat volatiles;
Or the polymorphism of 43 SNP site or genotypic material are preparing the application in wheat breed sort product in detection Wheat volatiles.
4., according to described application arbitrary in claim 1-3, it is characterized in that:
Described SNP site IWA530 is sequence 1 the 51st Nucleotide;
Described SNP site IWA2783 is sequence 2 the 101st Nucleotide;
Described SNP site IWA5405 is sequence 3 the 101st Nucleotide;
Described SNP site IWA5186 is sequence 4 the 101st Nucleotide;
Described SNP site IWA4153 is sequence 5 the 101st Nucleotide;
Described SNP site IWA4154 is sequence 6 the 101st Nucleotide;
Described SNP site IWA4155 is sequence 7 the 101st Nucleotide;
Described SNP site IWA4031 is sequence 8 the 43rd Nucleotide;
Described SNP site IWA695 is sequence 9 the 101st Nucleotide;
Described SNP site IWA4525 is sequence 10 the 101st Nucleotide;
Described SNP site IWA4884 is sequence 11 the 101st Nucleotide;
Described SNP site IWA2938 is sequence 12 the 101st Nucleotide;
Described SNP site IWA2442 is sequence 13 the 101st Nucleotide;
Described SNP site IWA4323 is sequence 14 the 101st Nucleotide;
Described SNP site IWA2961 is sequence 15 the 101st Nucleotide;
Described SNP site IWA5285 is sequence 16 the 101st Nucleotide;
Described SNP site IWA5106 is sequence 17 the 101st Nucleotide;
Described SNP site IWA2037 is sequence 18 the 101st Nucleotide;
Described SNP site IWA8303 is sequence 19 the 26th Nucleotide;
Described SNP site IWA662 is sequence 20 the 101st Nucleotide;
Described SNP site IWA5618 is sequence 21 the 101st Nucleotide;
Described SNP site IWA6279 is sequence 22 the 101st Nucleotide;
Described SNP site IWA3710 is sequence 23 the 101st Nucleotide;
Described SNP site IWA2124 is sequence 24 the 101st Nucleotide;
Described SNP site IWA3835 is sequence 25 the 101st Nucleotide;
Described SNP site IWA3011 is sequence 26 the 101st Nucleotide;
Described SNP site IWA1691 is sequence 27 the 101st Nucleotide;
Described SNP site IWA4854 is sequence 28 the 101st Nucleotide;
Described SNP site IWA5375 is sequence 29 the 101st Nucleotide;
Described SNP site IWA4805 is sequence 30 the 101st Nucleotide;
Described SNP site IWA5326 is sequence 31 the 101st Nucleotide;
Described SNP site IWA1755 is sequence 32 the 101st Nucleotide;
Described SNP site IWA2698 is sequence 33 the 101st Nucleotide;
Described SNP site IWA6526 is sequence 34 the 101st Nucleotide;
Described SNP site IWA7177 is sequence 35 the 101st Nucleotide;
Described SNP site IWA224 is sequence 36 the 61st Nucleotide;
Described SNP site IWA8380 is sequence 37 the 51st Nucleotide;
Described SNP site IWA3312 is sequence 38 the 101st Nucleotide;
Described SNP site IWA2476 is sequence 39 the 101st Nucleotide;
Described SNP site IWA2506 is sequence 40 the 101st Nucleotide;
Described SNP site IWA4187 is sequence 41 the 101st Nucleotide;
Described SNP site IWA1437 is sequence 42 the 101st Nucleotide;
Described SNP site IWA304 is sequence 43 the 61st Nucleotide.
5. one kind is carried out the method for assortment to multiple wheat to be measured, comprise the steps: to detect respectively 43 SNP site genotype described in multiple Wheat volatiles to be measured, whether identical according to described multiple wheats to be measured 43 SNP site genotype, determine that in described multiple wheat to be measured, which belongs to a class.
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CN109706263A (en) * 2019-02-22 2019-05-03 四川农业大学 Chain SNP marker and application with wheat stripe rust resisting ospc gene QYr.sicau-1B-1
CN111793712A (en) * 2020-07-28 2020-10-20 湖北省农业科学院粮食作物研究所 Application of single nucleotide polymorphism substance of wheat SNP IWA586 site
CN114107555A (en) * 2022-01-25 2022-03-01 华智生物技术有限公司 SNP molecular marker combination for detecting purity of wheat variety and application thereof
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CN114574613A (en) * 2022-02-11 2022-06-03 西北农林科技大学 Wheat-pseudo-goose grass whole genome liquid phase chip and application
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