CN116606916B - Method for identifying, screening or controlling wheat spike number per spike based on SNP locus - Google Patents
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Abstract
The application discloses a method for identifying, screening or controlling wheat spike number per spike based on SNP locus, which realizes related operation on wheat spike number per spike phenotype based on specific genotype of single nucleotide polymorphism locus named A583G in wheat genome. The application provides a new method for molecular marker assisted selective breeding of wheat, and has important significance in scientific research and practice of cultivating high-yield wheat varieties.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a wheat phenotype screening and identifying method based on a single nucleotide polymorphism site of wheat, which can be applied to wheat breeding scientific research and practice.
Background
Wheat (Triticum aestivum l.) is one of the most important food crops in our country. The plant type plays a role in the yield, quality and light energy utilization rate of the plant, and the plant height, spike shape, tillering number and tillering angle play an important role in plant type composition. The improvement of the ear shape is an important way of ultrahigh-yield breeding, the ear shape is one of important indexes of parent selection and offspring screening, and the structure of the ear determines the yield of wheat. Therefore, the agronomic characters of the wheat ears are improved, and the improvement of the agronomic characters plays an important role in improving the yield.
In the current research, studies on ear development in wheat have focused mainly on genetic localization and marker development. Hu et al used the Chinese wheat variety Yanzhan 1 as a common parent to construct RIL populations for QTL analysis, found and verified 8 stable QTLs, and also verified the small ear per spike QTL (QSPS-2A.4) in natural populations. Li et al construct a DH population of Kechengmai1/Chuanmai42 and a high-density genetic map through a wheat 55K SNP chip for spike QTL detection. 27 QTLs were detected that correlated with total number of spikelets per ear and number of fertile spikelets per ear. Wherein QTsn/fsn. Cib-3D is a co-located QTL for total and fertile spikelet numbers, explained phenotypic variation between 5.97-23.28%, and further validated the effect of this QTL in two populations of different genetic backgrounds using the developed KASP marker kasp_ax-110914105. And 3 new QTLs controlling wheat spike number were identified on wheat 2B and 2D chromosomes using 55K chips, accounting for 19.59% -26.57% of the phenotypic variation. However, since most QTLs have a small phenotypic contribution rate and poor repeatability between years and environments, QTLs are difficult to apply to genetic improvement of wheat spikelet numbers per spike.
CAPS markers are also called PCR-RFLP (restriction fragment length polymorphism polymerase chain reaction), are co-dominant molecular markers based on PCR, and reveal restriction length variation information of specific PCR fragments, the basic principle is that target DNA is amplified by PCR, and amplified products are digested and cut into fragments with different sizes by specific endonucleases, and are directly resolved on gel electrophoresis. Restriction enzyme sites of different alleles are distributed differently, resulting in DNA fragment bands of different lengths. The method has the advantages of avoiding complicated transfer and hybridization steps in RFLP analysis and maintaining the accuracy of RFLP analysis. However, since there are fewer cases where the SNP is located exactly at the restriction enzyme site, a dCAPS marker has been proposed, that is, a polymorphism similar to the CAPS marker is generated by introducing mismatched bases into the amplification primer on the basis of the CAPS marker, and introducing a new restriction enzyme action site in combination with the SNP site.
At present, development of single nucleotide polymorphism sites related to wheat phenotypes, especially wheat spike-per-spike number type is needed urgently, and important scientific research and application values are achieved.
Disclosure of Invention
The application aims to provide a method for identifying, screening or controlling the number of small ears per ear of wheat based on SNP loci.
In order to solve the technical problems, the technical scheme adopted by the application is as follows.
A breeding method for controlling the number of wheat ears per ear based on the specific genotype of single nucleotide polymorphism site in wheat genome.
As a preferred embodiment of the present application, the single nucleotide polymorphism site corresponds to the 583 base at the 5' -end of the sequence shown in SEQ ID NO. 1.
As a preferable technical scheme of the application, when the nucleotide at the single nucleotide polymorphism site is G/G homozygous, the wheat is corresponding to genotype I; the nucleotide at the single nucleotide polymorphism site is A/A homozygous and corresponds to genotype II wheat; the number of spikes per ear of genotype I wheat is less than the number of spikes per ear of genotype II wheat.
The method for identifying the wheat spike number per spike based on the SNP locus comprises the following steps: carrying out PCR amplification and identification on any section of DNA fragment containing the SNP locus in the genome DNA of the wheat to be detected; the SNP locus corresponds to the 583 base from the 5' end of the sequence shown in SEQ ID NO. 1; then determining the genotype of the wheat to be tested based on the amplification product in the step A; and finally, identifying the spike number character of the wheat to be tested according to the genotype of the wheat to be tested.
As a preferred technical solution of the present application, the method comprises the steps of:
A. carrying out PCR amplification on any section of DNA fragment containing the following SNP loci in the genome DNA of the wheat to be detected, and carrying out enzyme digestion on the PCR amplification product; the SNP locus corresponds to the 583 base from the 5' end of the sequence shown in SEQ ID NO. 1;
B. determining the genotype of the wheat to be detected, wherein when the nucleotide at the SNP locus is G/G homozygous, the corresponding genotype is I; when the nucleotide at the SNP locus is A/A homozygous, the corresponding genotype is II; determining the spike number character of the wheat to be tested according to the genotype of the wheat to be tested and the following standard: the number of spikes per ear of genotype I homozygous wheat is less than/the candidate is less than the number of spikes per ear of genotype II homozygous wheat.
As a preferred technical scheme of the application, in the step A, the specific primer pair for PCR amplification is primer pair 1F and 1R composed of SEQ ID NO.2 and SEQ ID NO.3, and/or primer pair 2F and 2R composed of SEQ ID NO.4 and SEQ ID NO. 5; the amplification and cleavage comprises the steps of: taking wheat genome DNA as a template and taking primers 1F and 1R as primer pairs for amplification to obtain a PCR product; the PCR product is used as a template, and primers 2F and 2R are used as primer pairs for amplification to obtain the PCR product; and (3) performing enzyme digestion by using a restriction enzyme PstI to obtain an enzyme digestion product.
In the step B, if the enzyme digestion product is a DNA fragment, the nucleotide at the SNP locus is A/A homozygous, and the wheat to be detected is genotype II; if the enzyme digestion products are two DNA fragments, the nucleotide at the SNP locus is G/G homozygote, and the wheat to be detected is genotype I.
A kit for detecting a single nucleotide polymorphism of a SNP site in the wheat genome corresponding to base 583 of the sequence shown in SEQ ID No.1 from the 5' end; the nucleotide at the SNP locus is G or A; when the nucleotide at the SNP locus is G/G homozygous, the corresponding genotype is I; the nucleotide at the SNP locus is A/A homozygous, and the corresponding genotype is II.
As a preferred embodiment of the present application, the kit comprises primer pairs 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and primer pairs 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5.
A primer pair for detecting a single nucleotide polymorphism of a SNP site in a wheat genome, wherein the SNP site corresponds to a583 base from the 5' -end of a sequence shown in SEQ ID NO. 1; the nucleotide at the SNP locus is G or A; the primer pair comprises primer pairs 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and primer pairs 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in: based on the technology of the application, the wheat with relatively high spike number per spike can be found by detecting the SNP. The application provides a new method for molecular marker assisted selective breeding of wheat, and has important significance in cultivating high-yield stable-yield wheat varieties and researching.
Drawings
FIG. 1 shows the results of amplification of 24 varieties of wheat TaJMJ12-5A genes in natural populations by using F1 and R1. The results of the detection of the control and 24 varieties of wheat are shown in the figure, and the varieties of 1-24 are Drysdale, ji Mai, ji wheat 32, ji Mai, ji wheat 41, ji wheat 6, ji wheat 9, ji wheat I, ji-to 5099, pest 26, jinguang 2148-7, jinmai 13, jinmai 17, jinmai 33, jinmai 39, jinmai 44, jinmai 47, jinmai 50, jinmai 51, jinmai 53, jinmai 54, jin Mai and Jinmai 63 in sequence.
FIG. 2 shows the results of gene detection of a portion of the wheat TaJMJ12-5A gene in the natural population. The results of the detection of the control and 24 varieties of wheat are shown in the figure, and the varieties of 1-24 are Drysdale, ji Mai, ji wheat 32, ji Mai, ji wheat 41, ji wheat 6, ji wheat 9, ji wheat I, ji-to 5099, pest 26, jinguang 2148-7, jinmai 13, jinmai 17, jinmai 33, jinmai 39, jinmai 44, jinmai 47, jinmai 50, jinmai 51, jinmai 53, jinmai 54, jin Mai and Jinmai 63 in sequence.
Detailed Description
The following examples illustrate the application in detail. The raw materials and the equipment used by the application are conventional commercial products, and can be directly obtained through market purchase. It should be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It should also be understood that the term "and/or" as used in the present specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations. As used in the present description and the appended claims, the term "if" may be interpreted as "when..once" or "in response to a determination" or "in response to detection" depending on the context. Similarly, the phrase "if a determination" or "if a [ described condition or event ] is detected" may be interpreted in the context of meaning "upon determination" or "in response to determination" or "upon detection of a [ described condition or event ]" or "in response to detection of a [ described condition or event ]". Furthermore, the terms "first," "second," "third," and the like in the description of the present specification and in the appended claims, are used for distinguishing between descriptions and not necessarily for indicating or implying a relative importance. Reference in the specification to "one embodiment" or "some embodiments" or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," and the like in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless expressly specified otherwise.
The wheat materials used in the examples below were all from the national crop germplasm library (website: http:// isccaas. Com. Cn/jiguoku/zhungzhiku. Htm) and the material information was found in the Chinese crop germplasm information network (website: http:// icgr. Caas. Net. Cn). Since wheat material is a cultivar, it is generally defaulted to highly homozygous plant material, and the genotypes are homozygous.
Example 1 discovery of A583G SNP in TaJMJ12-5A Gene of wheat and establishment of genotyping method of wheat based on TaJMJ12-5A Gene
1. Discovery of A583G SNP in wheat TaJMJ12-5A gene
Through a large number of experiments, the inventor of the application discovers a SNP locus on a wheat TaJMJ12-5A gene (the nucleotide sequence of which is shown as SEQ ID NO: 1), and the SNP locus is named as A583G SNP. The A583G SNP is located at 583 position from the 5' end of SEQ ID NO.1, and the genotypes are AA homozygous and GG homozygous. Since genomic DNA is a double-stranded DNA molecule composed of two single-stranded DNA molecules complementary in opposite directions, a DNA molecule encoding a protein is generally designated as a sense DNA molecule; the DNA molecule complementary to the reverse direction of the sense DNA molecule is designated as antisense DNA molecule. The genotypes of the A583G SNPs were all genotypes of sense DNA.
Wheat is classified into two genotypes according to the difference of the TaJMJ12-5A genes: taJMJ12-5A-G (hereinafter referred to as genotype I) and TaJMJ12-5A-a (hereinafter referred to as genotype II).
2. Primer set A and primer set B were synthesized for amplification of target sequences including the A583G SNP
Primer set a and primer set b for amplifying a target sequence including the a583G SNP were designed and synthesized. Primer pair A consists of primer F1 and primer R1. The primer pair B consists of a primer F2 and a primer R2.
The nucleotide sequences of the respective primers are specifically as follows:
primer F1:5'-GGAGGGGGACTGTCCGG-3' (SEQ ID NO: 2)
Primer R1:5'-CGGTGCGGTGTAGTCATTCGCG-3' (SEQ ID NO: 3)
Primer F2:5'-CAGGACACAACGGTTG-3' (SEQ ID NO: 4)
Primer R2:5'-ACGTCACTTCTCCTCTG-3' (SEQ ID NO: 5)
The target sequence amplified by the primer pair A is 14016-14032 from the 5' end of the TaJMJ12-5A gene (13981CAATAGGTGC ACTATAGCCA CGCCCGCCCG TCGTAGGAGG GGGACTGTCC GGTCGTCTGG 14040).
3. Establishment of genotyping method of wheat based on TaJMJ12-5A gene
1. Extracting genome DNA of wheat (Kenong 199) to be detected.
2. And (3) taking the genomic DNA of the wheat to be detected in the step (1) as a template, and carrying out PCR amplification by using a primer pair A consisting of a primer F1 and a primer R1 to obtain a PCR amplification product P1.
The reaction system was 10. Mu.L, consisting of 3.6. Mu.L of ddH 2 O, 5. Mu.L of 2 XTaq enzyme Mix, 0.2. Mu.L of an aqueous solution of primer F1 (concentration: 10. Mu. Mol/L), 0.2. Mu.LPrimer R1 aqueous solution (concentration: 10. Mu. Mol/. Mu.L), and 1. Mu.L of genomic DNA of wheat to be tested (concentration: 20 ng/. Mu.L).
2×Taq enzyme Mix is a product of Nanjinouzan corporation with a catalog number of P131.
The reaction conditions are as follows: 3min at 95 ℃; 15s at 95 ℃, 15s at 68 ℃, 1min at 72 ℃,35 cycles; 72 ℃ for 10min; preserving at 16 ℃.
3. After the step 2 is completed, a dilution of the PCR amplification product P1 (formed by mixing 1 part by volume of the PCR amplification product P1 with 9 parts by volume of water) is used as a template, and the primer pair B consisting of the primer F2 and the primer R2 is used for PCR amplification to obtain a PCR amplification product P2.
The reaction system was 10. Mu.L, consisting of 3.6. Mu.L of ddH 2 O, 5. Mu.L of 2 XTaq enzyme Mix, 0.2. Mu.L of an aqueous solution of primer F2 (concentration: 10. Mu. Mol/L), 0.2. Mu.L of an aqueous solution of primer R2 (concentration: 10. Mu. Mol/L), and 1. Mu.L of a dilution of the PCR amplification product P1.
The reaction conditions are as follows: 3min at 95 ℃; 15s at 95 ℃, 15s at 56 ℃, 15s at 72 ℃ and 35 cycles; 72 ℃ for 10min; preserving at 16 ℃.
4. Performing enzyme digestion on the PCR amplification product P2 obtained in the step 3 by using restriction enzyme PstI to obtain an enzyme digestion product; the enzyme digestion products are subjected to 4% agarose gel electrophoresis detection, and the following judgment is carried out: if the enzyme digestion product is in a band type A (two bands are shown, namely 94bp and 15bp respectively), the SNP of the wheat A583G to be detected is GG homozygote, namely the genotype of the wheat to be detected based on the TaJMJ12-5A gene is genotype I; if the enzyme digestion product is in the band type B (a band is displayed and is 109 bp), the SNP of the wheat A583G to be tested is in the AA homozygote type, namely the genotype of the wheat to be tested based on the TaJMJ12-5A gene is in the genotype II.
Example 2 analysis and verification of the correlation of the genotype of the wheat TaJMJ12-5A Gene with the wheat ear count per ear
1. Genotyping of the individual wheat TaJMJ12-5A genes in the Natural population
Genotyping was performed on each wheat variety in the natural population using the method of step three of example 1. The natural population consisted of 296 wheat varieties (hexaploids each). The names of wheat varieties are shown in Table 1.
The results of the partial detection are shown in FIG. 1 (M is DNA Marker, the other lanes are different wheat varieties, wherein G is genotype I wheat variety, and A is genotype II wheat variety).
Genotypes based on the TaJMJ12-5A gene are shown in Table 1: the genotype of 212 wheat varieties based on the TaJMJ12-5A gene is genotype I, and the genotype of 85 wheat varieties based on the TaJMJ12-5A gene is genotype II.
TABLE 1
Note that: i is genotype I and II is genotype II.
2. Detection of spike number per spike characteristics
Wheat varieties are planted in different environments respectively, and the average spike number per spike of wheat of two genotypes is counted after harvest. The statistical results are shown in Table 2.
TABLE 2
Note that: p value is the significance level of the correlation analysis, "" means P <0.05, "" means P <0.01, "" means P <0.0001.
The genotype of the wheat TaJMJ12-5A gene in the natural population and the number of spikelets per spike were subjected to correlation analysis by using a Tassel2.1 software selection single-line model+population structure (GLM+Q) method, and the results are shown in Table 2. The results show that the number of spikes per ear of "wheat of genotype I" is < "wheat of genotype II" in a natural population of 296 wheat varieties; the "<" is statistically <. The remaining three environments, although mean data type I < type II, were statistically greater than 0.05 and therefore not accounted for. Studies on natural populations have shown that genotype I is an excellent genotype for increasing the spike number per spike of wheat.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present application, and are intended to be included in the scope of the present application.
Claims (5)
1. The method for identifying the wheat spike number per spike based on the SNP locus is characterized by comprising the following steps of: the method comprises the following steps:
A. carrying out PCR amplification on any section of DNA fragment containing the following SNP loci in the genome DNA of the wheat to be detected, and carrying out enzyme digestion on the PCR amplification product; the SNP locus corresponds to the 583 base from the 5' end of the sequence shown in SEQ ID NO. 1;
B. determining the genotype of the wheat to be detected, wherein when the nucleotide at the SNP locus is G/G homozygous, the corresponding genotype is I; when the nucleotide at the SNP locus is A/A homozygous, the corresponding genotype is II; determining the spike number character of the wheat to be tested according to the genotype of the wheat to be tested and the following standard: the number of spikes per ear of genotype I homozygous wheat is less than/the candidate is less than the number of spikes per ear of genotype II homozygous wheat.
2. The method for identifying wheat spikelet number per spike based on SNP loci according to claim 1, wherein: in the step A, the specific primer pair for PCR amplification is a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5; the amplification and cleavage comprises the steps of: taking wheat genome DNA as a template and taking primers 1F and 1R as primer pairs for amplification to obtain a PCR product; the PCR product is used as a template, and primers 2F and 2R are used as primer pairs for amplification to obtain the PCR product; and (3) performing enzyme digestion by using a restriction enzyme PstI to obtain an enzyme digestion product.
3. The method for identifying wheat spikelet number per spike based on SNP loci according to claim 1, wherein: in the step B, if the enzyme digestion product is a DNA fragment, the nucleotide at the SNP locus is homozygous A/A, and the wheat to be detected is genotype II; if the enzyme digestion products are two DNA fragments, the nucleotide at the SNP locus is G/G homozygote, and the wheat to be detected is genotype I.
4. A kit for detecting a single nucleotide polymorphism of a SNP site in the wheat genome corresponding to base 583 of the sequence shown in SEQ ID No.1 from the 5' end; the nucleotide at the SNP locus is G or A; when the nucleotide at the SNP locus is G/G homozygous, the corresponding genotype is I; when the nucleotide at the SNP locus is A/A homozygous, the corresponding genotype is II;
the kit comprises primer pairs 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and primer pairs 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5;
the kit further comprises the restriction enzyme PstI.
5. A primer pair for detecting a single nucleotide polymorphism of a SNP site in a wheat genome, wherein the SNP site corresponds to a583 base from the 5' -end of a sequence shown in SEQ ID NO. 1; the nucleotide at the SNP locus is G or A; the primer pair comprises a primer pair 1F and 1R consisting of SEQ ID NO.2 and SEQ ID NO.3, and a primer pair 2F and 2R consisting of SEQ ID NO.4 and SEQ ID NO. 5.
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