A method for predicting malt pyrofiltration (PYF) performance based on barley gene expression during malting process and application thereof

Abstract

The application provides a malt PYF performance prediction method based on barley gene expression in a malting process and an application thereof, belongs to the field of biology, and can solve technical problems of traditional malt PYF detection methods, such as complicated operation steps, long time consumption, and narrow application range. The prediction method specifically comprises the following steps: candidate gene screening, tracking gene screening, reverse transcription reaction, multiple PCR reaction primer design, RNA extraction, reverse transcription cDNA template preparation, multiple PCR reaction, PCR amplification product electrophoresis and expression analysis, and malt PYF performance prediction. By comparing the gene expression differences of standard malt and PYF malt in the malting process, tracking genes are selected in the large production process, and then by comparing the expression amounts of the standard malt and the to-be-detected malt and combining the evaluation standard, the malt PYF performance can be rapidly and accurately analyzed. The application can be applied to the field of malt PYF performance prediction.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and in particular relates to a method for predicting the PYF performance of malt based on barley gene expression during the malting process and its application. Background Technology

[0002] Premature yeast flocculation (PYF) refers to the phenomenon where yeast flocculates and settles before sufficient fermentation time has been reached during beer fermentation. This slows down sugar degradation and acetaldehyde reduction during fermentation, resulting in poor maturity of the finished beer and affecting its fermentation quality, ultimately causing beer quality problems. PYF is a quality issue unique to the beer industry, caused by factors in the malt that induce premature yeast flocculation. Therefore, testing the PYF performance of malt is a standard indicator in the beer industry.

[0003] Currently, the detection of malt PYF (polyylated yeast fermentation) generally involves preparing wort from the malt, adding yeast for fermentation, and then detecting the number of suspended yeasts in the fermentation liquid. This method requires 40-48 hours of fermentation time, and with the wort preparation and yeast count detection, the total testing time is 3-4 days, which is quite long. Furthermore, this method detects the finished malt, lacking predictability for the malt production process and making timely adjustments to the malt production process impossible.

[0004] Therefore, it is evident that developing a method for predicting malt PYF performance that can be analyzed from the malt production process, with short processing time, high detection efficiency, and high accuracy, is of positive significance for guiding the adjustment of malt production processes and improving malt quality. Summary of the Invention

[0005] This invention addresses the technical problems of traditional malt PYF detection methods, which are cumbersome, time-consuming, and only applicable to finished malt products with a narrow scope of application. It proposes a PYF performance prediction method that can analyze the malt production process and features short detection time, high detection efficiency, and high accuracy.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for predicting malt PYF performance based on barley gene expression during the malting process includes the following steps:

[0008] Transcriptome analysis was conducted to identify gene expression differences between standard malt and PYF malt during the malting process, and several genes with significant expression differences were selected as candidate genes.

[0009] The large-scale production process of the standard malt and PYF malt was tracked, and the candidate genes with the most obvious change trends were further selected as the tracking genes.

[0010] Based on the tracked gene sequence, a set of primers for reverse transcription and a set of primers for multiplex PCR were designed.

[0011] Total RNA extraction from malt to be tested and standard malt;

[0012] Using the extracted total RNA as a template, the reverse transcription primer set was used as a primer to prepare a cDNA template through reverse transcription.

[0013] Using the reverse transcription reaction product as a template, the multiplex PCR primer set was used as primers to perform multiplex PCR reaction. Electrophoresis and expression level analysis were performed on the multiplex PCR reaction products of the test malt and standard malt to predict the PYF performance of malt.

[0014] In one embodiment, the candidate gene screening is performed by transcriptomic analysis of the differences in gene expression during the germination process of standard malt and PYF malt in the malting process.

[0015] In one embodiment, the screening criteria for the tracking gene are:

[0016] The change in gene expression is greater than or equal to 2.0 or less than 0.5;

[0017] The change in gene expression is defined as the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

[0018] In one embodiment, the tracking genes include 13 genes, including AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5.

[0019] In one embodiment, the reverse transcription primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, resulting in 13 primers as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26.

[0020] In one embodiment, the multiplex PCR primer set was designed with 13 primers for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25.

[0021] In one embodiment, the PYF performance of malt was evaluated using the following criteria after electrophoresis and expression level analysis of the multiplex PCR products of the test malt and standard malt:

[0022] Principal component analysis was used to obtain the principal component scores of the standard malt and the malt to be tested, and the absolute value of the difference between the two was taken.

[0023] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%;

[0024] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%.

[0025] When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%.

[0026] In one embodiment, the performance evaluation criteria for malt PYF are obtained through the following method:

[0027] Gene expression analysis was performed on the tracking genes of different PYF malts to obtain the principal component scores of gene expression.

[0028] The principal component scores of gene expression are compared with those of standard malt to establish the PYF performance evaluation criteria for the malt.

[0029] The present invention also provides an application of the malt PYF performance prediction method based on barley gene expression during the malting process described in any of the above embodiments in the prediction of malt PYF performance.

[0030] In one embodiment, the malt includes finished malt, barley, and barley malt that germinates during the malting process.

[0031] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0032] 1. The present invention proposes a method for predicting the performance of malt PYF based on barley gene expression during the malting process. This method compares the gene expression differences between standard malt and PYF malt during the malting process and selects tracking genes in the large-scale production process. Then, it compares the expression levels of standard malt and the malt to be tested and combines them with evaluation criteria to achieve accurate prediction of malt PYF performance.

[0033] 2. The malt PYF performance prediction method based on barley gene expression during the malting process proposed in this invention establishes a dual standard including "tracking gene screening standard and malt PYF performance evaluation standard" to improve the accuracy of the prediction results, which can realize rapid and accurate analysis of malt PYF performance from the malt production process.

[0034] 3. The malt PYF performance prediction method based on barley gene expression during the malting process proposed in this invention can predict the malt PYF situation in advance during the malting stage, which has certain guiding significance for actual production. Detailed Implementation

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention provides a method for predicting malt PYF performance based on barley gene expression during the malting process, comprising the following steps:

[0037] S1. By analyzing the differences in gene expression between standard malt and PYF malt during the malting process through transcriptome analysis, several genes with significant expression differences were selected as candidate genes.

[0038] S2. Track the large-scale production process of standard malt and PYF malt, and further select the candidate genes with the most obvious change trend as the tracking genes;

[0039] S3. Design reverse transcription primer sets and multiplex PCR primer sets based on the tracked gene sequences;

[0040] S4. Total RNA extraction from malt to be tested and standard malt;

[0041] S5. Using the extracted total RNA as a template and the reverse transcription primer set as primers, a reverse transcription reaction was performed to prepare a cDNA template.

[0042] S6. Using the reverse transcription reaction product as a template and the multiplex PCR primer set as primers, a multiplex PCR reaction was performed. The multiplex PCR reaction products of the test malt and standard malt were electrophoresed and their expression levels were analyzed to predict the PYF performance of malt.

[0043] In one specific implementation, candidate gene screening is performed by transcriptomic analysis of gene expression differences between standard malt and PYF malt during malting, especially during the germination process.

[0044] The above embodiments provide a method for predicting the PYF performance of malt based on barley gene expression during the malting process. This method compares the gene expression differences between standard malt and PYF malt during the malting process, selects tracking genes by combining the large-scale production process, and then compares the expression levels of standard malt and the test malt with the evaluation criteria to achieve accurate prediction of malt PYF performance. Compared with traditional methods, this method is less time-consuming, has a wider range of applications, and has higher practical application value.

[0045] In one specific implementation, the screening criteria for the tracking gene are as follows:

[0046] The change in gene expression is greater than or equal to 2.0 or less than 0.5;

[0047] Among them, the change in gene expression is the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

[0048] In the above implementation, the purpose of setting the screening criteria for tracking genes at a gene expression change of greater than or equal to 2.0 or less than 0.5 is mainly to identify genes with significant differences in expression before and after the change as characteristic genes. Generally, a change in expression greater than 2 times is considered a significant difference. This could be an increase of more than 2 times or a decrease to less than half of the original value, hence the standard of greater than or equal to 2.0 or less than 0.5.

[0049] In one specific implementation, the tracking genes include 13 genes, including AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5.

[0050] The functions of the 13 genes mentioned in the above implementation are as follows:

[0051]

[0052] In one specific embodiment, the reverse transcription primer set was designed for 13 tracking genes AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5, resulting in 13 primers as shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26, respectively.

[0053] In one specific embodiment, a multiplex PCR primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5. These primers are shown as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25, respectively.

[0054] In one specific embodiment, the PYF performance of malt was predicted by electrophoresis and expression level analysis of the multiplex PCR reaction products of the malt to be tested and the standard malt:

[0055] (1) Through principal component analysis, the principal component scores of the standard malt and the malt to be tested are obtained, and the absolute value of the difference between the two is taken.

[0056] When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%;

[0057] When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%.

[0058] When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%.

[0059] In one specific implementation, the performance evaluation criteria for malt PYF are obtained through the following method:

[0060] (1) Gene expression analysis was performed on the tracking genes of different PYF malts to obtain the principal component scores of gene expression;

[0061] The different PYF malts mentioned in step (1) above refer to malts with different PYF values ​​(specifically including PYF-90% malt, PYF-50% malt and PYF-30% malt). By analyzing the gene expression of malts with different PYF values, the resulting principal component scores and the judgment criteria based on these scores are more accurate.

[0062] Furthermore, the reason why this invention selects the principal component score of gene expression as the analysis index is that, in order to comprehensively evaluate the expression of multiple genes, the principal component analysis method is used to reduce the dimensionality of the expression data of multiple genes, thereby obtaining a comprehensive quantitative score of multiple genes.

[0063] (2) The principal component scores of gene expression are compared with the principal component scores of gene expression of standard malt to establish the performance evaluation criteria for malt PYF.

[0064] The present invention also provides an application of the malt PYF performance prediction method based on barley gene expression during the malting process described in any of the above embodiments in the prediction of malt PYF performance.

[0065] In one specific embodiment, the malt includes, but is not limited to, finished malt, barley, and barley malt that germinates during the malting process.

[0066] To more clearly and in detail introduce the method for predicting malt PYF performance based on barley gene expression during the malting process and its application provided by the embodiments of the present invention, the following will describe it in conjunction with specific embodiments.

[0067] Example 1

[0068] This embodiment provides the screening process for candidate genes and tracking genes in a method for predicting malt PYF performance based on barley gene expression during the malting process, specifically as follows:

[0069] (1) Studies have shown that the problem of PYF malt production is related to the stress response of barley caused by external stimuli during planting and germination. Therefore, this invention analyzed the gene expression differences between standard malt and PYF malt during the malting process, especially during seed germination, and found several genes with large expression differences, which were used as candidate genes (about 13).

[0070] (2) Based on the results of step (1), by tracking the large-scale production process (i.e., during the malting process, green malts after soaking are taken. To ensure that the vitality of green malts does not change, the samples taken should be immediately subjected to RNA extraction or stored at -80℃), 13 genes with the most obvious change trends are selected as tracking genes. The screening criteria for tracking genes are: the gene expression change range is greater than or equal to 2.0 or less than 0.5, and the gene expression change range is the ratio of the gene amount of PYF malt to the gene expression amount of standard malt (see Table 1 for relevant data).

[0071] Table 1. Statistical analysis of gene expression in standard malt and PYF malt.

[0072]

[0073]

[0074] Example 2

[0075] This embodiment provides the process for determining the malt PYF performance evaluation criteria in a malt PYF performance prediction method based on barley gene expression during the malting process, specifically as follows:

[0076] Gene expression was analyzed and detected in three different PYF malts (PYF-90% malt, PYF-50% malt, and PYF-30% malt), and principal component scores of gene expression were obtained. The relevant standards were determined by comparing the scores with those of standard malt (see Table 2 for relevant data).

[0077] Table 2 Principal component scores of standard malt and PYF malt

[0078]

[0079] As shown in the table above, by comparing the principal component scores of gene expression in malts with different PYF values ​​with those in standard malts, it was found that the absolute value of the difference between the principal component scores of gene expression in PYF-90% malt and standard malt was less than 0.5, the absolute value of the difference between the principal component scores of gene expression in PYF-50% malt and standard malt was greater than 0.5 but less than 5, and the absolute value of the difference between the principal component scores of gene expression in PYF-30% malt and standard malt was greater than 5.

[0080] Example 3

[0081] This embodiment provides a method for predicting the PYF performance of malt based on barley gene expression during the malting process. Specifically, this embodiment predicts the PYF performance of a newly purchased batch A of barley:

[0082] (1) Selection of monitoring genes: Thirteen genes including AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WRKY5 were selected as monitoring genes.

[0083] (2) Sample collection: During the wheat processing, take the green malt after soaking (Note: In order to ensure that the vitality of the green malt does not change, the sample taken should be extracted for RNA immediately or stored at -80℃).

[0084] (3) Green malt grinding: To ensure that the RNA in green malt is not degraded, green malt is ground and pulverized using liquid nitrogen;

[0085] (4) RNA extraction:

[0086] The crushed green malt was then subjected to RNA extraction by adding Trizol (Note: Trizol is a total RNA extraction reagent that can directly extract total RNA from cells or tissues).

[0087] Take 50-100 mg of sample, add 1 ml of Trizol, lyse by shaking, and centrifuge at 12000 g for 5 min at 4 °C. Transfer the supernatant to a Phasemarker tube and let stand for 5 min. Add 0.2 ml of chloroform, shake manually for 15 s, and let stand for 10 min. Centrifuge at 12000 g for 10 min at 4 °C. Take the supernatant (450-550 μL) to an EP tube, add 250 μL of 96% ethanol, and mix with a pipette tip. Transfer the mixture to a silicone tube, centrifuge at 12000 g for 1 min, and remove the waste liquid. Add 700 μL of WB1, centrifuge at 12000 g for 1 min, and remove the waste liquid. Add 500 μL of WB2, centrifuge at 12000g for 1 min, and remove the runoff waste liquid; add 500 μL of WB2, centrifuge at 12000g for 1 min, and transfer the silicone tube to a 1.5 ml EP tube; add 50 μL of nuclease-free water, let stand for 1 min, and centrifuge at 12000g for 1 min; collect the centrifuged RNA solution and store it at -80℃;

[0088] (5) RT-PCR reverse transcription:

[0089] Using the purified barley cell RNA obtained in step (4) as a template, the first strand of cDNA was synthesized using the Beckman Coulter GenomeLab™ GeXP starter kit with downstream primers of the designed multiple primers as specific primers (the sequences of which are shown in Table 3) and total RNA from barley cells as a template. The reaction system was 10 μL, and the parameters for the first strand of cDNA synthesis were set as follows: 48℃ for 1 minute; 42℃ for 60 minutes; 95℃ for 5 minutes.

[0090] Then, the DNA polymerase from Beckman Coulter and the GenomeLab™ GeXP starter kit were used. Using the synthesized first-strand cDNA as a template and the upstream primers of the 13 multiplex primers as specific primers (their sequences are shown in Table 3), RT-PCR amplification was performed. The RT-PCR amplification parameters were set as follows: 95℃ pre-denaturation for 10 minutes; 94℃ denaturation for 30 seconds; annealing at 56℃ for 30 seconds; extension at 71℃ for 1 minute, for 35 cycles, finally obtaining the reverse transcription amplified product of the expressed gene.

[0091] Table 3 Upstream and downstream primer sequences

[0092]

[0093]

[0094] (6) Gene expression level analysis:

[0095] The reverse transcription products were quantitatively analyzed using capillary electrophoresis. 1 μl of the PCR multiplex product was added to the well of a plate containing 39 μl of a mixture of 95% deionized formamide (SLS) and 400 bp marker, mixed with a pipette, and then covered with a drop of paraffin oil.

[0096] In addition, 250 μl of separation buffer was added to each well of the buffer plate. After all preparations were completed, capillary electrophoresis was performed. After electrophoresis, the expression levels of different genes at different stages were obtained.

[0097] (7) PYF performance prediction:

[0098] PCA analysis was performed on the gene expression of sample A and standard barley (the results are shown in Table 4):

[0099] Table 4. PCA analysis of gene expression in batch A malt and standard malt.

[0100]

[0101]

[0102] Principal component analysis shows that the principal component score of standard malt is -1.563, and the principal component score of malt A is -1.628. The absolute value of the difference between the two is 0.065, which is less than 0.5. Therefore, the PYF value of malt A is 99%.

[0103] Example 4

[0104] This embodiment provides a method for predicting the PYF performance of malt based on barley gene expression during the malting process. Specifically, this embodiment predicts the PYF performance of a newly purchased batch B barley:

[0105] The specific prediction method is the same as in Example 3, the only difference being the batch of malt.

[0106] PCA analysis was performed on the gene expression of batch B and standard barley (the results are shown in Table 5):

[0107] Table 5. PCA analysis of gene expression in batch B malt and standard malt.

[0108]

[0109]

[0110] Principal component analysis shows that the principal component score of standard malt is -1.563, while that of malt B is 0.345. The absolute value of the difference between the two is 1.908, which is greater than 0.5 and less than 5. The PYF value of malt B is 62%.

[0111] Example 5

[0112] This embodiment provides a method for predicting the PYF performance of malt based on barley gene expression during the malting process. Specifically, this embodiment predicts the PYF performance of a newly purchased batch of C barley:

[0113] The specific prediction method is the same as in Example 3, the only difference being the batch of malt.

[0114] PCA analysis was performed on the gene expression of batch C and standard barley (the results are shown in Table 6):

[0115] Table 6. PCA analysis of gene expression in batches C malt and standard malt.

[0116]

[0117]

[0118] Principal component analysis showed that the principal component score of standard malt was -1.563, while that of malt C was 3.378. The absolute value of the difference between the two was 5.121, which is greater than 5.0. Therefore, the PYF value of malt C was 41%.

[0119] Verification test

[0120] To further verify the accuracy of the malt PYF performance prediction method based on barley gene expression during the malting process, traditional evaluation methods were used for result verification, as follows:

[0121] The traditional evaluation method (which involves preparing malt into wort, adding yeast for fermentation, and then detecting the number of suspended yeast cells in the fermentation broth) was used, and the results were compared with those of Examples 3-5. The results are as follows:

[0122] The traditional evaluation methods are as follows:

[0123] (1) Preparation of wort: Take a certain amount of malt, crush it, add water for saccharification, and then filter to obtain saccharified wort;

[0124] (2) Yeast inoculation and fermentation: Take a certain amount of yeast mud and place it in sterilized wort, shake to aerate, and then ferment at 20℃ for 48 hours;

[0125] (3) Yeast counting: Take a certain amount of fermentation broth and count the yeast using a yeast counter. Obtain the yeast concentration in the fermentation broth;

[0126] (4) PYF value calculation: PYF value = number of yeast cells in the sample / number of yeast cells in the standard sample × 100%.

[0127] Table 7 Comparison of results from traditional evaluation methods and Examples 3-5

[0128]

[0129] As shown in Table 7, compared with traditional evaluation methods, the method of the present invention can accurately predict the performance of malt PYF by comparing the gene expression differences between standard malt and PYF malt during the malting process, selecting tracking genes in the large-scale production process, and then comparing the expression levels of standard malt and the test malt in combination with evaluation standards.

Claims

1. A method for predicting malt PYF performance based on barley gene expression during the malting process, characterized in that, Includes the following steps: Transcriptome analysis was conducted to identify gene expression differences between standard malt and PYF malt during the malting process, and several genes with significant expression differences were selected as candidate genes. The large-scale production process of the standard malt and PYF malt was tracked, and the genes with the most obvious change trends were further selected from the candidate genes as tracking genes. The tracking genes include 13 genes, including AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17 and WRKY5. Based on the tracked gene sequence, a set of primers for reverse transcription and a set of primers for multiplex PCR were designed. Total RNA extraction from malt to be tested and standard malt; Using the extracted total RNA as a template, the reverse transcription primer set was used as a primer to prepare a cDNA template through reverse transcription. Using the reverse transcription product as a template, the multiplex PCR primer set was used as primers for multiplex PCR. Electrophoresis and expression level analysis were performed on the multiplex PCR products of the malt to be tested and standard malt to predict malt PYF performance. The malt PYF performance was determined using the following criteria: Gene expression analysis was performed on the tracking genes of standard malt and malts with different PYF values. Principal component analysis was used to obtain the principal component scores of standard malt and malts to be tested, and the absolute value of the difference between the two was taken. The different PYF malts refer to malts with different PYF values, specifically including PYF-90% malt, PYF-50% malt and PYF-30% malt. When the absolute value of the difference between the two is less than 0.5, the PYF value of the malt to be tested is ≥90%; When the absolute value of the difference between the two is greater than 0.5 and less than 5.0, the PYF value of the malt to be tested is 50-90%. When the absolute value of the difference between the two is greater than 5.0, the PYF value of the malt to be tested is <50%.

2. The method for predicting malt PYF performance based on barley gene expression during the malting process according to claim 1, characterized in that, The candidate gene screening was conducted by transcriptome analysis of the differences in gene expression during the germination process of standard malt and PYF malt in the malting process.

3. The method for predicting malt PYF performance based on barley gene expression during malting according to claim 1, characterized in that, The screening criteria for the tracking genes are as follows: The change in gene expression is greater than or equal to 2.0 or less than 0.5; The change in gene expression is defined as the ratio of the principal component score of PYF malt gene expression to the principal component score of standard malt gene expression.

4. The method for predicting malt PYF performance based on barley gene expression during malting according to claim 1, characterized in that, The reverse transcription primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5. Thirteen primers were designed and are shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, SEQ ID NO.24, and SEQ ID NO.26, respectively.

5. The method for predicting malt PYF performance based on barley gene expression during malting according to claim 4, characterized in that, The multiplex PCR primer set was designed for 13 tracking genes: AP1, WRKY70, OSM1, GA2ox3, thioredoxin, serpin, RPP13, RbohE, RbohB2, A2, IAA16, BGU17, and WRKY5. Thirteen primers were designed and are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, SEQ ID NO.23, and SEQ ID NO.25, respectively.

6. The application of the method for predicting malt PYF performance based on barley gene expression during the malting process as described in any one of claims 1-5 in the prediction of malt PYF performance.

7. The application of the malt PYF performance prediction method based on barley gene expression during malting process according to claim 6 in malt PYF performance prediction, characterized in that, The malt includes finished malt, barley, and barley malt that germinates during the malting process.

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