CAPS molecular marker related to corn stem rot resistance gene ZmIAA19 and application of CAPS molecular marker

Abstract

The invention discloses a CAPS molecular marker related to a corn stem rot resistance gene ZmIAA19 and application of the CAPS molecular marker, and belongs to the technical field of plant breeding. The CAPS molecular marker is named as DNCAP79, the molecular marker is a fragment obtained through PCR amplification by using a CAPS primer pair and taking a corn genome as a template, the full length of the fragment is 143bp, the 32th basic group is an SNP locus, the locus in a stem rot-sensitive material is T, cannot be cut by PvuII, and is a fragment of 143bp through enzyme digestion detection, and the locus in a stem rot-resistant material is G; and the gene can be cut into two fragments of 111bp and 32bp by PvuII. The molecular marker provided by the invention can be used for cultivating stem rot resistant transgenic plants, provides a new technical means and method for screening and creating a stem rot resistant corn new material, and has important value in the field of plant breeding.

Description

Technical Field

[0001] This invention relates to a CAPS molecular marker associated with the maize stalk rot resistance gene ZmIAA19 and its application. It belongs to the field of plant breeding technology. Background Technology

[0002] Maize is an important crop for food, feed, and ethanol production, playing a vital role in economic development. Abiotic stress is a major limiting factor affecting maize yield, severely impacting its growth and development and causing large-scale yield reductions. Maize stalk rot is one of the major diseases in most major producing areas, and has long been a significant constraint on improving maize yield per unit area. Therefore, identifying genes related to maize stalk rot resistance, and subsequently screening and creating new stalk rot-resistant maize varieties, is of great importance to improving maize's resistance to stalk rot.

[0003] When subjected to biological stress, plants undergo a series of physiological changes, such as regulating the levels of endogenous hormones, in response to Fusarium graminearum infection. At the same time, Fusarium graminearum infection can induce the expression of plant stem rot resistance-related genes, activating their regulatory networks to respond to biological stress and minimize the damage caused by Fusarium graminearum infection to plants.

[0004] Aux / IAA is a negative regulator in the auxin signaling pathway. It binds to the transcription factor ARF to form a heterodimer, thereby negatively regulating the expression of auxin-regulated genes and playing a crucial role in the entire plant auxin signaling process. Current reports indicate that the aux1 mutant in plants such as Arabidopsis thaliana can enhance resistance to Fusarium oxysporum; however, there are few reports on the role of the Aux / IAA gene in maize stalk rot resistance. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a molecular marker related to the maize stalk rot resistance gene ZmIAA19 and its application.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] This invention identifies the ZmIAA19 (iaa19 - Aux / IAA-transcriptionfactor 19) gene, which is associated with maize stalk rot resistance. It analyzes the sequence variations in the coding region of the gene in different maize inbred lines, and then performs association analysis with the stalk rot resistance of the inbred lines to identify associated loci and develop molecular markers.

[0008] First, this invention proposes a CAPS molecular marker associated with the maize stalk rot resistance gene ZmIAA19, named DNCAP79. This molecular marker is a fragment obtained by PCR amplification using the maize genome as a template, with a full length of 143 bp. The 32nd base is an SNP site. In susceptible stalk rot materials, this site is T, which cannot be cleaved by PvuII, and enzyme digestion results in a 143 bp fragment. In resistant stalk rot materials, this site is G, which can be cleaved by PvuII into two fragments of 111 bp and 32 bp. The sequences of the CAPS primer pair are shown in SEQ ID NO. 3 and 4.

[0009] The primer pairs used to amplify the CAPS molecular marker are also within the scope of protection of this invention, and the sequences of the primer pairs are shown in SEQ ID NO.3 and 4.

[0010] Furthermore, this invention also proposes the use of the aforementioned CAPS molecular marker and primer pair in molecular marker-assisted breeding for maize resistance to stalk rot.

[0011] Furthermore, this invention also proposes a method for identifying resistance to maize stalk rot, comprising:

[0012] The primers described above were used to test the corn samples to be tested, and the stem rot resistance of the plant samples to be tested was determined based on the test results.

[0013] Preferably, the method includes: extracting genomic DNA from the maize sample to be tested, performing PCR amplification using the primer pair, digesting the amplification product with PvuII restriction endonuclease, wherein the stem rot-susceptible material cannot be digested by PvuII restriction endonuclease to obtain a fragment of 143 bp in length, and the stem rot-resistant material can be digested by PvuII restriction endonuclease to obtain two fragments of 111 bp and 32 bp in length.

[0014] Preferably, the nucleotide sequence of the 143bp fragment is shown in SEQ ID NO.5; the nucleotide sequence of the 111bp fragment is shown in SEQ ID NO.6; and the nucleotide sequence of the 32bp fragment is shown in SEQ ID NO.7.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Based on the maize stalk rot resistance-related gene ZmIAA19, the present invention analyzes the sequence variations in the CDS region of the ZmIAA19 gene in different maize inbred lines, conducts an association analysis with the stalk rot resistance phenotypic indicators of the inbred lines, mines the associated loci, analyzes the restriction enzyme sites of the loci and selects restriction endonucleases, and develops a CAPS molecular marker related to the plant stalk rot resistance gene ZmIAA19. The detection of the mutation type of this CAPS molecular marker in plants can be used to detect the stalk rot resistance of plants. The CAPS molecular marker provided by the present invention can be applied to improving the germplasm resources of maize, cultivating maize varieties resistant to stalk rot, and increasing the yield and quality of maize, which has important value in the field of maize planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the genotype detection result of the molecular marker DNCAP79 provided in Example 2 of the present invention;

[0019] Among them, M is the DNA Marker, and 1-6 are the PCR products of Ji853, Mo17, Dan99L, Zhong106, B73, and HD568 respectively; b: M is the DNA Marker, and the enzyme digestion products of PvuII-HF for wells 1-11 are Qi319, Mo17, R01-4-2, B73, PH4CV, DNY089, DNY052, Ye478, H340, P02-9-1, P07-6-2, PH6WC, DNY176, and DNY092 respectively;

[0020] Figure 2 It is the verification result of the molecular marker DNCAP79 in some inbred lines provided in Example 3 of the present invention;

[0021] Among them, M is the DNA Marker, and the enzyme digestion products of PvuII for wells 1-24 are PHVA9, DNY085, DNY089, DNY032, DNY041, DNY113, HF12202, DNY052, Dong412, Dong409, Jun92-8, DNY098, DNY110, DNY045, DNY139, ICI 893, DNTD512, Line 14, DNY007, DNY184, DN-1-2, T07, PH207, and DNY072 respectively. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0023] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0024] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0025] Example 1: Study on the correlation between the ZmIAA19 gene and maize resistance to stalk rot

[0026] I. Experimental Methods

[0027] Material selection: The core materials were the highly resistant stalk rot inbred lines DN1722 and Qi319 and the highly susceptible inbred lines H340 and Ye478. At the same time, 317 maize inbred lines with broad genetic bases (Table 1) and 103 inbred lines with different resistance (Table 2) were also involved.

[0028] Table 1. Resistance levels of the tested inbred lines to stem rot

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] Table 2 Evaluation of stalk rot resistance levels of 103 maize inbred lines

[0036]

[0037]

[0038] Note: HR indicates high resistance, R indicates resistance, MR indicates moderate resistance, S indicates susceptibility, and HS indicates susceptibility.

[0039] Transcriptome sequencing and differential gene screening: Transcriptome sequencing was performed on DN1722 and H340 cells inoculated with Fusarium graminearum (12h, 24h) and uninoculated cells. Differentially expressed genes were screened using |log2foldchange|≥1 and P<0.05 as the criteria.

[0040] qRT-PCR validation: Primers were designed for candidate genes, and their expression levels were detected after inoculation with resistant materials to validate the reliability of transcriptome data.

[0041] Bioinformatics analysis: Analyzing the conserved domains, physicochemical properties, secondary and tertiary structures, subcellular localization, and other characteristics of candidate genes using online tools.

[0042] Sequence variation and association analysis: The CDS region of 103 inbred lines ZmIAA19 was amplified, and polymorphism was analyzed using DNASPv6.0. Combined with the stem rot resistance phenotype, association analysis was performed using TASSEL5.0.

[0043] II. Experimental Results

[0044] 1. Identification and functional localization of the ZmIAA19 gene:

[0045] Transcriptome screening identified three differentially expressed genes associated with stem rot resistance. Among them, Zm00001eb212890 (iaa19-Aux / IAA-transcription factor 19) is a candidate gene for stem rot resistance. Located on chromosome 5, it is 1492 bp in length and named ZmIAA19, belonging to the 19th member of the IAA gene family. Its sequence is shown in SEQ ID NO.1. Its CDS sequence is 546 bp long, encoding 182 amino acids, and its sequence is shown in SEQ ID NO.2.

[0046] This gene is mainly involved in auxin response, plant hormone signal transduction, and fungal defense response pathways. Phylogenetic analysis shows that its function is related to abiotic and biotic stresses.

[0047] 2. The relationship between ZmIAA19 expression pattern and resistance:

[0048] qRT-PCR results showed that ZmIAA19 expression was significantly downregulated at 12h and 24h after inoculation in disease-resistant materials (DN1722 and Qi319) (P<0.01). The relative expression levels in DN1722 were -2.75 and -2.01, respectively, while those in Qi319 were -2.56 and -1.34, respectively. In susceptible materials (H340 and Ye478), the gene expression was significantly downregulated at 12h and significantly upregulated at 24h (P<0.01). The relative expression levels in H340 were -2.21 at 12h and 2.15 at 24h, while those in Ye478 were -3.04 at 12h and 1.66 at 24h.

[0049] III. Conclusion

[0050] The ZmIAA19 gene participates in the defense response of maize against stalk rot by regulating its expression pattern (downregulated in resistant materials and upregulated in susceptible materials). The SNP79 site variation in its CDS region can be used as a key indicator for resistance screening. The developed DNCAP79 marker can be effectively applied to the screening and breeding of maize germplasm resistant to stalk rot.

[0051] Example 2: Sequence Variation Analysis of the Maize ZmIAA19 Gene

[0052] 1. Primers designed in this invention (Table 3) were used to amplify the DNA sequences of 103 maize inbred lines (Table 2) with different resistance to stalk rot. The CDS region of the ZmIAA19 gene was extracted using Snap Gene software, and the sequence variation analysis of the CDS region of the ZmIAA19 gene of 102 maize inbred lines was performed using DNA SPv 6.0 software.

[0053] Table 3 Primers for ZmIAA19 gene sequence

[0054]

[0055] 2. ZmIAA19 haplotype analysis

[0056] In this invention, 19 SNP sites and 2 InDel sites were detected in the CDS region of the ZmIAA19 gene of 103 maize inbred lines with different stalk rot resistance levels. The SNP sites were located at positions 36, 47, 78, 79, 82, 90, 115, 158, 162, 273, 283, 314, 354, 375, 410, 433, 496, 498, and 516, respectively, while the InDel sites were located at positions 57-65 and 142-147 (Table 4). The polymorphic sites were genotyped based on the type and number of nucleotide variations. Using DNA SPv6.0 software, 12 haplotypes were detected, with a haplotype polymorphism of 0.6815. The major haplotypes HAP1, HAP2, and HAP11 accounted for 78.6% of the tested materials, while the remaining haplotypes were rare variations. The HAP1 haplotype includes 50 inbred lines, mostly highly susceptible and susceptible materials such as Ye 478; the HAP2 haplotype includes 29 inbred lines, mostly resistant and moderately resistant materials; the HAP3 haplotype includes 4 inbred lines; and the HAP11 haplotype includes 2 inbred lines, composed of the highly resistant material Qi 319 and the resistant material Danhuang 25. Based on the above results, the 29 materials in HAP2, mostly resistant to or moderately resistant to stem rot, are preliminarily identified as superior haplotypes (Table 5).

[0057] Table 4. SNPs, InDel values, and haplotypes in the CDS region of the ZmIAA19 gene.

[0058]

[0059] Table 5 Self-crossing lines corresponding to ZmIAA19 haplotype

[0060]

[0061] 2. Analysis of changes in amino acid levels in the ZmIAA19 gene

[0062] In this invention, 10 synonymous mutation SNP sites, 9 non-synonymous mutation SNP sites, and 2 InDel sites were found in the CDS region of the ZmIAA19 gene in 103 maize inbred lines. Non-synonymous mutations may alter the structure and physiological function of the protein (Table 6). The changes are as follows: SNP47 changes from serine (Ser, S) to threonine (Thr, T); SNP79 changes from serine (Ser, S) to alanine (Ala, A); SNP115 changes from valine (Val, V) to leucine (Leu, L); SNP158 changes from aspartic acid (Asp, D) to alanine (Ala, A); SNP283 changes from glycine (Gly, G) to serine (Ser, S); SNP314 changes from alanine (Ala, A) to valine (Val, V); SNP410 changes from alanine (Ala, A) to glycine (Gly, G); SNP433 changes from valine (Val, V) to isoleucine (Ile, I); and SNP496 changes from isoleucine (Ile, I) to valine (Val, V). InDel InDel 57-65 encodes amino acids missing three amino acids: valine (V), isoleucine (Ile, I), and asparagine (Asn, N). InDel 142-147 encodes amino acids missing two amino acids: aspartic acid (Asp, D) and histidine (His, H).

[0063] Table 6. Amino acid changes corresponding to SNPs of nonsynonymous mutations in the ZmIAA19 gene.

[0064]

[0065] 3. Nucleotide diversity analysis of ZmIAA19 gene

[0066] This invention combines the results of artificial inoculation stalk rot phenotypic identification of maize inbred lines with the SNP nucleotide diversity and haplotype diversity analysis of the ZmIAA19 gene sequence in 103 maize inbred lines using DNAspv6.0 software. Tajima's D value was 0.37776, nucleotide diversity π = 0.00862, and haplotype diversity was 0.6810, all of which did not reach a highly significant level, indicating that the ZmIAA19 gene belongs to neutral evolution in the maize inbred line population.

[0067] Example 3 Development of CAPS functional markers for the ZmIAA19 gene

[0068] 1. Association analysis between ZmIAA19 gene sequence variation and maize stalk rot resistance

[0069] Based on the results of previous artificial inoculation phenotypic identification of maize inbred lines in our laboratory, and the 19 SNP sites and 2 InDel sites in the CDS region of the ZmIAA19 gene, association analysis was performed using TASSEL 5.0 software. The results showed that at the P<0.05 level, SNP47, SNP273, SNP516, SNP78, SNP79, SNP162, SNP375, and InDel142-147 were significantly correlated with field phenotypic indicators of stalk rot resistance. The phenotypic contribution rates of synonymous mutation sites SNP273, SNP162, SNP78, SNP516, and SNP375, and non-synonymous mutation sites SNP79, SNP47, and InDel142-147 were 18.21%, 15.07%, and 9.53%, respectively, with SNP79 showing the highest contribution rate (Table 7).

[0070] Table 7. Results of correlation analysis between ZmIAA19 gene sequence variations and maize resistance to stalk rot.

[0071]

[0072] 2. Design of CAPS functional markers for the ZmIAA19 gene

[0073] Based on the results of CDS region association analysis of the ZmIAA19 gene, this invention selected the SNP79 (T / G) site, which was significantly correlated with the field stem rot resistance phenotype index (P<0.01) and had the largest phenotypic contribution rate, for CAPS marker development. The optimal restriction enzyme site and restriction endonuclease were analyzed using the online website CAPS Finder to develop the functional marker. The marker transformed from SNP79 (T / G) was named DNCAP79. PCR amplification showed a full length of 143 bp (shown in SEQ ID NO.5), with the 32nd base being the mutation site. If the stem rot-susceptible material had the T site, it could not be cleaved by PvuII, and the restriction enzyme detection showed a 143 bp band. If the stem rot-resistant material had the G site, it could be cleaved by PvuII into two fragments: 111 bp (shown in SEQ ID NO.6) and 32 bp (shown in SEQ ID NO.7). Figure 1 ).

[0074] Example 4: Verification of DNCAP79 Functional Markers

[0075] This invention uses 317 maize inbred lines with varying resistance to stalk rot, previously evaluated in our laboratory, as test materials (Table 1) to validate the DNCAP79 marker. Using total genomic DNA from different maize inbred lines as templates, PCR amplification was performed using marker primers. The amplified products were recovered and digested with PvuII restriction endonuclease.

[0076] The results are as follows Figure 2 As shown, out of 317 maize inbred lines, 137 could be digested with Pvull (Table 8), with digestion products of 111 bp and 32 bp in size. Among them, 111 materials showed moderate to high resistance genotypes, and 26 materials showed susceptible genotypes. The concordance rate between enzyme-resistant genotypes and phenotypes was 81.02% (Table 9). In conclusion, DNCAPS79 can effectively identify SNP79 (T / G) sites and can be used to screen inbred lines resistant to stalk rot.

[0077] Table 8 Evaluation of stalk rot resistance levels of 317 maize inbred lines

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Table 9 Selection efficiency analysis of DNdCAPS603

[0085]

[0086] In summary, the molecular marker DNCAP79 developed based on the maize ZmIAA19 gene in this invention can be used to identify maize stalk rot resistance, and then to screen or breed stalk rot resistant maize varieties.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A CAPS molecular marker associated with the maize stalk rot resistance gene ZmIAA19, characterized in that, The CAPS molecular marker is named DNCAP79. The molecular marker is a fragment obtained by PCR amplification using maize genome as a template with CAPS primer pairs. The full length of the fragment is 143 bp, where the 32nd base is an SNP site. In stem rot-susceptible materials, this site is T, which cannot be cleaved by PvuII, and the enzyme digestion detection shows a 143 bp fragment. In stem rot-resistant materials, this site is G, which can be cleaved by PvuII into two fragments of 111 bp and 32 bp. The sequences of the CAPS primer pairs are shown in SEQ ID NO. 3 and 4.

2. The use of the CAPS molecular marker as described in claim 1 in marker-assisted breeding of maize for resistance to stalk rot.

3. A primer pair for amplifying the CAPS molecular marker of claim 1, characterized in that, The sequences of the primer pairs are shown in SEQ ID NO.3 and 4.

4. The use of the primer pair according to claim 3 in molecular marker-assisted breeding of maize for resistance to stalk rot.

5. A method for identifying resistance to maize stalk rot, characterized in that, include: The primers described in claim 3 are used to test the corn sample to be tested, and the stem rot resistance of the plant sample to be tested is determined based on the test results.

6. The method according to claim 5, characterized in that, include: Genomic DNA was extracted from the maize sample to be tested and PCR amplification was performed using the primer pair described in claim 3. The amplification product was digested with PvuII restriction endonuclease. The stem rot-susceptible material could not be digested by PvuII restriction endonuclease, resulting in a fragment of 143 bp in length. The stem rot-resistant material could be digested by PvuII restriction endonuclease, resulting in two fragments of 111 bp and 32 bp in length.

7. The method as described in claim 6, characterized in that, The nucleotide sequence of the 143 bp fragment is shown in SEQ ID NO.5; the nucleotide sequence of the 111 bp fragment is shown in SEQ ID NO.6; and the nucleotide sequence of the 32 bp fragment is shown in SEQ ID NO.7.

Images