Closely linked SNP molecular markers, primer sets and detection methods for the Ht3 gene for resistance to corn leaf blight

Abstract

The present invention relates to the field of molecular biology technology, and in particular, to a closely linked SNP molecular marker, primer set and detection method for the corn northern leaf blight resistance Ht3 gene. The molecular markers include KA002081 and KA002084; the disease resistance allele locus of KA002081 is A; the disease resistance allele locus of KA002084 is G. In the reference gene sequence of corn variety B73, KA002081 is located at 154681279bp on chromosome 8, and KA002084 is located at 155270367bp on chromosome 8. The molecular marker can accurately detect the corn northern leaf blight resistance Ht3 gene, and can be used to identify the corn northern leaf blight resistance Ht3 genotype and assist breeding, which is of great significance to the improvement of corn northern leaf blight resistance. The detection method performs genotyping on SNP molecular markers, and has the advantages of simple operation, low cost, short detection cycle, stable markers, etc.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a closely linked SNP molecular marker, a primer set and a detection method for a corn northern blight resistance Ht3 gene. Background Art

[0002] Diseases are an important factor limiting corn yield and quality, causing serious losses to corn production. Corn leaf blight (NCLB) is caused by the fungus Exserohilum turcicum (Chang and Fan, 1986) and is one of the most destructive foliar diseases in most corn-growing areas in the world. Corn grown in areas with high humidity and moderate temperatures is more susceptible to NCLB outbreaks. During the grain filling period, NCLB causes leaf necrosis, disrupts photosynthesis, and thus reduces grain yield (Raymundo and Hooker, 1981). Although the use of fungicides can prevent and control NCLB, the cost is high and reduces farmers' income. Therefore, developing molecular markers for corn leaf blight resistance genes and conducting molecular-assisted selection breeding are the most environmentally friendly and cost-effective methods to reduce yield losses caused by NCLB.

[0003] NCLB resistance is a very complex trait, including both qualitative and quantitative resistance. So far, quality resistance genes such as Ht1, Ht2, Ht3 and Htn1 have been discovered. The first quality resistance gene Ht1 discovered in the inbred lines GE440 and Ladyfinger popcorn in 1959 is partially dominant and located on chromosome 2L (Bentolila et al., 1991). Ht1 can significantly inhibit the formation of conidia during the chlorosis process (Hilu and Hooker, 1964, 1965). Ht2 has a similar resistance phenotype and genetic structure to Ht1 and is located on chromosome 8L (Hooker, 1977). Ht3 is a dominant single gene independent of Ht1 and Ht2, which was introduced into the maize genome from Tripsacumfloridanum (Hooker, 1981). Recently, Ht2 and Ht3 were considered to be identical and allelic to the previously cloned gene Htn1 (Yang et al., 2021). Although plants containing Ht2, Ht3, and Htn1 all showed delayed damage, plants containing Ht2 or Ht3 still showed yellowing (Hooker, 1977, 1981; Welz and Geiger, 2000; Yang et al., 2021). This suggests that there are other genes involved in the NCLB resistance function of Htn1. In addition, histological studies have shown that plants with different resistance genes (Ht1, Ht2, Ht3, Htn1) show different symptoms, indicating that the resistance mechanisms conferred by these resistance genes are not the same (Navarro et al., 2020).

[0004] Previous studies have shown that quantitative trait loci (QTLs) for NCLB resistance are distributed on 10 chromosomes of maize (Balint-Kurti et al., 2010; Welz and Geiger, 2000; Wisseret et al., 2006; Zwonitzeret et al., 2010). Although a large number of NCLB resistance QTLs have been found in different populations, few QTLs have been finely mapped or cloned. So far, ZmWAK-RLK1 at the Htn1 locus is the only QTL gene for resistance to corn leaf blight cloned by map-based cloning (Hurni et al., 2015). Further studies have found that ZmWAK-RLK1-mediated NCLB resistance is associated with reduced benzoxazole content (Yang et al., 2019).

[0005] Conventional molecular marker-assisted breeding has low throughput, high cost, and long time consumption, and cannot efficiently support breeding selection. Taking conventional SSR molecular marker detection as an example, it takes about 3-4 days to complete the detection and data analysis of 2 molecular markers in a backcross population of 1,000 plants. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a closely linked SNP molecular marker, a primer set and a detection method for a corn northern blight resistance Ht3 gene.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] The invention provides a closely linked SNP molecular marker of a corn northern blight resistance Ht3 gene, including KA002081 and KA002084.

[0009] Furthermore, the allele locus of KA002081 is T or A; the allele locus of KA002084 is C or G.

[0010] Furthermore, the disease resistance allele locus of KA002081 is A; and the disease resistance allele locus of KA002084 is G.

[0011] Further, in the reference gene sequence of corn variety B73, KA002081 is located at 154681279bp on chromosome 8, and KA002084 is located at 155270367bp on chromosome 8.

[0012] The present invention also provides application of the tightly linked SNP molecular marker of the corn northern leaf blight resistance Ht3 gene in corn breeding.

[0013] The present invention also provides a primer set, which is used for amplifying the corn genome and detecting the tightly linked SNP molecular marker of the corn northern blight resistance Ht3 gene.

[0014] Further, the primer set for detecting KA002081 includes a first forward primer as shown in the sequence SEQ ID NO.1, a second forward primer as shown in the sequence SEQ ID NO.2, and a reverse primer as shown in the sequence SEQ ID NO.3;

[0015] The primer set for detecting KA002084 includes a first forward primer as shown in the sequence SEQ ID NO.4, a second forward primer as shown in the sequence SEQ ID NO.5, and a reverse primer as shown in the sequence SEQ ID NO.6.

[0016] The present invention also provides a method for detecting the genotype of the corn leaf blight resistance gene Ht3, using the above primer set to respectively detect the disease resistance allele loci of KA002081 and the disease resistance allele loci of KA002084 in the genome of the corn sample;

[0017] When the disease resistance allele locus of KA002081 is A, and the disease resistance allele locus of KA002084 is G, the corn sample is determined to have the Ht3 genotype.

[0018] Furthermore, the primer set is used to detect the genome of the corn sample through a KASP reaction.

[0019] The present invention also provides a method for molecular marker-assisted breeding of corn leaf blight resistance, comprising the following steps:

[0020] S1, obtaining a first corn plant containing a marker site of a corn leaf blight resistance gene Ht3 in its genome;

[0021] S2, hybridizing the first corn plant with the second corn plant to obtain a progeny plant;

[0022] S3. Using the above method, detect the genotype of the corn leaf blight resistance gene Ht3 in the progeny plants, and screen out the progeny plants with the Ht3 genotype to complete the breeding.

[0023] The beneficial effects of the present invention are:

[0024] (1) The SNP molecular marker tightly linked to the corn leaf blight resistance Ht3 gene of the present invention can accurately detect the Ht3 gene in the corn gene; at the same time, because it is tightly linked to the Ht3 gene, it can still reliably mark Ht3 after recombination and has good stability;

[0025] (2) The SNP molecular marker tightly linked to the corn leaf blight resistance Ht3 gene of the present invention can be used to identify whether corn has resistance to leaf blight at the DNA level, without the need for further identification after plant development, thereby improving the identification efficiency;

[0026] (3) The closely linked SNP molecular markers of the maize northern blight resistance Ht3 gene of the present invention are all based on the Douglas Sdentifie "Array Tape" high-throughput SNP molecular marker detection platform. TM ) was developed, which can select breeding populations quickly, at low cost and accurately, greatly improving breeding efficiency and is of great significance to improving resistance to corn leaf blight;

[0027] (4) The method of the present invention for detecting the genotype of the corn leaf blight resistance gene Ht3 uses a special primer set to perform genotyping on the SNP molecular marker, which has the advantages of simple operation, low cost, short detection cycle, and stable markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the diseased leaf area of ​​each corn plant in Example 1 of the present invention; wherein, Figure 1 A in the middle is a schematic diagram of the diseased leaves of A619, A619Ht3 and L3162. Figure 1 B is the percentage of diseased leaf area (DLA) of each type of plant, Figure 1 C is a schematic diagram of diseased leaves with a DLA percentage ranging from 0 to 100%;

[0029] Figure 2 The resistance to corn leaf blight of maize inbred lines A619, A619Ht3 and L3162 in Example 1 of the present invention is shown; wherein, Figure 2 A in the middle is the gene locus and corresponding disease resistance of each recombinant in the BC4F1 population; Figure 2 B in the middle is the gene loci and corresponding disease resistance of each recombinant in the BC6F1 population; Figure 2 C in the middle is the gene loci of each recombinant in the BC8F1 population and the corresponding disease resistance; Figure 2 D stands for BC 10 The gene loci of each recombinant in the F1 population and the corresponding disease resistance; Figure 2 E stands for BC 10 Four pairs of near-isogenic lines of F3 and their DLA;

[0030] Figure 3 In Example 2 of the present invention, BC4F1, BC6F1, BC8F1, BC 10 F1 and BC 10 Bar graph showing the results of genetic effect detection of the Ht3 gene locus in F3. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0032] The maize large leaf spot resistance Ht3 gene tightly linked SNP molecular markers of the present invention include KA002081 and KA002084. Specifically, for the reference gene sequence (AGPV5) of maize variety B73, the physical position of KA002081 is located at 154681279bp on chromosome 8, and the physical position of KA002084 is located at 155270367bp on chromosome 8.

[0033] The molecular markers KA002081 and KA002084 of the present invention are closely linked SNP molecular markers of the Ht3 gene. SNP (single nucleotide polymorphisms) refers to single nucleotide polymorphisms. SNP molecular markers have the characteristics of high genetic stability. After multiple generations, they can still accurately mark Ht3, have high reliability, and are more suitable for assisted breeding.

[0034] The allele locus of KA002081 is T or A; the allele locus of KA002084 is C or G.

[0035] Preferably, the disease resistance allele locus of KA002081 is A; and the disease resistance allele locus of KA002084 is G.

[0036] The tightly linked SNP molecular marker of the corn northern leaf blight resistance Ht3 gene of the present invention can be applied to corn breeding.

[0037] The primer set of the present invention is used to amplify the above-mentioned corn northern blight resistance Ht3 gene tightly linked SNP molecular marker.

[0038] Among them, the primer set used to amplify KA002081 includes a first forward primer as shown in sequence SEQ ID NO.1, a second forward primer as shown in sequence SEQ ID NO.2, and a reverse primer as shown in sequence SEQ ID NO.3; the primer set used to amplify KA002084 includes a first forward primer as shown in sequence SEQ ID NO.4, a second forward primer as shown in sequence SEQ ID NO.5, and a reverse primer as shown in sequence SEQ ID NO.6.

[0039] Among them, the types of the above sequences are all DNA.

[0040] The method for detecting the Ht3 genotype of the corn leaf blight resistance gene of the present invention uses the above-mentioned primer set to respectively detect the disease resistance allele site of KA002081 and the disease resistance allele site of KA002084 in the genome of the corn sample; when the disease resistance allele site of KA002081 is A and the disease resistance allele site of KA002084 is G, it is determined that the corn sample has the Ht3 genotype.

[0041] Preferably, the specific detection steps are:

[0042] S1: A primer set is used to perform KASP reaction detection on the genome of corn samples; wherein two pairs of forward primers are connected to different fluorescent sequences respectively.

[0043] S2: Only when the fluorescence signals corresponding to the fluorescence sequences connected to markers KA002081 and KA002084 are detected at the same time, the corn sample is judged to have the genotype of the northern leaf blight resistance gene Ht3.

[0044] Preferably, the fluorescent sequence is a fluorescent sequence commonly used in the art, namely FAM and HEX fluorescent sequences. FAM and HEX fluorescent sequences are connected to the 5' end of two sets of forward primers, respectively, wherein FAM is connected to the first forward primer and HEX is connected to the second forward primer.

[0045] The method for molecular marker-assisted breeding of corn leaf blight resistance of the present invention comprises the following steps:

[0046] S1, obtaining a first corn plant containing a marker site of a corn leaf blight resistance gene Ht3 in its genome;

[0047] S2, hybridizing the first corn plant with the second corn plant to obtain a progeny plant;

[0048] S3. Use the above method to detect the Ht3 genotype of the corn leaf blight resistance gene in the progeny plants, and screen out the progeny plants with the Ht3 genotype to complete the breeding.

[0049] The technical solution of the present invention is explained and illustrated by means of specific embodiments below.

[0050] Example 1

[0051] This example discovered the tightly linked SNP molecular marker of the corn leaf blight resistance Ht3 gene through experiments. The specific experimental process is as follows:

[0052] This example uses a pair of near-isogenic lines A619Ht3 and A619, which were introduced from the Eastern Grains and Oilseeds Research Center of the Canadian Ministry of Agriculture and Agri-Food (Ma et al., 2014). After years of multi-point inoculation and natural disease identification in Liaoning Province, A619Ht3 has high resistance to corn leaf blight, while A619 is highly sensitive to corn leaf blight ( Figure 1 ), the homozygosity of the two genomes was 98.84%. A backcross population of A619Ht3 (resistant parent) and L3162 (susceptible parent) was constructed for preliminary QTL detection and initial positioning.

[0053] The BC2F1 populations of A619, A619Ht3 and A619Ht3 / L3162 were genotyped using maize Maize3K and Maize6K chips. Through whole genome and regional association analysis, the Ht3 locus was preliminarily located on bin8.06, flanked by SNP markers PZE-108095959 and PZE-108110343. With reference to the B73 physical map (AGPv5), the physical distance was 11.23 Mb, as shown in Table 1.

[0054] Table 1 SNP molecular markers significantly associated with resistance to corn leaf blight at bin8.06 locus

[0055]

[0056] Based on the above preliminary positioning of the Ht3 locus, a fine positioning strategy based on the recombinant progeny verification method was continued to narrow down the Ht3 locus.

[0057] As shown in Table 2, this example developed 18 KASP markers for genotyping segregating populations for fine mapping.

[0058] Table 218 KASP tag information

[0059]

[0060] a. Physical positioning according to the B73 reference genome sequence (AGPV5).

[0061] b. FAM fluorescent sequence forward primer.

[0062] c. HEX fluorescent sequence forward primer.

[0063] After 5 rounds of fine mapping, the Ht3 locus was finally located in the 577kb region between KA002081 and KA002084 on chromosome 8, with a physical distance of 11.23Mb. The specific positioning process is as follows:

[0064] like Figure 2As shown, in the first round of fine mapping, six KASP markers (A001802, A000823, A001155, A001807, A000827 and A001808) were used to detect recombination events in the 11.23MbHt3 region of the BC2F1 population. The seven BC2F1 recombinants obtained were backcrossed twice with the susceptible parent L3162 to obtain the corresponding BC4F1 population. Using another five newly developed KASP markers (A007452, A007453, A007455, A007456 and A007457), the detailed recombination breakpoints of the seven recombinants were determined. 1678 BC4F1 progeny were planted in the field and their diseased leaf area (DLA) percentages were determined. At the same time, all individuals were genotyped using markers in the heterozygous Ht3 region to distinguish between homozygous (L3162 / L3162) and heterozygous (A619Ht3 / L3162) offspring. Using the genotypic and phenotypic data, the average percentage of DLA in homozygous and heterozygous genotypes in each BC4F1 offspring can be estimated. The t-test was used to calculate the significant difference in diseased leaf area (DLA) between each genotype. The results of the assay are shown in Figure 2. Figure 2 As shown in A.

[0065] according to Figure 2 As can be seen in Figure A, the significant difference in DLA between the two genotypes indicates that there is an Ht3 site in the A619Ht3 donor segment, otherwise, there is no Ht3 site. Recombinant IV has a significant difference between homozygous and heterozygous genotypes in its BC4F1 progeny (P<0.05), so there is an Ht3 site on the A619Ht3 donor segment, and the corresponding parental recombinant is inferred to be NCLB resistant (R); while recombinants VI and VII have no significant difference (P>0.05), so there is no Ht3 site, and the corresponding recombinants are inferred to be NCLB susceptible (S). Recombinants V (with Ht3) and VII (without Ht3) define it as the left border marker of A001807. At the same time, recombinants I (with Ht3) and VI (without Ht3) define the right border marker of A007455. These recombinants clearly restricted the Ht3 locus to the donor region flanked by markers A001807 and A007455, with a physical distance of 2.17 Mb (AGPv5).

[0066] The BC4F1 recombinant was selected and backcrossed twice with L3162 to produce its BC6F1 progeny. Genotyping was performed using 11 markers in the 2.17Mb region, including 7 newly developed markers (KA002076, KA002079, KA002081, KA002082, KA002084, KA002085, and KA002088) to determine the exact crossover interval. Similarly, markers in the heterozygous Ht3 region were used to distinguish between homozygous and heterozygous BC6F1 progeny. Figure 2 As shown in B, the Ht3 locus is located in the A001807 / KA002076 or KA002081 / KA002085 interval.

[0067] The BC6F1 recombinant was backcrossed twice with L3162 to obtain 7 BC8F1 offspring, containing a total of 2540 individuals. Figure 2 As shown in C, through the analysis of genotype and phenotype data, the new recombinant (IV) in the heterozygous region downstream of marker KA002076 showed significant differences in DLA between homozygous and heterozygous BC8F1 offspring, which excluded the A001807 / KA002076 interval as a candidate region. Similarly, the third round of fine mapping located the Ht3 locus to the regions on both sides of markers KA002081 and KA002085, with a physical distance of 838 kb (AGPv5).

[0068] like Figure 2 As shown in D, the BC8F1 recombinant was backcrossed twice with L3162 to obtain 3200 BC10F1 plants. Through the analysis of genotype and phenotypic data, the new recombinants (IV and VI) between KA002082 and KA002084 showed that Ht3 was missing from the heterozygous region, thereby moving the right boundary marker from KA002085 to KA002084. Therefore, Ht3 was finally divided on both sides of markers KA002081 and KA002084, with a physical distance of 577 kb (AGPv5).

[0069] On the basis of the above experiments, the Ht3 gene locus of corn resistance to northern leaf spot was further verified.

[0070] BC 10 The F1 offspring heterozygous recombinants were self-fertilized twice to obtain BC 10 In F3, four pairs of near isogenic lines (NILs) were screened to evaluate their DLA. Figure 2 As shown in Figure E, the first three pairs of NILs (I-III) showed significant differences in DLA between NILs with and without A619Ht3 donors, but there was no significant difference in pair 4. Therefore, Ht3 is definitely restricted to the interval on both sides of markers KA002081 and KA002084, with a physical distance of 577 kb (AGPv5).

[0071] Example 2

[0072] This example tests the genetic effect of the Ht3 locus.

[0073] The donor material and its offspring homozygous Ht3 / Ht3 genotypes have always maintained a high resistance to chlorosis and reduced lesion area. 10The DLA of the Ht3 homozygous and heterozygous genotypes in the F1 population. The difference in DLA between the homozygous ht3 / ht3 and heterozygous Ht3 / ht3 genotypes in each generation is calculated. The last two columns calculate the BC of the two homozygous genotypes ht3 / ht3 and Ht3 / Ht3, respectively. 10 The DLA of F3 was calculated by paired two-tailed t-test, with P value < 0.05 indicating a significant difference, and P value > 0.05 indicating no significant difference.

[0074] By calculating BC4F1, BC6F1, BC8F1 and BC 10 The difference in diseased leaf area (DLA) between heterozygous and homozygous genotypes in the F1 population was used to calculate the genetic effect of the Ht3 / ht3 genotype.

[0075] like Figure 3 As shown, the average DLA values ​​of BC4F1 for ht3 / ht3 and Ht3 / ht3 genotypes were 20.27% and 16.45%, BC6F1 were 29.43% and 21.58%, BC8F1 were 40.54% and 32.10%, and BC 10 The values ​​for F1 were 42.20% and 35.88%, respectively. As expected, the DLA values ​​of Ht3 / ht3 heterozygotes were lower than those of ht3 / ht3 homozygotes.

[0076] The genetic effect of Ht3 / ht3 was 3.82% in the BC4F1 population, 7.86% in the BC6F1 population, 8.44% in the BC8F1 population, and 1.3% in the BC 10 It was 6.32% in the F1 population.

[0077] The average DLA values ​​of the susceptible offspring (ht3 / ht3) and the resistant offspring (Ht3 / Ht3) were 37.53% and 22.11%, respectively. Therefore, the genetic effect of the homozygous Ht3 / Ht3 genotype was estimated to be 16.5%.

[0078] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A SNP molecular marker tightly linked to the Ht3 gene for resistance to corn leaf blight, characterized in that: Including KA002081 and KA002084; KA002081 is located at 154681279bp on chromosome 8 of the maize gene sequence, and KA002084 is located at 155270367bp on chromosome 8; The allele locus of KA002081 is T or A; the allele locus of KA002084 is C or G; the disease resistance allele locus of KA002081 is A; the disease resistance allele locus of KA002084 is G; The corn gene sequence is the reference gene sequence AGPv5 of corn variety B73; The primer set for detecting KA002081 includes a first forward primer as shown in the sequence SEQ ID NO.1, a second forward primer as shown in the sequence SEQ ID NO.2, and a reverse primer as shown in the sequence SEQ ID NO.3; The primer set for detecting KA002084 includes a first forward primer as shown in the sequence SEQ ID NO.4, a second forward primer as shown in the sequence SEQ ID NO.5, and a reverse primer as shown in the sequence SEQ ID NO.

6.

2. Use of the tightly linked SNP molecular marker of the corn northern blight resistance Ht3 gene according to claim 1 in corn breeding.

3. A primer set, characterized in that: The primer set is used to amplify the corn genome and detect the closely linked SNP molecular marker of the corn northern blight resistance Ht3 gene according to claim 1; The primer set for detecting KA002081 includes a first forward primer as shown in the sequence SEQ ID NO.1, a second forward primer as shown in the sequence SEQ ID NO.2, and a reverse primer as shown in the sequence SEQ ID NO.3; The primer set for detecting KA002084 includes a first forward primer as shown in the sequence SEQ ID NO.4, a second forward primer as shown in the sequence SEQ ID NO.5, and a reverse primer as shown in the sequence SEQ ID NO.

6.

4. A method for detecting the genotype of the corn leaf blight resistance gene Ht3, characterized in that: The primer set of claim 3 is used to detect the disease resistance allele loci of KA002081 and the disease resistance allele loci of KA002084 in the genome of the corn sample respectively; When the disease resistance allele locus of KA002081 is A, and the disease resistance allele locus of KA002084 is G, the corn sample is determined to have the Ht3 genotype.

5. A method for detecting the genotype of the corn leaf blight resistance gene Ht3 according to claim 4, characterized in that: The primer set is used to detect the genome of the corn sample through the KASP detection method.

6. A method for detecting the genotype of the corn leaf blight resistance gene Ht3 according to claim 5, characterized in that: The fluorescence sequences used in the KASP detection method are FAM fluorescence sequence and HEX fluorescence sequence.

7. A method for molecular marker-assisted breeding of corn leaf blight resistance, characterized in that: The following steps are involved: S1, obtaining a first corn plant having marker sites KA002081 and KA002084 of a corn leaf blight resistance gene Ht3 in its genome; S2, hybridizing the first corn plant with other corn plants to obtain progeny plants; S3. Use the method described in claim 5 or 6 to detect the genotype of the corn leaf blight resistance gene Ht3 in the progeny plants, and screen out the progeny plants with the Ht3 genotype to complete the breeding.

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