A dCAPS molecular marker related to low temperature tolerance of maize seedling stage and application thereof

Abstract

The application discloses a dCAPS molecular marker related to corn seedling low-temperature resistance and application thereof, relates to the technical field of plant molecular breeding, and the dCAPS molecular marker comprises a nucleotide sequence composed of a base at the 31st position shown in SEQ ID NO. 1 and upstream and downstream bases of the base, and the polymorphism of the base at the 31st position shown in SEQ ID NO. 1 is A or C; when the polymorphism is A, the corn to be detected is a low-temperature resistant corn variety; and when the polymorphism is C, the corn to be detected is a low-temperature sensitive corn variety. The molecular marker can be used for quickly and accurately identifying the genotype of the corn low-temperature resistance at the seedling stage, has the advantages of co-dominance, simple operation, low cost, independence from precise instruments and the like, and can effectively accelerate the breeding process of the corn low-temperature resistant variety.

Description

Technical Field

[0001] This invention relates to the field of plant molecular breeding technology, specifically to a dCAPS molecular marker related to low-temperature tolerance in maize seedlings and its application. Background Technology

[0002] Maize (Zea mays L.) is an important food crop originating from tropical low-latitude regions. It is not only a direct source of food for humans, but also an important foundation for livestock feed and industrial raw materials. As a warm-loving C4 crop, maize is extremely sensitive to temperature and remains highly susceptible to low-temperature damage. Low-temperature damage can cause abnormalities in cell membrane structure, photosynthetic organs, and reproductive organs, affecting the physiological metabolism of maize plants and producing harmful substances that impair growth and development, leading to reduced yields. Maize's sensitivity to low temperatures is mainly due to weakened photosynthetic capacity and metabolic disorders. Short-term exposure of maize seedlings to low temperatures can lead to reduced photosynthetic activity and affect the transport of assimilates. Therefore, it is necessary to cultivate cold-resistant maize varieties.

[0003] Existing studies have shown that there are significant differences in cold tolerance among different maize inbred lines. It is generally believed that maize cold tolerance is a quantitative trait inherited trait. Traditional identification of maize cold tolerance mainly relies on field phenotypic observation and physiological index measurement. However, these methods have disadvantages such as being complex to operate, time-consuming, and greatly affected by environmental factors, making it difficult to meet the needs of large-scale breeding screening. In contrast, molecular marker technology analyzes at the molecular genome level, which can quickly, efficiently, and accurately identify target traits and is not affected by the environment. Phenotypic selection based on molecular marker genotypes can overcome the disadvantages of traditional breeding being time-consuming and labor-intensive.

[0004] Single nucleotide polymorphism (SNP) markers, due to their abundant genetic polymorphisms in the genome and high frequency of occurrence within genes, offer advantages such as high efficiency, accuracy, and economy. They can significantly shorten the breeding cycle and serve as a powerful supplement to traditional breeding techniques. A relatively economical and convenient method for detecting SNPs is through cleaved amplified polymorphic sequences (CAPS) markers or derived CAPS markers. Both CAPS (segmentation-dependent restriction enzyme digestion) and dCAPS (dCAPS) markers combine PCR amplification with restriction enzyme digestion. Restriction endonucleases recognize and digest target SNP sequences, followed by electrophoretic typing. CAPS markers can be developed for SNP sequences with naturally occurring restriction sites, while dCAPS markers can be developed for SNPs without naturally occurring restriction sites by introducing mutated bases. However, the probability of naturally occurring SNPs being distinguished by restriction endonucleases is low, limiting the number of CAPS markers that can be developed. Nevertheless, dCAPS, by introducing mismatched bases into primers and binding to the SNP to create restriction sites not originally present in the sequence, theoretically allows for the detection of polymorphisms at all SNP sites. Both CAPS and dCAPS markers offer advantages such as co-dominance, site specificity, ease of operation, rapid detection, low cost, and independence from sophisticated instruments, making them suitable for plant genotyping, mapping, genetic diversity analysis, and variety identification.

[0005] In the study of molecular markers for maize cold tolerance, some studies have attempted to discover cold-related genes through genome-wide association analysis (GWAS) and quantitative trait locus (QTL) analysis. However, most studies have stopped at the preliminary localization or candidate gene stage. There are currently no reports of developing molecular markers that co-segregate with maize cold tolerance using dCAPS molecular marker technology. Therefore, it is necessary to conduct further research on dCAPS molecular markers for maize cold tolerance in order to provide a more convenient screening method for breeding cold-tolerant maize varieties. Summary of the Invention

[0006] To address the lack of simple, economical, and efficient specific molecular markers for identifying low-temperature tolerance in maize in existing technologies, the present invention aims to provide a dCAPS molecular marker related to low-temperature tolerance in maize seedlings and its application.

[0007] The present invention achieves the above objectives through the following technical solutions: This invention provides a dCAPS molecular marker related to low temperature tolerance in maize seedlings. The dCAPS molecular marker includes a nucleotide sequence consisting of the base at position 31 as shown in SEQ ID NO.1 and its upstream and downstream bases. The polymorphism of the base at position 31 as shown in SEQ ID NO.1 is A or C.

[0008] The present invention also provides an application of the dCAPS molecular marker as described above in identifying the low-temperature resistance of maize. When the 31st base of the dCAPS molecular marker as shown in SEQ ID NO.1 is A, the maize to be tested is a low-temperature resistant maize variety. When the 31st base of the dCAPS molecular marker as shown in SEQ ID NO.1 is C, the maize to be tested is a low-temperature sensitive maize variety.

[0009] As a further optimization of the present invention, the dCAPS molecular marker sequence of the low-temperature resistant maize variety is shown in SEQ ID NO.4, and the dCAPS molecular marker sequence of the low-temperature sensitive maize variety is shown in SEQ ID NO.5.

[0010] This invention also provides a method for identifying the low-temperature tolerance of maize using the dCAPS molecular markers described above, comprising the following steps: S1. Extract DNA from the corn to be tested; S2. Using the sequence containing the base site of the dCAPS molecular marker and its upstream and downstream bases as an amplification template, PCR amplification is performed using primers for verifying the dCAPS molecular marker to obtain an amplification product containing the site of the dCAPS molecular marker. S3. Genotyping the amplification products to obtain the dCAPS molecular marker type of the maize to be tested.

[0011] As a further optimization of the present invention, the nucleotide sequence of the primer for verifying the dCAPS molecular marker is shown in SEQ ID NO.2-3: SEQ ID NO.2: Upstream primer: 5'-CCGGTCAATTCCTCTTTTTTCGCTGGGTAC-3'; SEQ ID NO.3: Downstream primer: 5'-GGCAAGTTGAAGGGCGACTGGGA-3'.

[0012] As a further optimization of the present invention, the genotyping detection method is to obtain the enzyme digestion product by digesting the amplification product with restriction endonuclease, detect the enzyme digestion product by agarose gel electrophoresis, and perform genotyping based on the image. If the enzyme digestion product is a single band, the dCAPS molecular marker type is A; if the enzyme digestion product is two bands, the dCAPS molecular marker type is C.

[0013] As a further optimization of the present invention, if the dCAPS molecular marker type of the maize to be tested is A, then the maize to be tested is a low-temperature resistant maize variety; if the dCAPS molecular marker type of the maize to be tested is C, then the maize to be tested is a low-temperature sensitive maize variety.

[0014] As a further optimization of the present invention, the restriction endonuclease is restriction endonuclease Kpn I.

[0015] The present invention has the following beneficial effects: 1) This invention screened and obtained an SNP site closely linked to maize's low-temperature tolerance, and developed it into a dCAPS molecular marker. The dCAPS molecular marker can rapidly and accurately identify the genotype of maize's low-temperature tolerance trait during the seedling stage. It has the advantages of codominance, simple operation, low cost, and no dependence on precision instruments, which can effectively accelerate the breeding process of maize low-temperature tolerant varieties. 2) This invention obtains maize genomic DNA amplification products by using primers to verify the dCAPS molecular marker as shown in SEQ ID NO.2-3. After digestion with restriction endonuclease Kpn I, if the digestion product is a single band, the dCAPS molecular marker type is A; if the digestion product is two bands, the dCAPS molecular marker type is C. The identification results are intuitive and reliable, and can be directly interpreted by agarose gel electrophoresis without sequencing. The operation is simple and low-cost, and it is suitable for large-scale germplasm resource screening and molecular marker-assisted breeding. Attached Figure Description

[0016] Figure 1 This is an agarose gel electrophoresis image of the dCAPS molecular markers used in this invention to identify the low-temperature sensitive genotype maize material (HapB) and the low-temperature resistant genotype maize material (HapA). Figure 2 This is a phenotypic comparison diagram of the low-temperature sensitive genotype maize material (B73) and the low-temperature resistant genotype maize material (Mo17) of the present invention; Figure 3 This is a record table of haplotype and low-temperature treatment phenotypic examination of partially inbred lines of the IBM population of the present invention; Figure 4 This is an agarose gel electrophoresis image of the haplotype of the partially inbred IBM population of the present invention; Figure 5 It is the Mo17 of the NIL group of this invention. HapA and Mo17 HapB Phenotypic comparison diagrams of low temperature treatment experiments, where A is before treatment, B is after 3 days of treatment at 4℃, and C is a close-up view of the leaves numbered 1-6 in B. Figure 6 This is an agarose gel electrophoresis image identifying the haplotype of dCAPS molecular markers in maize inbred lines. Figure 7 This is a record of the haplotype and low-temperature treatment phenotype of dCAPS molecular markers in maize inbred lines. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] 1. Materials Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0019] 2. Method 2.1. Acquisition of tightly linked SNP sites for low-temperature tolerance in maize and development of dCAPS molecular markers A SNP site closely linked to low-temperature tolerance in maize was obtained through screening. This SNP site is located at the 5177825th base on chromosome 8 of the maize B73 inbred line (as shown in SEQ ID NO.1, the 31st base), and its polymorphism is A or C.

[0020] Specific primers were designed to target the sequence differences at this SNP site, and dCAPS molecular markers were developed. The specific primer sequences are shown in SEQ ID NO.2-3, specifically: SEQ ID NO.2: Upstream primer: dCAPS-F1-KpnI: 5'-CCGGTCAATTCCTCTTTTTTCGCTGGGTAC-3'; SEQ ID NO.3: Downstream primer: dCAPS-R1-KpnI: 5'-GGCAAGTTGAAGGGCGACTGGGA-3'; Two haplotype maize materials were identified: the low-temperature tolerant maize inbred line Mo17 (HapA haplotype, with Y base A in the HapA haplotype) and the low-temperature sensitive maize inbred line B73 (HapB haplotype, with Y base C in the HapB haplotype). Genomic DNA was extracted from the two haplotype maize materials and used as templates for PCR amplification. PCR amplification was performed using the specific primers and restriction endonuclease KpnI designed above to obtain different gene fragments. The PCR amplification system consisted of: a total volume of 20 μL, genomic DNA of 2 μL, forward and reverse primers of 0.5 μL each, 2×Es Taq DNA Polymerase Reaction mix of 10 μL, and ddH2O of 7 μL. The PCR amplification program was as follows: denaturation at 95℃ for 3 min; 32 cycles of denaturation at 95℃ for 30 s, 58℃ for 30 s, and 72℃ for 15 s; extension at 72℃ for 5 min. The PCR amplification product was digested with the restriction endonuclease Kpn I. The 10 μL digestion system consisted of: 5 μL PCR product, 0.3 μL Kpn I (KpnI HF, catalog number: NEB, R3142S), 1 μL rCutSmart Buffer, and 3.7 μL ddH2O. The digestion product was obtained by digestion at 37°C for 8 hours. Take 6 μL of the above enzyme digestion product and perform genotyping using 3% agarose gel electrophoresis. If the enzyme digestion product contains only one 142bp band, the maize material to be tested is a low-temperature tolerant maize line; if the enzyme digestion product contains two bands, 112bp and 30bp, the maize material to be tested is a low-temperature sensitive maize line.

[0021] Experimental results: such as Figure 1 As shown, the DNA fragment amplified by the above primer sequence from the low-temperature resistant maize material (Mo17) produced only one 142bp band after digestion with restriction endonuclease Kpn I; the DNA fragment amplified by the above primer sequence from the low-temperature sensitive maize material (B73) produced two bands of 112bp and 30bp after digestion with restriction endonuclease Kpn I; and the heterozygous maize material produced three bands of 142bp, 112bp, and 30bp after digestion.

[0022] 2.2 Validation of SNP sites in the IBM population like Figure 2-4As shown, 134 recombinant inbred lines of the IBM population constructed by multiple generations of self-pollination after crossing Mo17 and B73 were subjected to low-temperature treatment (4℃ for three days) and genotypic analysis was performed. The results showed that there were 80 haplotypes HapA and 54 haplotypes HapB. Among them, 13 haplotypes of HapA were sensitive to low temperature and 67 were tolerant to low temperature, with a genotypic-phenotypic concordance rate of 83.75%; haplotypes of HapB had 36 low-temperature sensitive types and 18 tolerant to low temperature, with a genotypic-phenotypic concordance rate of 66.67%. This indicates that this SNP locus makes a significant contribution to the low-temperature tolerance phenotype of maize Mo17.

[0023] 2.3 Validation of SNP sites in the Mo17 NIL population with haplotype HapB F1 generation was obtained by crossing maize inbred lines Mo17 and B73 as parents. Six consecutive backcrosses were conducted using F1 as the female parent and recurrent parent Mo17 as the male parent. In each backcross generation, individual plants carrying the B73-derived haplotype HapB were retained through genotyping. After six rounds of backcrossing, the target individual plants were self-pollinated and purified. Homozygous individuals with the HapB haplotype were selected by genotyping to construct near-isogenic lines (NILs) with a background of Mo17 and the target segment infused with the HapB haplotype. HapA and Mo17 HapB Two-leaf-one-heart seedlings were treated at 4℃ for three days, and their phenotypes were observed after three days.

[0024] like Figure 5 As shown, the experimental results indicate that Mo17 with the HapA haplotype exhibits resistance to low temperatures, while Mo17 with the HapB haplotype is sensitive to low temperatures and shows obvious leaf damage phenotype. This suggests that in Mo17, the HapA haplotype plays a decisive role in its low-temperature resistance phenotype.

[0025] 2.4. Screening of low-temperature tolerance in maize inbred lines at the seedling stage using dCAPS molecular markers Genotyping and phenotypic identification were performed on 70 collected maize inbred lines. Among them, 35 haplotypes of HapA were identified, with 33 exhibiting cold tolerance in the seedling stage (94.3% phenotype). 29 haplotypes of HapB were identified, with 23 exhibiting cold sensitivity and 6 exhibiting cold tolerance (79.3% phenotype). Six heterozygous genotypes were identified, with 4 exhibiting cold tolerance and 2 being cold sensitivity. Partial genotyping results are available in [link to genotyping results]. Figure 6 The corresponding phenotypic statistics can be found in [link to statistics]. Figure 7 This indicates that the HapA haplotype is widely present in the collected natural inbred lines and is significantly correlated with low-temperature stress in these inbred lines.

[0026] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A dCAPS molecular marker related to low temperature tolerance at the seedling stage of maize, characterized in that, The dCAPS molecular marker comprises a nucleotide sequence consisting of the base at position 31 and its upstream and downstream bases as shown in SEQ ID NO.1, wherein the polymorphism of the base at position 31 as shown in SEQ ID NO.1 is A or C.

2. Use of a dCAPS molecular marker according to claim 1 for identifying cold tolerance in maize, characterized by: When the dCAPS molecular marker, as shown in SEQ ID NO.1, has an A base at position 31, the maize to be tested is a low-temperature resistant maize variety; when the dCAPS molecular marker, as shown in SEQ ID NO.1, has a C base at position 31, the maize to be tested is a low-temperature sensitive maize variety.

3. Use according to claim 2, characterized in that, The dCAPS molecular marker sequence of the low-temperature resistant maize variety is shown in SEQ ID NO.4, and the dCAPS molecular marker sequence of the low-temperature sensitive maize variety is shown in SEQ ID NO.

5.

4. A method for identifying the low-temperature tolerance of maize using the dCAPS molecular marker as described in claim 1, characterized in that: Includes the following steps: S1. Extract DNA from the corn to be tested; S2. Using the sequence containing the base site of the dCAPS molecular marker and its upstream and downstream bases as an amplification template, PCR amplification is performed using primers for verifying the dCAPS molecular marker to obtain an amplification product containing the site of the dCAPS molecular marker. S3. Genotyping the amplification products to obtain the dCAPS molecular marker type of the maize to be tested.

5. The method according to claim 4, characterized in that, The nucleotide sequences of the primers used to verify the dCAPS molecular marker are shown in SEQ ID NO.2-3: SEQ ID NO.2: Upstream primer: 5'-CCGGTCAATTCCTCTTTTTTCGCTGGGTAC-3'; SEQ ID NO.3: Downstream primer: 5'-GGCAAGTTGAAGGGCGACTGGGA-3'.

6. The method according to claim 4, characterized in that, The genotyping detection method involves obtaining the digested products by digesting the amplification products with restriction endonucleases, detecting the digested products using agarose gel electrophoresis, and performing genotyping based on the images. If the digested product is a single band, the dCAPS molecular marker type is A; if the digested product is two bands, the dCAPS molecular marker type is C.

7. The method according to claim 6, characterized in that, If the dCAPS molecular marker type of the maize to be tested is A, then the maize to be tested is a low-temperature resistant maize variety; if the dCAPS molecular marker type of the maize to be tested is C, then the maize to be tested is a low-temperature sensitive maize variety.

8. The method according to claim 6, characterized in that, The restriction endonuclease is Kpn I.

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