CN113930537B - Wheat grain peroxidase activity associated site qPOD-6B mark - Google Patents
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- 235000021307 Triticum Nutrition 0.000 title claims abstract description 75
- 230000000694 effects Effects 0.000 title claims abstract description 69
- 102000003992 Peroxidases Human genes 0.000 title claims abstract description 60
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims abstract description 60
- 235000013339 cereals Nutrition 0.000 title claims description 10
- 239000003550 marker Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 108700028369 Alleles Proteins 0.000 claims abstract description 7
- 210000000349 chromosome Anatomy 0.000 claims abstract description 7
- 230000035772 mutation Effects 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 230000002068 genetic effect Effects 0.000 abstract description 12
- 238000009395 breeding Methods 0.000 abstract description 7
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- 235000013312 flour Nutrition 0.000 abstract description 7
- 230000009456 molecular mechanism Effects 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 3
- 235000013305 food Nutrition 0.000 abstract description 3
- 108700022207 wheat peroxidase Proteins 0.000 abstract description 3
- 238000012216 screening Methods 0.000 description 10
- 238000003205 genotyping method Methods 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 244000098338 Triticum aestivum Species 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
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- 230000000875 corresponding effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000012098 association analyses Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 238000007726 management method Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000007264 Triticum durum Nutrition 0.000 description 1
- 241000209143 Triticum turgidum subsp. durum Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 229940114124 ferulic acid Drugs 0.000 description 1
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 description 1
- 235000001785 ferulic acid Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
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- 230000001590 oxidative effect Effects 0.000 description 1
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- 235000009048 phenolic acids Nutrition 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
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- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- 235000011844 whole wheat flour Nutrition 0.000 description 1
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Abstract
The invention belongs to the field of wheat molecular breeding, and in particular relates to a peroxidase activity related site in wheat grainsqPOD‑6BAnd (5) marking. The genetic marker is named AX-109420494 and is positioned on a short arm of a wheat 6B chromosome; a mutation of an allele of G/T at base 36 of the sequence; when the 36 th base is G, the gene is a genotype with high POD activity; when the 36 th base is T, the gene is a genotype with low POD activity. The potential excellent sites and genes obviously related to the activity of the wheat peroxidase are fully excavated and positioned, and the method has important theoretical and practical significance for clarifying the POD activity genetic molecular mechanism of the wheat and developing corresponding functional markers, changing the POD activity by a genetic approach and improving the appearance quality of the flour food.
Description
Technical Field
The invention belongs to the field of wheat molecular breeding, and in particular relates to a peroxidase activity related site in wheat grainsqPOD-6BAnd (5) marking.
Background
With the advances in grain breeding technology and the development of breeding concepts, technicians gradually extend the breeding goals of wheat from high yield and disease resistance to improvement of wheat quality. Quality has become an important goal for wheat production and breeding, and is also an important factor affecting the competitiveness between industries. Among the determining factors of the quality of wheat, the color and luster of the flour and the products thereof are one of the important indexes for evaluating the quality of wheat, and are also important factors for restricting the quality of flour and products thereof in China, and the color improvement is an important content of the genetic improvement of the quality of wheat at present.
Peroxidase (POD) as an oxidative active substance tends to have a relatively visual and pronounced effect on the relevant color phenotype. The method takes hydrogen peroxide as a substrate in plant tissues to catalyze oxidation reactions of various reducing agents: RH (relative humidity) 2 +H 2 O 2 →2H 2 O+R. In particular to wheat, peroxidase (POD) in wheat grains can catalyze the oxidation of main phenolic acids such as ferulic acid and generate chromophoric groups, which is one of the main reasons that flour and flour products often have double changes of browning and bleaching in the processing and storage processes.
Although environmental factors have some influence on the POD activity, the POD activity is still controlled to some extent mainly by genetic factors. For example, some statistics show that POD activity of common wheat is significantly higher than durum wheat (P < 0.05), and that POD activity varies 3-10 times in different varieties of wheat kernels. Therefore, the genetic molecular mechanism for regulating POD activity is deeply researched from genetic approaches, and the method has important scientific theory and practical application significance for cultivating new wheat varieties and regulating the appearance quality of the final flour food.
Disclosure of Invention
On the basis of analysis and analysis of whole genome association of wheat, the application aims to provide a series of genetic markers related to POD activity of peroxidase in wheat grains, so as to lay a certain technical foundation for research on molecular mechanism of POD activity regulation in the wheat grains and cultivation of new wheat varieties finally.
The technical scheme adopted by the application is described in detail below.
Wheat kernel peroxidase activity associated siteqPOD-6BA marker, the genetic marker being designated AX-109420494, located on the short arm of the wheat 6B chromosome and having the nucleotide sequence:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAA[G/T]CTTCACCAAACCTCGCCTTCCGACAAGAACCAATT；
a mutation of an allele of G/T at base 36 of the sequence; when the 36 th base is G, the genotype with high POD activity is obtained, and the base sequence (71 bp) is shown as SEQ ID No.1, and is specifically as follows:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAAGCTTCACCAAACCTCGCCTTCCGACAAGAACCAATT；
when the 36 th base is T, the genotype with low POD activity is obtained, and the base sequence (71 bp) is shown in SEQ ID No.2, and the specific steps are as follows:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAATCTTCACCAAACCTCGCCTTCCGACAAGAACCAATT。
related site for peroxidase activity of wheat seedsqPOD-6BA marker, a KASP marker developed based on the polymorphism (nucleotide polymorphism) of the genetic marker, can be effectively used for detection and judgment of POD activity in wheat grains;
the specific KASP marker primer sequences were designed as:
AX-109420494-F1:
5’-GAAGGTGACCAAGTTCATGCT-CAAGCCACTCCCCACCAAGTACAAT-3’，
AX-109420494-F2:
5’-GAAGGTCGGAGTCAACGGATT-CAAGCCACTCCCCACCAAGTACAAG-3’，
AX-109420494-R12:
5’-GAAAATTGGTTCTTGTCGGAAGGCGA-3’；
the specific application is as follows:
AX-109420494-F1, when combined with AX-109420494-R12, is used to identify low POD activity wheat of genotype T;
AX-109420494-F2, when combined with AX-109420494-R12, was used to identify high POD activity wheat of genotype G.
The KASP marker is applied to wheat breeding and is used for distinguishing the POD activity, and particularly, when the KASP marker is used for detection, the POD activity of the wheat variety with the genotype G is obviously higher than that of the wheat variety with the genotype T; that is, genotype G is an excellent allele of POD activity in wheat kernels and can be used to screen wheat varieties with high POD activity.
In the early research process, the inventor utilizes the natural population constructed in the early stage and combines 660K SNP chip genotype data to obtain 224706 effective SNPs marker loci through preliminary screening. On the other hand, the inventors performed phenotypic measurement of POD activity on 207 natural population wheat varieties planted 3-year-old within 1 year using spectrophotometry, and performed Genome-wide association analysis (Genome-wide association study) on POD activity in wheat kernels by using a "Q+K" mixed linear model. Results screening of SNPs marker loci from the previous screening gave a significant association of AX-109420494 markers on the 6B chromosome short arm. Sequence analysis showed that base 36 of the tag sequence was an allelic mutation of G/T, which resulted in nucleotide polymorphism in the sequence. Genotyping analysis was performed using polymorphisms at this site, and when the base at this site was G, POD activity in wheat whole wheat flour was higher.
Based on the sequence analysis result, the inventor further develops and designs a KASP marker, and can effectively screen wheat with high POD activity and G genotype by using the marker, thereby laying a certain technical foundation for the cultivation of new varieties of wheat.
In general, it has been considered that genes controlling POD activity are located on a plurality of chromosomes such as wheat 2A, 2B, 2D, 4B, 7D and 7A. Therefore, the method fully digs and locates the potential excellent sites and genes obviously related to the activity of the wheat peroxidase, and has important theoretical and practical significance for clarifying the POD activity genetic molecular mechanism of the wheat and developing corresponding functional markers, and for changing the POD activity by a genetic approach method and improving the appearance quality of the flour food.
Drawings
FIG. 1 shows BLUP values for POD Activity under different circumstancesManhattan plot of SNP distribution on wheat genome: the dotted line is-log 10 PA threshold line of =3;
FIG. 2 is a KASP marker genotyping chart: FAM (lower right circle) fluorescence shows that the genotype is T; HEX fluorescence (upper left) shows that the genotype is G; fluorescence corresponding to the right triangle shows that the fluorescent probe is heterozygous at the locus; fluorescence corresponding to the lower left square is a blank control without template DNA;
FIG. 3 is a histogram of POD activity distribution for the KASP marker genotyping of common wheat:
FIG. 4 is a box plot of POD activity distribution for the KASP marker genotyping of common wheat: * Representation ofP<0.05。
Detailed Description
The present application is further illustrated below with reference to examples. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.
Partial early experimental background:
in the early research work, the inventor constructs a natural population based on different wheat varieties planted in 3 places (Yuan Yang (YY), zhu (SQ) and Kai (KF)) in the same year, and after sowing, management and harvesting according to a normal management mode, based on the natural population, the inventor preliminarily screens and identifies effective SNPs in the natural population by using 660K SNP chip genotype data to obtain 224,706 marking sites. The relevant marker loci in this application are within this screening range.
Further, the inventors measured the POD activity phenotype of 207 wheat varieties in natural populations by spectrophotometry, and based on these measurements and the aforementioned SNPs locus screening results, the inventors performed genome-wide association analysis of SNPs loci correlated with POD activity in wheat kernels using a "Q+K" mixed linear model, in order to screen for potentially superior loci and genes significantly correlated with wheat peroxidase activity.
Example 1
In the screening process, adopting STRUCTURE to calculate the group STRUCTURE of wheat variety and utilizing Bayesian model to divideAnd (3) carrying out group linkage disequilibrium analysis by adopting a TASSEL (total internal framework) according to the group structure. At a threshold value set to-log 10 PIn the case of=3, and with SNP sites detected under different environments as significant sites, some of the results of the studies are summarized in fig. 1 and 2.
Analysis shows that the AX-109420494 marker related to significance is obtained by screening on a 6B chromosome short arm, and 6.90-7.47% of phenotypic variation can be explained.
Further sequence analysis results show that the 36 th base of the marker sequence has a G/T allele mutation, and the specific nucleotide sequence is as follows:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAA[G/T]CTTCACCAAACCTCGCCTTCCGACAAGAACCAATT；
specifically:
when the 36 th base is G, the gene is of high POD activity, as shown in AGTGACTTCATCAAGCCACTCCCCACCAAGTACAAGCTTCACCAAACCTCGCCTTCCGACAAGAACCAATT of SEQ ID No.1
When the 36 th base is T, the genotype with low POD activity is shown as SEQ ID No. 2:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAATCTTCACCAAACCTCGCCTTCCGACAAGAACCAATT。
example 2
Based on the nucleotide sequence of the SNP locus AX-109420494 marker determined in the screening in the embodiment 1, the inventor further designs a KASP marker, combines the genotyping analysis result of part of common wheat varieties collected by the inventor and the POD activity of wheat materials with different genotypes to perform further function verification and screening of the wheat varieties, thereby laying a certain foundation for improving the quality of the wheat. The detailed experimental procedure is outlined below.
Primer design
The primers were designed for this marker (AX-109420494) sequence as follows:
AX-109420494-F1:
5’-GAAGGTGACCAAGTTCATGCT-CAAGCCACTCCCCACCAAGTACAAT-3’，
AX-109420494-F2:
5’-GAAGGTCGGAGTCAACGGATT-CAAGCCACTCCCCACCAAGTACAAG-3’，
AX-109420494-R12:
5’-GAAAATTGGTTCTTGTCGGAAGGCGA-3’。
(II) genotyping detection
First, 3 primers each having a concentration of 10umol/L were mixed with water at a ratio of 12 (AX-109420494-F1): 12 (AX-109420494-F2): 30 (AX-109420494-R12): 46 (H) 2 O) mixing to obtain a Primer Mix;
then, PCR reaction is carried out, and a 10 mu l reaction system is designed as follows:
2×KASP Master Mix，5ul；
Primer Mix，1.4ul；
MgCl 2 ，0.08ul；
DNA（100ng/ul），1ul；
H 2 O，2.52ul；
the reaction procedure is: 95 ℃ for 15min;95 ℃ for 20 seconds, 65 ℃ to 55 ℃ and 1min for 10 cycles (each cycle is reduced by 1 ℃); 95 ℃,20 s,57 ℃, 1min,30 cycles; 37 ℃ for 1min; genotyping was performed after the end of the reaction.
207 parts of the common wheat material collected by the inventors were genotyped using the above-mentioned KASP marker and reaction system, reaction program. The specific genotyping results, i.e., POD activity results, are shown in FIG. 3 and Table 1 below.
TABLE 1 KASP marker genotyping and POD Activity distribution in 207 parts wheat Material
Continuing the table:
continuing the table:
(note that the detection result of SNPs of Taishan No. 5 is doubtful, so this is indicated by ".
The results of table 1 above were further statistically categorized (excluding the "thai mountain No. 5" results) and the results are shown in table 2 below.
TABLE 2 comparative analysis of POD Activity between different genotypes of AX-109420494 in wheat Material
。
As can be seen in connection with FIG. 3, both allelic types are evident, which also demonstrates that the markers are effective in segregating the allelic types in different wheat varieties. Namely:
AX-109420494-F1, when combined with AX-109420494-R12, is used for identifying wheat with low POD activity of genotype T;
AX-109420494-F2 was used in combination with AX-109420494-R12 to identify wheat having high POD activity of genotype G.
Further combining the statistical results of tables 1 and 2, it can be seen from the analysis that the wheat material of genotype G accounts for about 47%, and 52% of the wheat material contains genotype T. POD activity concentration in wheat variety with genotype G is 410-1301U.min -1 ·g -1 In between, POD activity in wheat varieties with genotype T is concentrated at 366.93-1236.71 U.min -1 ·g -1 Between them. As can be seen from the results of FIG. 4, the POD activity of the wheat material with the marker genotype G is high; the POD activity in the wheat material with the genotype T is low, and the POD activity between the two genotypes reaches a significant difference level.
In view of the above, it can be considered that: the wheat variety with the genotype G has the POD activity remarkably higher than that of the wheat variety with the genotype T, which shows that the genotype G is an excellent allele for increasing the POD activity of wheat seeds, and the marker can be further verified to be used for screening the wheat variety with high POD activity and improving the quality character of the wheat.
SEQUENCE LISTING
<110> academy of agricultural sciences in Henan province
<120> wheat grain peroxidase Activity-associated site qPOD-6B marker
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 71
<212> DNA
<213> Triticum aestivum L.
<400> 1
agtgacttca tcaagccact ccccaccaag tacaagcttc accaaacctc gccttccgac 60
aagaaccaat t 71
<210> 2
<211> 71
<212> DNA
<213> Triticum aestivum L.
<400> 2
agtgacttca tcaagccact ccccaccaag tacaatcttc accaaacctc gccttccgac 60
aagaaccaat t 71
Claims (2)
1. Wheat kernel peroxidase activity associated siteqPOD-6BThe application of the marker in wheat cultivation is characterized in that the wheat grain peroxidase activity related sitesqPOD-6BThe marker is named AX-109420494, is positioned on a short arm of a wheat 6B chromosome, and has the nucleotide sequence as follows:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAA[G/T]CTTCACCAAACCTCGCCTTCCGACAAGAACCAATT；
a mutation of an allele of G/T at base 36 of the sequence; when the 36 th base is G, the gene is of a genotype with high POD activity, and the base sequence is shown as SEQ ID No. 1;
when the 36 th base is T, the genotype with low POD activity is obtained, and the base sequence is shown as SEQ ID No. 2.
2. Related site for peroxidase activity of wheat seedsqPOD-6BLabeled KASP markers in wheat kernel POD activityUse in a test, characterized in that the KASP tagged primer sequence is designed to:
AX-109420494-F1:
5’-GAAGGTGACCAAGTTCATGCT-CAAGCCACTCCCCACCAAGTACAAT-3’，
AX-109420494-F2:
5’-GAAGGTCGGAGTCAACGGATT-CAAGCCACTCCCCACCAAGTACAAG-3’，
AX-109420494-R12:
5’-GAAAATTGGTTCTTGTCGGAAGGCGA-3’；
when in application, the method comprises the following steps:
AX-109420494-F1, when combined with AX-109420494-R12, is used to identify low POD activity wheat of genotype T;
AX-109420494-F2, when combined with AX-109420494-R12, is used to identify high POD activity wheat of genotype G;
the wheat grain peroxidase activity related siteqPOD-6BThe marker is named AX-109420494, is positioned on a short arm of a wheat 6B chromosome, and has the nucleotide sequence as follows:
AGTGACTTCATCAAGCCACTCCCCACCAAGTACAA[G/T]CTTCACCAAACCTCGCCTTCCGACAAGAACCAATT；
a mutation of an allele of G/T at base 36 of the sequence; when the 36 th base is G, the gene is of a genotype with high POD activity, and the base sequence is shown as SEQ ID No. 1;
when the 36 th base is T, the genotype with low POD activity is obtained, and the base sequence is shown as SEQ ID No. 2.
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| CA3049172A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
| CN111118208A (en) * | 2020-03-06 | 2020-05-08 | 河南省农业科学院 | Molecular marker of wheat grain zinc content QTL qZn-5A |
| CN111733281A (en) * | 2020-08-06 | 2020-10-02 | 新疆农业大学 | Molecular marker for identifying peroxidase activity of wheat grains and application thereof |
| CN112266975A (en) * | 2020-11-27 | 2021-01-26 | 山东省农业科学院作物研究所 | Primer group and kit for detecting KASP (Kaempferi-N-linked immunosorbent assay) marker related to POD (peroxidase) activity of wheat grains and application of primer group and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3049172A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
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