CN108913798B - Wheat seedling stage frost resistance related CAPS (cleaved amplified polymorphic sequence) marker and method for identifying wheat seedling stage frost resistance - Google Patents

Wheat seedling stage frost resistance related CAPS (cleaved amplified polymorphic sequence) marker and method for identifying wheat seedling stage frost resistance Download PDF

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CN108913798B
CN108913798B CN201810693327.XA CN201810693327A CN108913798B CN 108913798 B CN108913798 B CN 108913798B CN 201810693327 A CN201810693327 A CN 201810693327A CN 108913798 B CN108913798 B CN 108913798B
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wheat
caps
seedling stage
frost resistance
wheat seedling
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张海萍
卢杰
常成
张璨
姜昊
董连生
邵辉
司红起
马传喜
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Anhui Agricultural University AHAU
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Abstract

The invention discloses a CAPS mark related to wheat seedling stage frost resistance and a method for identifying the wheat seedling stage frost resistance, and relates to the technical field of wheat molecular breeding, wherein the CAPS mark is named as CAPS-Ta-2-4, and the CAPS mark has the nucleotide sequence as follows: SEQ ID No. 1; the method for identifying the freezing resistance of the wheat seedlings by using the CAPS marker comprises the following steps of 1, extracting the genomic DNA of wheat to be detected; step 2, carrying out PCR amplification on the wheat genome DNA obtained in the step 1 by using the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplification product; step 3, carrying out enzyme digestion on the amplification product by using BtsaI endonuclease to obtain an enzyme digestion product; step 4, when the enzyme digestion product is 185bp and 70bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-A, the frost resistance of the corresponding wheat seedling stage is strong, and when the enzyme digestion product is 255bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-G, and the frost resistance of the corresponding wheat seedling stage is weak; the CAPS marker developed by the invention can provide a molecular marker for the genetic improvement of wheat freezing injury resistance.

Description

Wheat seedling stage frost resistance related CAPS (cleaved amplified polymorphic sequence) marker and method for identifying wheat seedling stage frost resistance
Technical Field
The invention relates to the technical field of wheat molecular breeding, in particular to a CAPS (cleaved amplified polymorphic sequence) marker related to wheat seedling stage freezing resistance and a method for identifying the wheat seedling stage freezing resistance by using the CAPS marker.
Background
About 40% of the world population is based on wheat as staple food, China is the largest world producing and consuming country, and therefore high and stable yield are always important targets for wheat breeding (who tiger et al 2018). However, the wheat suffers from low-temperature freeze injury (less than-5 ℃) in the seedling stage, and the high yield and the stable yield of the wheat are seriously influenced. The cultivation of wheat varieties with strong freezing resistance is an effective way to solve the problem.
Wheat freeze injury (FT) resistance is commonly controlled by multiple genes. The antifreeze-related genes reported in wheat at present are mainly located in chromosomes 1B, 2B, 1D, 2D, 4D, 5A and 7A, etc., and encode related transcription factors (e.g., CBF, bZIP, ICE, WRKY, NAC, etc.) and cold-inducible proteins (e.g., WCOR, WSC, aquaporins, etc.), respectively (Quellet et al 1998; Sakuma et al 2002; Ohno et al 2003; Kobayashi et al 2004; Qiu et al 2004; Vagujfalvi et al 2005; Forrest et al 2008; Sutton et al 2009; Takumi et al 2003; Mao et al 2012). However, the genetic mechanism of the wheat freezing resistance trait is complex, different materials and genetic backgrounds are different, and main effect genes for controlling freezing resistance also have differences. More sites and molecular markers closely related to wheat seedling stage freezing resistance are identified, so that the method is favorable for accelerating the process of polygene polymerization breeding and is also favorable for further clarifying a molecular regulation mechanism of wheat freezing resistance.
At present, methods for identifying target trait related sites/molecular markers mainly include parental-based linkage analysis and genome-wide association analysis. The former requires the construction of a mapping population, is time consuming, and the identification of sites/molecular markers is limited to the differences between the two parents. The latter is based on Linkage Disequilibrium (LD), identifies genotypes of hundreds or thousands of individuals through high-density molecular markers, screens out sites/molecular markers which are obviously related to phenotypic variation of target traits, and further analyzes the genetic effect of the sites/molecular markers on phenotypes (Tansxiajie et al 2011). With the development of high-density SNP chips (such as wheat 660K, 90K, 55K and the like), the association analysis method can efficiently detect all loci influencing target characters and molecular markers closely linked with the loci.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a CAPS mark related to wheat seedling stage frost resistance and a method for identifying the wheat seedling stage frost resistance by using the CAPS mark.
The method mainly uses a natural population consisting of 125 wheat varieties with rich genetic background as a test material, carries out genotype scanning on the natural population by using a high-density 90K SNP chip, carries out field seedling frost resistance identification on the natural population at three stages of 12-17 days in 2015, 19 days in 2016, 1-19 days in 2016 and 16 days in 2016, adopts TASSEL 5.0 software to carry out correlation analysis of genotype and phenotype, and as a result, the natural population is remarkably correlated to a new seedling frost-resistant locus on a 2AL chromosome (712.3Mb), and further develops a CAPS marker (CAPS-Ta-2-4) based on SNP (wsnp _ BG605368A _ Ta-2-4) marker information closely linked with the locus, and verifies that the locus/marker is remarkably correlated to frost resistance by using the natural population consisting of 403 wheat varieties.
The invention is realized by the following technical scheme:
the invention provides a CAPS marker related to wheat seedling stage frost resistance, which is named as CAPS-Ta-2-4, and the nucleotide sequence of the CAPS marker is as follows: SEQ ID NO. 1.
The invention also provides a primer pair for detecting the CAPS marker, wherein the nucleotide sequence of the primer pair is as follows:
F：ATCCATCCGATTTCGTTAC SEQ ID NO.2；
R：GTGTTTGGGCTGTTCTCC SEQ ID NO.3。
the invention also provides a method for identifying the frost resistance of wheat seedlings by using the CAPS markers, which comprises the following steps:
step 1, extracting wheat genome DNA to be detected
Step 2, carrying out PCR amplification on the wheat genome DNA obtained in the step 1 by using the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplification product;
step 3, carrying out enzyme digestion on the amplification product by utilizing BtsaI endonuclease to obtain an enzyme digestion product
Step 4, when the enzyme digestion products are 185bp and 70bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-A, the average field freezing injury grade of the corresponding wheat seedling stage is less than or equal to 2.2, and the wheat seedling stage has stronger frost resistance; when the enzyme digestion product is 255bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-G, the average field freezing injury grade of the corresponding wheat seedling stage is more than or equal to 2.6, and the wheat seedling stage is weak in freezing resistance.
Compared with the prior art, the invention has the following advantages:
the method utilizes correlation analysis to identify a new locus which is obviously related to wheat seedling stage frost resistance on a wheat 2AL chromosome, an SNP marker which is tightly linked with the new locus is wsnp _ BG605368A _ Ta _2_4, a CAPS marker CAPS-Ta-2-4 is further developed based on the marker, and then a natural population consisting of 403 wheat varieties is utilized to verify that the new locus/marker is tightly related to wheat seedling stage frost resistance. The new locus identified by the invention lays an important foundation for cloning candidate genes, and the developed CAPS marker can provide a molecular marker for the genetic improvement of wheat freezing injury resistance.
Drawings
FIG. 1 is an electrophoretic test of CAPS-labeled CAPS-Ta-2-4 in wheat varieties with different freezing resistance.
In the figure: a represents BtsaI before enzyme digestion; b represents BtsaI after enzyme digestion; 1, 1023 middle education; 2, Yumai No. 9; 3, RL 6077; 4, Wanmai 38; 5, Huai Mai 0226; 6,03G 7; 7, TupatecoR M, Marker I.
Detailed Description
1. Identification of wheat seedling stage antifreeze phenotype
Continuous low-temperature weather appears in Anhui Huaibei in 2015 12-2016 2 months. According to the method, 403 wheat varieties planted in five-spread farms in Huaibei are identified by Freezing injury (FT) in seedling stage in three stages of 12-17 days in 2015, 1-19 days in 2016 and 16 days in 2 months, and the identification is marked as 15FT, 16FT-1 and 16FT-2 respectively. The average freezing phenotype value of the three periods is calculated and is marked as FT-M. The wheat field freezing injury grade record refers to the agricultural industry standard of the people's republic of China, and is divided into 5 grades:
Figure BDA0001713046350000031
2. extraction of wheat genome DNA by SDS-Tris saturated phenol method
(1) 2-3 wheat seeds were placed in a 2ml sterile centrifuge tube and ground to powder using an MP high throughput tissue grinder.
(2) Adding 1.2ml of DNABuffer (200mM Tris-Cl,250mM N)aCl,25mM EDTA,0.5％SDS,2％β-ME)。
(3)60 deg.C water bath for 45min, with intermittent shaking for sufficient extraction.
(4) Centrifuge at 12000rpm for 10min at room temperature.
(5) Transferring the supernatant to a new 2ml sterilized centrifuge tube, adding precooled equal volume of Tris saturated phenol/chloroform iso/pentanol (volume ratio 25: 24: 1), and mixing thoroughly on ice for 15min with intermittent shaking.
(6) Centrifuge at 12000rpm for 10min at room temperature.
(7) The supernatant was transferred to a new 2ml sterile centrifuge tube and steps (5), (6) were repeated to remove the protein sufficiently.
(8) Transferring the supernatant to a new 1.5ml sterilized centrifuge tube, adding 0.6 time isopropanol (300. mu.l) and 1/10 times volume (50. mu.l) NaAc (pH5.2), mixing well, standing on ice for 17min to precipitate DNA white precipitate.
(9) Centrifuging at 4 deg.C 10000rpm for 10 min.
(10) The supernatant was discarded, and the supernatant was rinsed 2 times with pre-cooled 70% ethanol, then 1 time with absolute ethanol, air-dried at room temperature, and dissolved overnight in 100. mu.l of 1 XTE buffer (or double distilled water) containing 2. mu.l of 10mg/ml RNase enzyme.
(11) The DNA concentration was measured on a NanoVue Plus microspectrophotometer and diluted uniformly to 50ng/ul of working solution for future use.
3. CAPS primer design, PCR amplification and detection
CAPS-Ta-2-4 primers (F: ATCCATCCGATTTCGTTAC; R: GTGTTTGGGCTGTTCTCC) were designed using Primer premier5.0 software. The CAPS marker PCR amplification system was 10. mu.L, including 10 XBuffer (containing 2.0mmol L-1 Mg)2+) mu.L of 1.0. mu.L, 0.8. mu.L of 2.5mmol L-1dNTPs, 0.1. mu.L of 5U. mu.L-1 EasyTaq DNA polymerase, 0.4. mu.L of 10. mu. mol L-1 upstream and downstream primers, 2.0. mu.L of template DNA (50-60 ng. mu.L-1), and 5.3. mu.L of ddH2O 5.3. Setting a PCR program: pre-denaturation at 94 ℃ for 5min, 35 cycles (denaturation at 94 ℃ for 30S, annealing at 57 ℃ for 30S, and extension at 72 ℃ for 30S), extension at 72 ℃ for 10min, and storage at 4 ℃. The PCR product was digested with BtsaI endonuclease (cleavage site: N/CACTG), and the digestion time and method were described in the website (http:// www.n)Com). The enzyme digestion product was analyzed and detected by 2.0% agarose gel electrophoresis, stained with GelStain fluorescent dye, and scanned and photographed by a BIO-RAD gel imaging system, as shown in FIG. 1.
4. Verification of antifreeze phenotype effect of CAPS marker on wheat seedling stage
Carrying out genotype analysis on a natural population consisting of 403 parts of wheat varieties by using CAPS-Ta-2-4 markers, wherein two genotypes of CAPS-Ta-2-4-A and CAPS-Ta-2-4-G coexist, wherein a material (containing A basic group) carrying the CAPS-Ta-2-4-A band type has stronger frost resistance, an amplification product can be cut by BtsaI endonuclease (the cutting site is N/CACTG), and 185bp and 70bp products are generated (wherein the fragment of the 70bp product is smaller, and an obvious band cannot be detected by agarose electrophoresis); the material carrying CAPS-Ta-2-4-G band type (containing G base) has weak freezing resistance, and an amplification product cannot be cut by BtsaI endonuclease to generate a 255bp product. the t-test result shows that in 403 wheat varieties, the difference of the varieties carrying two allelic genotypes in the antifreeze phenotype reaches an extremely significant level (P is less than 0.01) (Table 1), and the site/marker is verified to be significantly related to the antifreeze property of the wheat in seedling stage.
TABLE 1 test for freezing resistance between wheat varieties carrying two different allelic types
Figure BDA0001713046350000051
Note: 0.01 significant level
The method utilizes correlation analysis to identify a new locus which is obviously related to wheat seedling stage frost resistance on a wheat 2AL chromosome, an SNP marker which is tightly linked with the new locus is wsnp _ BG605368A _ Ta _2_4, a CAPS marker CAPS-Ta-2-4 is further developed based on the marker, and then a natural population consisting of 403 wheat varieties is utilized to verify that the new locus/marker is tightly related to wheat seedling stage frost resistance. The new locus identified by the invention lays an important foundation for cloning candidate genes, and the developed CAPS marker can provide a molecular marker for the genetic improvement of wheat freezing injury resistance.
Sequence listing
<110> agriculture university of Anhui
<120> wheat seedling stage frost resistance related CAPS mark and method for identifying wheat seedling stage frost resistance
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 255
<212> DNA
<213> wheat (Triticum aestivum L)
<220>
<221> misc_feature
<222> (187)
<223> n is a or g
<400> 1
atccatccga tttcgttaca cataactgga atatcatctt gcacttgatc acttcaccgg 60
cagtcactaa gtatgtgacg atccacatgt ctaaattgat ggagtgcaga gcgtgctgtt 120
cgtgaacaac cagaacacgg tggtgcgcga catcacgtcg gtaaacagca agttcttcca 180
catcgcnctg ctgcagagta agaatatgaa gttgatcaac atccagatca acgcgccgga 240
gaacagccca aacac 255
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Synthetic sequence)
<400> 2
atccatccga tttcgttac 19
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence (Synthetic sequence)
<400> 3
gtgtttgggc tgttctcc 18

Claims (1)

1. The method for identifying the frost resistance of the wheat seedling by using the CAPS marker related to the frost resistance of the wheat seedling is characterized in that the CAPS marker is named as CAPS-Ta-2-4, and the nucleotide sequence of the CAPS marker is as follows: SEQ ID NO.1, wherein the nucleotide sequence of the primer pair for detecting the CAPS marker is shown as SEQ ID NO.2 and SEQ ID NO. 3;
the method for identifying the freezing resistance of the wheat seedlings comprises the following steps:
step 1, extracting a wheat genome DNA to be detected;
step 2, carrying out PCR amplification on the wheat genome DNA obtained in the step 1 by using the primer sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplification product, wherein the nucleotide sequence of the amplification product is SEQ ID NO.1, and n in the sequence of the SEQ ID NO.1 is a or g;
step 3, carrying out enzyme digestion on the amplification product by using BtsaI endonuclease to obtain an enzyme digestion product;
step 4, when the enzyme digestion products are 185bp and 70bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-A, the average field freezing injury grade of the corresponding wheat seedling stage is less than or equal to 2.2, and the wheat seedling stage has stronger frost resistance; when the enzyme digestion product is 255bp, the genotype of the corresponding amplification product is named CAPS-Ta-2-4-G, the average field freezing injury grade of the corresponding wheat seedling stage is more than or equal to 2.6, and the wheat seedling stage is weak in freezing resistance.
CN201810693327.XA 2018-06-29 2018-06-29 Wheat seedling stage frost resistance related CAPS (cleaved amplified polymorphic sequence) marker and method for identifying wheat seedling stage frost resistance Active CN108913798B (en)

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