CN112266975B - Primer group and kit for detecting KASP marker related to POD activity of wheat grains and application of kit - Google Patents

Primer group and kit for detecting KASP marker related to POD activity of wheat grains and application of kit Download PDF

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CN112266975B
CN112266975B CN202011356893.5A CN202011356893A CN112266975B CN 112266975 B CN112266975 B CN 112266975B CN 202011356893 A CN202011356893 A CN 202011356893A CN 112266975 B CN112266975 B CN 112266975B
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pod
wheat
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wheat grains
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CN112266975A (en
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翟胜男
刘建军
李豪圣
曹新有
刘成
宋健民
刘爱峰
程敦公
赵振东
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CROP Research Institute of Shandong Academy of Agricultural Sciences
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract

The invention provides a primer group for detecting an Excalibur _ c95720_329-KASP marker related to the POD activity of wheat grains, a kit and application, and belongs to the technical field of molecular genetic breeding. The Excalibur _ c95720_329-KASP labeled primer group for detecting the POD activity of the wheat grains provided by the invention has a good typing effect, can be used for quickly detecting the POD-2D allele, and provides a molecular tool and a theoretical basis for efficiently and accurately improving the POD activity of the wheat grains by utilizing molecular marker-assisted breeding.

Description

Primer group and kit for detecting KASP marker related to POD activity of wheat grains and application of kit
Technical Field
The invention belongs to the technical field of molecular genetic breeding, and particularly relates to a primer group for detecting an Excalibur _ c95720_329-KASP marker related to POD activity of wheat grains, a kit and application.
Background
Wheat is one of the most important food crops in the world, and is the staple food for about 35% of the population. With the improvement of living standard, quality improvement becomes an important target of wheat breeding in China, and the cultivation and popularization of high-quality wheat is an important content of the current agricultural supply-side innovation. The color of the flour and flour products is an important index for wheat quality evaluation, and has important influence on the quality of traditional foods such as noodles, steamed bread and the like. The gluten network structure determines the physicochemical and rheological properties of the dough, significantly affecting the flour processing quality.
Peroxidases (PODs) are a large family of proteins that are widely found in plants, animals, fungi, bacteria and yeasts. Dozens to hundreds of PODs are generally present in the same plant and participate in a series of growth and development processes from germination to apoptosis of the plant, such as seed germination, auxin metabolism, lignification and suberization, pest protection, stress resistance and the like.
POD is present in various parts of wheat grain, such as the epidermis, seed coat, embryo and endosperm. The POD activity of wheat grains is relatively high and is 3, 6 and 7 times of that of oats, rice and corns respectively. The POD of the wheat grains has double functions of browning and bleaching on flour and the color of products thereof, and the bleaching function is greater than the browning function. In the process of flour processing, POD catalyzes tyrosine crosslinking and arabinoxylan ferulic acid crosslinking among gluten proteins or increases protein and arabinoxylan crosslinking, thereby improving the gluten network structure and improving the flour processing quality. POD has been widely used as a biological food additive for bleaching dough, improving gluten strength, increasing bread volume, and the like. Therefore, basic research on the genetic activity of the POD of the wheat grains is strengthened, the endogenous POD activity of the wheat grains is improved through genetic improvement, the POD additive is replaced, the most economical, effective, healthy and safe way for obtaining the color and luster and the processing quality of the ideal flour product is provided, and important theoretical and practical significance is achieved.
The previous researches on the wheat POD mainly focus on the aspects of pathogenic bacteria defense and abiotic stress response, the basic researches on the activity inheritance of the wheat seed POD are less, only a few genes influencing the activity of the wheat seed POD are cloned, and the genetic improvement of the wheat quality and the molecular marker-assisted breeding process are restricted. Therefore, a method for rapidly detecting the POD activity of the wheat grains by utilizing molecular marker-assisted breeding is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides a primer group for detecting an Excalibur _ c95720_329-KASP marker related to the POD activity of wheat grains, a kit and application. The primer provided by the invention has a good group forming effect, can be used for rapidly detecting POD-2D alleles, and provides a molecular tool and a theoretical basis for efficiently and accurately improving the POD activity of wheat grains by utilizing molecular marker-assisted breeding.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer group for detecting an Excalibur _ C95720_329-KASP marker related to POD activity of wheat grains, which comprises a primer A, a primer B and a primer C;
the nucleotide sequence of the primer A is shown as SEQ ID NO. 1;
the nucleotide sequence of the primer B is shown as SEQ ID NO. 2;
the nucleotide sequence of the primer C is shown as SEQ ID NO. 3.
The invention provides a kit for detecting an Excalibur _ c95720_329-KASP marker related to POD activity of wheat grains, which comprises a primer group, KASP2 XMasterMix and water in the technical scheme.
The invention provides a method for detecting the POD activity of wheat grains based on the primer group in the technical scheme or the kit in the technical scheme, which comprises the following steps: taking the genomic DNA of the wheat to be detected as a template, and carrying out PCR amplification by using the primer group in the technical scheme to obtain an amplification product, and typing the wheat grains; and (3) judging the POD activity of the wheat grains according to the parting result, wherein the genotypes corresponding to the POD activity of the wheat grains from high to low are GG, GA and AA respectively.
Preferably, the site corresponding to the genotype is nucleotide 4031 in the POD-2D gene.
Preferably, every 5 μ L of the reaction system for PCR amplification comprises: 2 μ L of template DNA, 0.05 μ L of primer working solution, KASP2 × MasterMix 2.0 μ L and balance water.
Preferably, the concentration of primer A, primer B and primer C in the primer working solution is 100. mu.M, 100. mu.M and 100. mu.M, respectively.
Preferably, the KASP2 XMastermix comprises fluorescent probe A, fluorescent probe B, quenching probe A, quenching probe B, Taq enzyme, dNTP and Mg 2+ 。
Preferably, the reaction procedure of the PCR amplification is as follows: 95 ℃ for 15 min; at 95 ℃, 20s, 65-57 ℃, 1min, 9 cycles, each cycle reducing by 1.0 ℃; 95 ℃, 20s, 57 ℃, 60s, 32 cycles.
The invention provides a substance for detecting an Excalibur _ c95720_329-KASP marker related to the POD activity of wheat grains, or a primer group in the technical scheme, a kit in the technical scheme or an application of the method in the technical scheme in auxiliary wheat breeding, wherein the Excalibur _ c95720_329 is located at the 4031 th nucleotide in a POD-2D gene.
The invention provides an application of detecting an Excalibur _ c95720_329-KASP labeled substance related to the activity of a wheat grain POD or a primer group in the technical scheme, a kit in the technical scheme or a method in the technical scheme in the genetic improvement of POD activity, wherein the Excalibur _ c95720_329 is positioned at the 4031 th nucleotide in a POD-2D gene.
Has the beneficial effects that:
the invention provides a primer group for detecting an Excalibur _ C95720_329-KASP marker related to POD activity of wheat grains, which comprises a primer A, a primer B and a primer C. The primer group is based on two sequencing information of Excalibur _ c95720_329, is designed by using Polymarker software, has a good parting effect, and can be used for quickly detecting POD-2D alleles. 166 wheat varieties are used for verifying the effectiveness of the labeled primer group, and the result shows that GG, AA and GA genotypes are closely related to high POD activity, low POD activity and medium POD activity respectively. Therefore, the Excalibur _ c95720_329-KASP marked primer group can assist in screening wheat varieties with high or low grain POD activity, and provides a molecular tool and a theoretical basis for efficiently and accurately improving the wheat grain POD activity by utilizing molecular marker-assisted breeding.
Drawings
Fig. 1 is a genetic linkage diagram near wheat grain POD activity QTL qpod. saas-2 DL;
FIG. 2 is a diagram showing the detection result of the Excalibur _ c95720_329-KASP mark.
Detailed Description
The invention provides a primer group for detecting an Excalibur _ C95720_329-KASP marker related to POD activity of wheat grains, which comprises a primer A, a primer B and a primer C;
the nucleotide sequence of the primer A is shown as SEQ ID NO.1, and specifically comprises the following steps: 5' -GAAGGTGACCAAGTTCA TGCTCCGGACATCATGAGGGCA-3'; in the invention, 1-21 nucleotides at the 5' end of the primer A are FAM fluorescent label sequences.
The nucleotide sequence of the primer B is shown as SEQ ID NO.2, and specifically comprises the following steps: 5' -GAAGGTCGGAGTCAACG GATTCCGGACATCATGAGGGCG-3'; in the invention, 1-21 nucleotides at the 5' end of the primer B are HEX fluorescent label sequences.
The nucleotide sequence of the primer C is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-GCTCCAACTCATCCAGCGTA-3' are provided.
In the invention, the primer A and the primer B are upstream primers, and the 3' ends of the primer A and the primer B are allelic variant bases A/G marked by Excalibur _ c95720_ 329; the primer C is a downstream primer, and ensures the specificity of the POD-2D chromosome amplified by PCR.
The primer group is designed by using Polymarker software based on two sequencing sequence information of Excalibur _ c95720_329, has a good parting effect, can be used for rapidly detecting POD-2D alleles, so that the activity of POD of wheat grains is detected, and molecular tools and theoretical bases are provided for rapidly improving the POD activity of the wheat grains by molecular marker-assisted breeding and genetically improving the wheat quality.
The invention provides a kit for detecting an Excalibur _ c95720_329-KASP marker related to the POD activity of wheat grains, which comprises a primer group, KASP2 x MasterMix and water in the technical scheme. In the present invention, the primer set is preferably used in a primer working solution, the concentration of primer A is preferably 1M, the concentration of primer B is preferably 1M, and the concentration of primer C is preferably 1M. In the present invention, the KASP2 XMasterMix preferably comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, Taq enzyme, dNTP and Mg 2+ Practice of the inventionKASP2 XMASterMix used in the examples is a commercially available reagent manufactured by LGC corporation, having a product number of KBS-1016-. In the present invention, the water is preferably ultrapure water. The invention has no special requirement on the source of the ultrapure water, and can be prepared by adopting ordinary commercially available or self-made ultrapure water.
The invention also provides a method for detecting the POD activity of wheat grains based on the primer group in the technical scheme or the kit in the technical scheme, which comprises the following steps: taking the genomic DNA of the wheat to be detected as a template, and carrying out PCR amplification by using the primer group in the technical scheme to obtain an amplification product, and typing the wheat grains; and (3) judging the POD activity of the wheat grains according to the parting result, wherein the genotypes corresponding to the POD activity of the wheat grains from high to low are GG, GA and AA respectively.
The invention takes the wheat genome DNA to be detected as a template, and utilizes the primer group in the technical scheme to carry out PCR amplification to obtain an amplification product, and carries out typing on wheat grains. The invention has no special requirements on the method for obtaining the wheat genome DNA template, and can be obtained by adopting the conventional method in the field. In the present invention, the reaction system for PCR amplification preferably comprises, per 5. mu.L of the reaction system: 2 μ L of template DNA, 0.05 μ L of primer working solution, KASP2 × MasterMix 2.0 μ L and balance water. In the present invention, the concentration of primer A in the primer working solution is preferably 1M, the concentration of primer B is preferably 1M, and the concentration of primer C is preferably 1M. In the invention, the KASP2 XMASterMix comprises a fluorescent probe A, a fluorescent probe B, a quenching probe A, a quenching probe B, Taq enzyme, dNTP and Mg 2+ KASP2 XMaster Mix used in the examples of the present invention is a commercially available reagent manufactured by LGC corporation under the trade designation KBS-1016-. In the present invention, the water is preferably ultrapure water. The invention has no special requirements on the source of the ultrapure water, and can be prepared by adopting ordinary commercially available or self-made ultrapure water. In the present invention, the reaction procedure of the PCR amplification is preferably: 95 ℃ for 15 min; the temperature is 95 ℃, 20s, 65-57 ℃, 1min, 9 cycles, and each cycle is reduced by 1.0 ℃; 95 ℃, 20s, 57 ℃, 60s, 32 cycles. And obtaining an amplification product after PCR amplification is completed. The invention preferably carries out the genotype of the wheat grains according to the sequencing result of the amplification productAnd (4) parting. The corresponding site is 4031 th nucleotide in POD-2D gene. When the fluorescence signal data of the wheat amplification product to be detected is gathered at a position close to an X axis and is shown as blue through analysis of Kluster Caller software, the genotype of the Excalibur _ c95720_329 is the allele type AA connected with the FAM fluorescence label sequence; when the fluorescence signal data of the wheat amplification product to be detected is analyzed and gathered at a position close to a Y axis by Kluster Caller software and is shown to be red, the genotype of the SNP marker Excalibur _ c95720_329 is the allele GG connected with the HEX fluorescence label; when the fluorescence signal data of the wheat amplification product to be detected is gathered at the middle position of a diagonal line and is displayed as green through analysis of Kluster Caller software, the genotype of the SNP marker Excalibur _ c95720_329 is heterozygous genotype AG, and the diagram is shown in figure 2.
After the genotyping is obtained, the method judges the POD activity of the wheat grains according to the genotyping result, wherein the genotypes corresponding to the POD activity of the wheat grains from high to low are GG, GA and AA respectively. The method for detecting the activity of the POD of the wheat grains detects the activity of the POD of the wheat grains through the genotyping of the wheat grains, has accurate detection result, and can be used for quickly detecting the POD activity of the wheat grains.
The invention provides an application of an Excalibur _ c95720_329-KASP labeled substance related to POD activity of wheat grains or a primer group in the technical scheme, a kit in the technical scheme or a method in the technical scheme in auxiliary wheat breeding, wherein the Excalibur _ c95720_329 is positioned at the 4031 th nucleotide in POD-2D genes. The invention uses the Excalibur _ c95720_329-KASP marker for rapid detection of POD-2D allele, GG, AA and GA genotypes are closely related to high POD activity, low POD activity and medium POD activity respectively, and wheat variety materials with high or low POD activity of grains are screened in an auxiliary manner.
The invention provides an application of detecting an Excalibur _ c95720_329-KASP labeled substance related to the POD activity of wheat grains or a primer group in the technical scheme, a kit in the technical scheme or a method in the technical scheme in POD activity genetic improvement, wherein the Excalibur _ c95720_329 is located at the 4031 th nucleotide in a POD-2D gene. According to the invention, the screening of POD-2D gene GG genotype is strengthened by an Excalibur _ c95720_329-KASP marker, the POD activity of wheat grains is improved, and further the color and luster of a flour product and the processing quality are improved.
In order to further illustrate the invention, the following examples are given to describe in detail a primer set, a kit and an application for detecting an Excalibur _ c95720_329-KASP marker associated with POD activity of wheat grain, but they should not be construed as limiting the scope of the invention.
Example 1
Wheat grain POD activity QTL positioning analysis
176 strains of a 16RIL population of Gao Cheng/Zhou Mai and parents thereof (purchased from the national center for crop cultivation and preservation of Chinese academy of agricultural sciences) are planted in Henan Anyang and Anhui 28617xi in 2012-2013 and 2013-2014 respectively, the strains are designed into a random block, 3 rows of areas are designed, the row length is 1.5m, the row spacing is 25cm, 3 times of repetition, and the field management refers to a local conventional management mode to obtain the wheat grains under different environments. The 90K chip is used for carrying out whole genome scanning on 176 ligustrum sinense 8901/Zhoumai 16RIL population families and parents thereof, and markers which are free of polymorphism, have a marker deletion rate of more than 10 percent and have segregation of more than 30 percent are removed. And (3) optimizing the rest polymorphic markers by using the BIN-Mapping function of IciMapping V4.0 software for genetic map construction. Genetic map construction was performed using the JoinMap V4.0 software. Utilizing IciMapping V4.0 software, and adopting a complete composite interval mapping method (ICIM) to carry out QTL positioning on the POD activity of grains in different environments of RIL groups, wherein the LOD value is 2.5 as a threshold value.
Based on a 90K chip, the invention applies the total localization of the ligusticum city 8901/Zhoumai 16RIL population to 10 QTLs of PODs, wherein QPOD.saas-2DL on the 2D chromosome long arm stably exists in 2 environments (see figure 1), and explains 7.7-11.8% of phenotypic variation, which indicates that the locus has important influence on the POD activity of grains. Based on an IWGSC Refseq v1.0(http:// www.wheatgenome.org) database, sequence alignment analysis finds that a QPOD.saas-2DL locus interval SNP marker Excalibur _ c95720_329 is just POD-2D gene (TramesCS 2D02G583700), so that the marker is converted into a KASP marker for wheat grain POD active molecular marker-assisted selective breeding.
Example 2
KASP marker development for assisting in screening POD activity of wheat grains
The 51 st position of the flanking sequence of Excalibur _ c95720_329 is two polymorphic single nucleotides A or G of the SNP marker. Based on the information of the two sequencing sequences of Excalibur _ C95720_329, the Polymarker software was used to design an Excalibur _ C95720_329-KASP marker for the POD-2D gene, consisting of primer A, primer B and primer C.
The nucleotide sequence of the primer A is shown as SEQ ID NO.1, and the primer A specifically comprises the following components: 5' -GAAGGTGACCAAGTTCATGCTCCGGACATCATGAGGGCA-3'; in the invention, 1-21 nucleotides at the 5' end of the primer A are FAM fluorescent label sequences.
The nucleotide sequence of the primer B is shown as SEQ ID NO.2, and specifically comprises the following steps: 5' -GAAGGTCGGAGTCAACG GATTCCGGACATCATGAGGGCG-3'; in the invention, 1-21 nucleotides at the 5' end of the primer B are HEX; a fluorescent tag sequence.
The nucleotide sequence of the primer C is shown as SEQ ID NO.3, and specifically comprises the following steps: 5'-GCTCCAACTCATCCAGCGTA-3' is added.
Detecting the genotypes of the 8901/Zhoumai 16RIL population and the parental Excalibur _ c95720_329 of the ligusticum sinensis by using the KASP marker primer group, wherein if the fluorescence signal data of the amplified product of the wheat to be detected are gathered at the position close to the X axis and are shown as blue by the analysis of Kluster Caller software, the genotype of the Excalibur _ c95720_329 is an allelic type AA connected with a FAM fluorescence label sequence; if the fluorescence signal data of the wheat amplification product to be detected is analyzed and gathered at a position close to a Y axis by Kluster Caller software and is shown to be red, the genotype of the SNP marker Excalibur _ c95720_329 is the allele GG connected with the HEX fluorescence label; if the fluorescence signal data of the wheat amplification product to be detected is gathered at the middle position of a diagonal line and is displayed as green by the analysis of Kluster Caller software, the genotype of the SNP marker Excalibur _ c95720_329 is heterozygous genotype AG. The test uses the detection result of the wheat 90K chip as a reference, and the comparison result with the detection result of the KASP labeled primer group is shown in the table 1.
TABLE 1 genotype test results for the Ligusticum city 8901/Zhoumai 16RIL population and the parent Excalibur _ c95720_329
Figure BDA0002802811350000081
Figure BDA0002802811350000091
The results in Table 1 show that the typing effect of the Excalibur _ c95720_329-KASP marker is good, and the genotype results of the 176 Ligusticum sinense 8901/Zhoumai 16RIL populations are completely consistent with the 90K chip genotype typing results, which shows that the Excalibur _ c95720_329-KASP marker primer group can accurately detect the genotype of the site and greatly reduce the detection cost.
Example 3
The invention discloses a KASP marker validity verification for assisting in screening POD activity of wheat grains
166 parts of main wheat cultivars (from the national center for crop planting and preservation of Chinese agricultural academy of sciences) in Huang-Huai wheat areas of China are planted in the Henan Anyang and Anhui (28617); xi, random block design, 3 rows of areas, 1.5m of row length, 25cm of row spacing and 3 times of repetition in 2012-2013 and 2013-2014 respectively, and the wheat grains under different environments are obtained by field management according to a local conventional management mode.
166 varieties of wheat were typed for POD-2D gene using Excalibur _ c95720_329-KASP marker.
The activity of POD (peroxidase) of 166 grains of a representative wheat variety in Huang-Huai-Mai region of China is measured by using a guaiacol method and an enzyme-labeling instrument. Specific procedures are described in Wei et al [1] . POD Activity (U.min) -1 ·g -1 ) Δ a/t (t is measurement time) × total volume of extracted enzyme solution 5 mL/(mass of flour 0.5g × volume of enzyme taken at the time of measurement 0.005mL × 0.01), unit POD activity (U) is defined as an increase in absorbance at 470nm of reaction product per gram of flour per minute by 0.01. Each sample is repeatedly tested twice, if the difference of the two test results is more than 10%, a third test is requiredAnd (6) measuring.
The preparation method of the wheat flour comprises the following steps:
kernel hardness was determined using a single kernel grain characterizer SKCS 4100(Perten, Sweden). Using a near infrared analyzer Foss-Tecator 1241(Foss,
Figure BDA0002802811350000092
sweden) to determine the protein content and water content of the grain. Calculating the water adding amount required by wheat wetting according to the hardness grade: 14% of soft wheat (hardness index is less than 40), 15% of mixed wheat (hardness index is more than 40 and less than 60) and 16% of hard wheat (hardness index is more than 60). And (3) moistening wheat for 16-18 h at room temperature, and milling by using a Junior test mill (Brabender, Germany), wherein the flour yield is about 60%. The flour is stored at 4 ℃ and used for POD activity detection.
166 wheat varieties POD-2D genotype and POD activity are shown in detail in Table 2, and the results are analyzed in Table 3.
TABLE 2166 wheat variety seed POD activity and POD-2D genotype
Figure BDA0002802811350000101
Figure BDA0002802811350000111
Figure BDA0002802811350000121
Figure BDA0002802811350000131
Figure BDA0002802811350000141
Remarking: BLUP is the best linear unbiased prediction, correcting for 4 environmental phenotypes;
TABLE 3166 multiple comparison of wheat variety POD-2D genotype with POD activity
Figure BDA0002802811350000142
Note: a different letters after the mean POD activity indicate significant differences between genotypes
Table 3 shows that the typing results of the Excalibur _ c95720_329-KASP marker of the present invention are closely related to the POD activity, the GG genotype is significantly related to the high POD activity (P <0.05), the AA genotype is significantly related to the low POD activity (P <0.05), the GA genotype is at a moderate POD activity level, and the Excalibur _ c95720_329-KASP primer set and the detection line can be used for molecular marker-assisted selection targeting improvement of the POD activity of wheat.
From the above embodiments, the typing result of the Excalibur _ c95720_329-KASP marker of the present invention is closely related to the POD activity, and can assist in screening wheat varieties with high or low POD activity, so as to provide a molecular tool and a theoretical basis for efficiently and accurately improving the POD activity of wheat grains by molecular marker-assisted breeding.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Sequence listing
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Claims (6)

1. A method for detecting POD activity of wheat grains based on an Excalibur _ c95720_329-KASP labeled primer group comprises the following steps: taking the genomic DNA of the wheat to be detected as a template, and carrying out PCR amplification by using the primer group marked by Excalibur _ c95720_329-KASP to obtain an amplification product, and typing the wheat grains; judging the POD activity of the wheat grains according to the parting result, wherein the genotypes corresponding to the POD activity of the wheat grains from high to low are GG, GA and AA respectively;
the primer group consists of a primer A, a primer B and a primer C;
the nucleotide sequence of the primer A is shown as SEQ ID NO. 1;
the nucleotide sequence of the primer B is shown as SEQ ID NO. 2;
the nucleotide sequence of the primer C is shown as SEQ ID NO. 3.
2. The method of claim 1, wherein each 5 μ L of the PCR amplification reaction system comprises: 2 μ L of template DNA, 0.05 μ L of primer working solution, 2.0 μ L of KASP2 × Master Mix and the balance of water.
3. The method according to claim 2, wherein the primer A concentration in the primer working solution is 100. mu.M, the primer B concentration is 100. mu.M, and the primer C concentration is 100. mu.M.
4. The method of claim 2, wherein the KASP2 xmaster Mix comprises fluorescent probe a, fluorescent probe B, quench probe a, quench probe B, Taq enzyme, dntps and Mg 2+ 。
5. The method according to any one of claims 1 to 4, wherein the reaction procedure of PCR amplification is as follows: 95 ℃ for 15 min; at 95 ℃, 20s, 65-57 ℃, 1min, 9 cycles, each cycle reducing by 1.0 ℃; 95 ℃, 20s, 57 ℃, 60s, 32 cycles.
6. Use of the method according to any one of claims 1 to 5 for the genetic improvement of POD activity, wherein the Excalibur _ c95720_329 is located at the 4031 th nucleotide in the POD-2D gene.
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