Polymorphic molecular marker of corn drought character and application thereof

Abstract

The invention discloses a polymorphic molecular marker for corn drought traits and application of the polymorphic molecular marker, an ABA receptor ZmPYL8 is re-sequenced, the polymorphic molecular marker influencing corn drought resistance exists in a second intron through analysis, the polymorphic molecular marker is a ZmPYL8Hap2 molecular marker, and the nucleotide sequence of the polymorphic molecular marker is TTTTAAAAAAAAAAA. The kit is designed based on the ZmPYL8 intron region T5A12 drought resistance analysis marker, the drought resistance can be judged through conventional PCR electrophoresis, the cost is low, the kit is easy to master, the kit is suitable for different breeding stages, the breeding period can be shortened, the cost can be reduced, and the kit has important scientific significance and application value.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a polymorphic molecular marker for drought traits in maize and its application. Background Technology

[0002] Globally, corn ( Zea mays Maize (L.) is one of the most important food crops. Maize has a high water requirement during its growth period; however, the main maize-growing areas are mostly located in arid and semi-arid zones, where natural rainfall often fails to meet the needs of maize growth and development, making water a key factor limiting maize yield. Therefore, in-depth research into the drought resistance mechanisms of maize, the discovery of new drought-resistant genes and superior natural variations, and the breeding and promotion of new drought-resistant varieties are crucial for ensuring food security. In recent years, resequencing hundreds of inbred lines to discover potential natural variation sites, studying their functions, elucidating their drought resistance genetic and molecular mechanisms, developing drought resistance molecular markers, combining gene editing technology for germplasm creation, and applying them to drought-resistant breeding and improvement have become hot topics and key areas of plant drought resistance research. Identifying and obtaining drought-resistant genes that play a decisive role in crops is of great significance for crop drought resistance genetic improvement and molecular breeding practices.

[0003] Therefore, there is an urgent need to develop a drought resistance marker in the maize genome, and the use of drought resistance markers in crop stress resistance genetic improvement and breeding has great value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a polymorphic molecular marker for drought traits in maize and its application. This invention uses next-generation resequencing technology to analyze 197 inbred lines. ZmPYL8 Gene regions were sequenced, and sequence alignment analysis revealed a drought resistance marker composed of varying numbers of "T" and "A" bases. The "T" bases represent natural variations containing "polyU," and a higher number of "T" bases results in higher splicing efficiency. Specifically, the marker is TTTTTAAAAAAAAAAAA ("T5A"). 12 This invention identifies a key haplotype NIL. Hap2 (Contains ZmPYL8) Hap2 The molecular marker (TTTTTTAAAAAAAAAAAA) participates in the drought stress response of maize, and its introduction can improve the drought resistance of maize.

[0005] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a polymorphic molecular marker for the drought trait in maize, wherein the polymorphic molecular marker is located at... ZmPYL8the second intron region of the gene, the polymorphic molecular marker is ZmPYL8 Hap2 the molecular marker, wherein, the ZmPYL8 Hap2 The nucleotide sequence of the molecular marker is shown as SEQ ID NO: 1: TTTTTAAAAAAAAAAAA.

[0006] the above ZmPYL8 The DNA sequence is 2342 bp (B73) in full length; it should be clear that the above polymorphic molecular marker is located in ZmPYL8 the second intron region of the gene, and the sequence is composed of continuous T and A, and it is difficult to identify. Therefore, the judgment method of the marker is to perform PCR amplification only through variation of the promoter region, and the promoter variation is highly linked with the intron variation.

[0007] The application also provides application of the above polymorphic molecular marker in identifying or assisting in identifying drought-resistant corn varieties and breeding drought-resistant corn varieties.

[0008] The application also provides a primer pair for obtaining the above polymorphic molecular marker, and the primer pair is: ZmPYL8-Hap2-F: 5'-GCCTTGATTGCTTGGTACTT-3', as shown in SEQ ID No. 2; ZmPYL8-Hap2-R: 5'-ATCAACATTGACGCCTACCT-3', as shown in SEQ ID No. 3; The application also provides application of the above primer pair in identifying or assisting in identifying drought-resistant corn varieties.

[0009] The application also provides a kit for identifying or assisting in identifying drought-resistant corn varieties, and the kit contains the above primer pair.

[0010] The application also provides a method for identifying or assisting in identifying drought-resistant corn varieties, comprising the following steps: 1) extracting DNA in a corn variety to be tested, 2) performing PCR amplification by using the above primer pair or the above kit to obtain a PCR product; 3) performing amplification on the PCR product, and judging the electrophoresis result: When a pair of primers ZmPYL8-Hap2-F and ZmPYL8-Hap2-R amplifies a target band, it indicates that the corn variety contains ZmPYL8 Hap2 the molecular marker, namely, the variety is haplotype Hap2, and it indicates that the variety is strong in drought resistance; Alternatively, if a pair of primers, ZmPYL8-Hap2-F and ZmPYL8-Hap2-R, fails to amplify the target band, it indicates that the maize variety does not contain ZmPYL8. Hap2 Molecular markers indicate that this variety has weak drought resistance.

[0011] Furthermore, in step 2), PCR amplification was performed using 2×Taq Master Mix, and the reaction system is as follows: The reaction program was as follows: 95℃ for 3 min; 95℃ for 30 s; 57℃ for 30 s; 72℃ for 60 s; 72℃ for 5 min; 35 cycles.

[0012] The present invention also provides an application of the above-mentioned reagent kit in the breeding of drought-resistant maize varieties.

[0013] The beneficial effects of this invention are: 1. Provided clear and reproducible molecular markers for drought resistance. Based on second-generation resequencing of 197 maize inbred lines, in ZmPYL8 A polymorphic sequence consisting of varying numbers of "T" and "A" was identified in the second intron of the gene region, with the key molecular marker being ZmPYL8. Hap2 (TTTTTAAAAAAAAAAAA). This marker site is easily identified by routine PCR sequencing. The detection process is simple, highly sensitive, and reproducible, making it suitable for large-scale population screening.

[0014] 2. To reveal the functional relationship between the number of "polyU(T)" and splicing efficiency and drought resistance. The mark reflects ZmPYL8 Natural variation in "polyU" within introns shows that a higher number of "T" markers indicates higher splicing efficiency and is positively correlated with drought stress response. Compared to anonymized markers based solely on linkage disequilibrium, this invention provides functionally relevant markers that exhibit higher stability and predictive power under diverse genetic backgrounds and ecological conditions.

[0015] 3. Accelerate the drought-resistant breeding process and reduce breeding costs. Using the markers of this invention, unfavorable genotypes can be rapidly screened out in early and conventional generations, transforming the large-scale, long-cycle, and highly environmentally dependent phenotypic screening of drought identification in the field into high-throughput genotypic screening in the laboratory, significantly shortening the breeding cycle and saving experimental resources and land.

[0016] 4. Improve the accuracy of seed selection and the extent of drought resistance improvement Introducing ZmPYL8 into conventional selection, composite backcrossing, and population improvement. Hap2(TTTTTAAAAAAAAAAAA) favorable haplotype, which can effectively improve the drought resistance potential of candidate materials. The marker can be used as an anchor point for selection of the main effective site, and is used for superimposed breeding with genome-wide selection (GWAS) or other stress resistance sites.

[0017] In summary, the present application focuses on ZmPYL8 Intron region "polyU (T)" natural variation, provides a mechanism clear, simple, stable and reliable drought resistance molecular marker (ZmPYL8 Hap2 ), conventional PCR electrophoresis can determine drought tolerance type, low cost, easy to use, suitable for different breeding stages, can shorten the breeding period and reduce the cost, has important scientific significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure is a drought phenotype analysis diagram of high generation population materials; In the figure, A is a drought phenotype diagram of population materials; B is a comparison diagram of survival rate of population materials; C is a schematic diagram of transcript expression amount and proportion of population materials, D is a comparison diagram of in vitro leaf water loss rate of population materials, Figure 2 For the promoter variation significantly related to ZmPYL8_T01 splice proportion and InDel_993 variation in intron, In the figure, the upper half is a Manhattan plot In which, the red dot represents the significantly associated variation site; the middle part shows ZmPYL8 The gene ZmPYL8_ T01 And ZmPYL8_T02 Transcript, black and white boxes represent exons and non-coding regions, respectively, and the 5' and 3' ends of the gene are labeled to determine the position of the significantly associated site within the gene region; The lower half is a linkage disequilibrium (LD) analysis heat map of the locus, The color of each square represents the R 2 value between the corresponding variation pairs, and the color gradient reflects the size of the linkage disequilibrium coefficient R 2 (range from 0 to 1, the closer the value is to 1, the stronger the linkage disequilibrium degree).

[0019] Figure 3 Figure is a schematic diagram of drought resistance marker identified by maize resequencing analysis; In the figure, A is a comparison diagram of different transcripts, ZmPYL8 ​Wherein the white box represents the UTR region, the black box represents the exon region, the black line segment represents the intron region, and the red box position represents the position of the marker, that is ZmPYL8 the second intron of the gene region.

[0020] B is an information diagram of the comparison of partial second intron sequences of corn B73 material and CIMBL55 material, wherein red represents the sequence of the marker.

[0021] Figure 4 B73 and CIMBL55 material promoter activity diagram; In the figure, A is a vector diagram, B is a luciferase activity diagram of the promoter of corn B73 material and CIMBL55 material.

[0022] Figure 5 is a partial PCR product electrophoresis diagram of the sample to be tested; DETAILED DESCRIPTION

[0023] The present application will be further described in detail below with specific examples, so that those skilled in the art can understand.

[0024] Example 1 Investigation of drought phenotype of population material In order to explore ZmPYL8 the genetic variation of the gene, 197 corn materials were resequenced in this embodiment. By analyzing the sequence alignment results and the difference in drought resistance, (drought-resistant material CIMBL55) was selected as the donor parent (marked as Hap2), and non-drought-resistant material B73 was selected as the recipient parent (i.e. Hap1). Through the cross implemented in summer 2022, an F1 population was successfully established. By constructing near-isogenic lines (NIL) to F6 generation, pure NIL Hap1 and NIL Hap2 were obtained.

[0025] (1) Investigation of survival rate of population material under drought Pure NIL Hap1 (8T) and NIL Hap2 (5T) were sowed and grown in plug trays, transplanted to pots (length x width x height = 34 cm x 16 cm x 12 cm) at the 1-leaf stage, and the same volume of nutrient soil was filled into each pot. NIL Hap1 and NIL Hap2 materials were planted in half, a total of 24 plants were planted in each pot, of which 12 plants of each material were planted, and 12 replicates were set. Watering was stopped at the 3-leaf stage of corn, and drought treatment was started. When the corn plants showed changes such as lodging, soft stems, and wilting leaves, watering was started. After 5 days of rehydration, the survival rate of each replicate was calculated.

[0026] Through analysis, it was found that under drought stress, NILHap2 Plant ratio NIL Hap1 The plants had a higher survival rate, indicating that NIL Hap2 Significantly improved the drought resistance of corn. Figure 1 A, B).

[0027] (2) Group materials ZmPYL8_T02 Proportional survey The pure NIL will be separated. Hap1 and NIL Hap2 Total RNA was extracted from the plants and reverse transcribed into cDNA. The cDNA was then detected by RT-qPCR. ZmPYL8_T01 and ZmPYL8_T02 The expression level, calculated ZmPYL8_T02 / ZmPYL8_T01 That is ZmPYL8_T02 The proportion. It should be noted that NIL... Hap1 It contains "8T", NIL Hap2 The more "T"s in the formula, the higher the splicing efficiency. This is known as NIL. Hap1 There are more ZmPYL8_T02 Conversely, NIL Hap2 There are fewer ZmPYL8_T02 .

[0028] Therefore, by calculation ZmPYL8_T02 The proportion of materials in a group is used to determine its drought resistance.

[0029] The results showed that NIL Hap2 in materials ZmPYL8_T02 The proportion decreased significantly, indicating that more materials bearing the drought-resistant marker "5T" were used. ZmPYL8_T01 This makes corn more drought-resistant.

[0030] (3) Investigation on water loss rate of detached leaves of group materials Pure NIL Hap1 (8T) and NIL Hap2 (5T) Sow seeds in seedling trays and transplant them into pots (length × width × height = 34cm × 16cm × 12cm) at the 1-leaf stage. Fill each pot with the same volume of potting soil and add NIL. Hap1 and NIL Hap2 The plants were planted in a 50 / 50 ratio with 8 replicates. At the 4-leaf stage of maize, healthy plants of both overexpression and wild-type varieties were selected, with 6 plants selected from each material. Five leaves from the same part of the plant were cut and weighed using a 0.01% balance; the initial weight was recorded as W0. All leaves were then left to air dry indoors, and weighed every hour, with the weights recorded as W1, W2…W… n After the leaves were detached and dried for 12 hours, weighing was stopped. The leaf water loss rate at each time point was calculated as follows: (W0 - W...) n) / W0.

[0031] By analyzing, it was found that NIL Hap2 has lower leaf water loss rate, and compared with the "NIL Hap2 " plant, it is more drought-resistant, indicating that the "NIL Hap2 " plant has stronger drought resistance ability. Figure 3

[0032] Example 2 Analysis of the relationship between intron variation and promoter variation of ZmPYL8 gene The phenotype data includes ZmPYL8 The expression amount (transcripts per million, TPM) and splicing ratio of the ZMPYL8_T01 and ZMPYL8_T02 transcripts of the gene. ZmPYL8 The SNPs in the gene and flanking regions were obtained by the following steps: extracting the relevant SNPs from the corn association panel data set, aligning them to the APGv5 reference genome for coordinate correction, and then screening by minimum allele frequency (MAF>0.05). Subsequently, whole genome association analysis (GWAS) was carried out in TASSEL5 using a mixed linear model (MLM) that includes population structure (evaluated by principal component analysis (PCA), the first three principal components were calculated by all SNPs) and kinship (K) matrix (calculated by the identity by state (IBS) method) - i.e. PCA+K model - to control false positive associations; then based on the GWAS results and the preset significance threshold, the significantly associated SNPs were determined. Finally, the linkage disequilibrium (LD) coefficient (R 2 ) between all SNPs in the ZmPYL8 gene and flanking regions was calculated using the LD analysis module of TASSEL5.

[0033] The splicing ratio of ZmPYL8_T01 was associated with genetic variation Figure 2 , the upper Manhattan plot revealed multiple genetic variation sites significantly associated with ZmPYL8_T01 splicing efficiency. The lower part is the linkage disequilibrium (LD) analysis of the locus.

[0034] The above results show that ZmPYL8 participate in plant drought stress response, and the material with drought resistance marker has higher drought resistance. Under drought stress, compared with NIL Hap1 , the NIL Hap2 plant has higher survival rate and lower leaf water loss rate, and the above results show that the NIL Hap2 material improves the drought resistance of corn. At the same time, it also shows that the marker also has the potential to realize stable yield under drought stress.​

[0035] Example 3: Screening of polymorphic molecular markers for drought traits in maize This embodiment uses drought-resistant inbred line CIMBL55 and drought-sensitive inbred line B73 as experimental materials, and identifies maize inbred lines through resequencing and sequence alignment. ZmPYL8 Promoter region mutations and intron mutations, like Figure 4 As shown, compared to B73, the CIMBL55 material has a 0.76K insertion upstream of the promoter region. New vectors were constructed by combining the B73 and CIMBL55 promoter sequences (2kb upstream of the ATG) and placing them upstream of the firefly luciferase. The LUC / REN ratio was then measured after transforming maize protoplasts to determine the difference between the two promoters. The results showed no significant difference between the two promoters, indicating that the 0.76K insertion does not affect the expression of luciferase. ZmPYL8 Key sites for the proportion of gene transcripts.

[0036] Studies have shown that UA-rich sequences can affect the efficiency of intron splicing site recognition in plants; therefore, we focused on analyzing variations in the intron region. The location of this marker is... ZmPYL8 The second intron of the gene region. Specifically, the drought resistance marker (i.e., ZmPYL8) of the drought-resistant material CIMBL55 is located anterior to the 5' end of branch point A. Hap2 The nucleotide sequence of the molecular marker is shown in SEQ ID NO:1: TTTTTAAAAAAAAAAAA, which is haplotype Hap2; the sequence marker of this marker in sensitive material B73 is (ZmPYL8). Hap1 The nucleotide sequence of the molecular marker is shown in SEQ ID NO:4 (TTTTTTTTAAA), which is haplotype Hap1.

[0037] Based on the above ZmPYL8 Hap2 Molecular marker design of primer pairs: ZmPYL8-Hap2-F: 5'-GCCTTGATTGCTTGGTACTT-3', as shown in SEQ ID No. 2; ZmPYL8-Hap2-R: 5'-ATCAACATTGACGCCTACCT-3', as shown in SEQ ID No. 3; Example 4 Kits for identifying or assisting in the identification of drought-resistant varieties of maize; primer pairs mentioned above: ZmPYL8-Hap2-F: 5'-GCCTTGATTGCTTGGTACTT-3', as shown in SEQ ID No. 2; ZmPYL8-Hap2-R: 5'-ATCAACATTGACGCCTACCT-3', as shown in SEQ ID No. 3.

[0038] Example 5: Electrophoretogram of PCR products of maize high generation NIL population The drought-resistant material CIMBL55 was used as the male parent, and the sensitive material B73 was used as the female parent, and the hybridization was F1 generation. Each generation of heterozygotes was continuously self-crossed to F5, and part of the tested varieties were selected from the heterozygotes. The above kit was used for detection, and the specific experimental steps were as follows: Gene amplification was performed using 2x Taq Master Mix, and the reaction system was as follows: The reaction program was as follows: 95℃ 3min; 95℃ 30s; 57℃ 30s; 72℃ 60s; 72℃ 5min; 35 cycles.

[0039] The results show that the size of the amplified target band is 635bp. When the target band appears, it indicates that the variety contains the drought-resistant marker, that is, the variety is haplotype Hap2, and it indicates that the variety has strong drought resistance. Figure 5 ).

[0040] The other parts not specifically described are prior art. Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A polymorphic molecular marker for drought trait in maize, characterized in that: The polymorphic molecular marker is ZmPYL8 Hap2 The molecular marker, wherein the ZmPYL8 Hap2 The nucleotide sequence of the molecular marker is shown as SEQ ID NO:

1.

2. Use of the polymorphic molecular marker of claim 1 in identifying or assisting in identifying drought-resistant maize varieties and breeding drought-resistant maize varieties.

3. A primer pair for obtaining the polymorphic molecular marker of claim 1, characterized in that: The primer pair is: ZmPYL8-Hap2-F: 5'-GCCTTGATTGCTTGGTACTT-3', ZmPYL8-Hap2-R: 5'-ATCAACATTGACGCCTACCT-3'.

4. Use of the primer pair of claim 3 in identifying or assisting in identifying drought-resistant maize varieties.

5. A kit for identifying or aiding in the identification of drought resistant varieties of maize, characterized in that: The kit contains the primer pair of claim 3.

6. A method of identifying or aiding in the identification of drought resistant varieties of maize, characterized by: The kit contains the primer pair of claim 3. The kit contains the primer pair of claim 3. The kit contains the primer pair of claim 3. 1) Extracting DNA from the maize varieties to be tested, When a pair of primers ZmPYL8-Hap2-F and ZmPYL8-Hap2-R amplifies the target band, it indicates that the corn variety contains ZmPYL8 Hap2 The molecular marker, i.e. the variety is haplotype Hap2, indicates that the variety is strong in drought resistance; Or, when a pair of primers ZmPYL8-Hap2-F and ZmPYL8-Hap2-R cannot amplify the desired band, it indicates that the corn variety does not contain ZmPYL8 Hap2 Molecular markers indicate that the variety is weakly drought-resistant.

7. The method of claim 6, wherein: 2) PCR amplification using the primer pair of claim 3 or the kit of claim 5 to obtain PCR products; 3) PCR product amplification, electrophoresis results for judgment: In step 2), PCR amplification uses 2x Taq Master Mix for gene amplification, and the reaction system is as follows: Reaction program: 95℃ 3min; 95℃ 30s; 57℃ 30s; 72℃ 60s; 72℃ 5min; 35 cycles.

8. Use of the kit of claim 5 in breeding drought-resistant maize varieties.

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