Method for breeding corn germplasm with high southern rust resistance

Abstract

The invention relates to the technical field of molecular assisted breeding, in particular to a breeding method of corn germplasm with high southern rust resistance, which comprises the following steps: hybridizing excellent receptor variety and southern rust-resistant corn germplasm to obtain F1 generation; performing backcross on the F1 generation for 2-3 generations by using the recurrent parent, selecting strains which are high in background recovery rate, short in flanking linkage burdensome breaking distance and free of transgenic pollution through molecular marker detection, and selecting an advanced generation backcross improved line after comprehensive evaluation; performing a field experiment, and observing the performance; and comprehensively evaluating agriculture, quality and southern rust resistance, and selecting final stable corn germplasm resistant to southern rust. The backcross generation can be greatly shortened, breeding is accelerated, the breeding period is greatly shortened, and the efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular assisted breeding, and in particular to a method for breeding corn germplasm with high resistance to southern rust. Background Art

[0002] Corn is a staple food crop and a major source of feed and light industrial energy. Southern corn rust is an airborne disease that can easily break out over a large area when conditions are right, causing devastating damage to corn production. In recent years, due to global warming and other factors such as cropping systems, the incidence of corn rust pathogens has gradually increased, and the impact on corn production has become increasingly serious. The most economical and effective way to control the disease is to develop corn lines that carry resistance genes.

[0003] At present, the breeding of maize varieties resistant to southern rust mainly relies on experience and phenotypic selection for improvement. The traditional backcross breeding process is as follows: Figure 1 As shown, there are problems such as long breeding cycle, low efficiency and poor predictability, and the accuracy is not high, which may lead to the loss of some excellent traits and affect the final crop quality.

[0004] In the existing technology, molecular assisted breeding is also used to shorten the breeding cycle and improve accuracy. The existing technology shortens the breeding cycle by shortening the time for detecting genes: relying on experience and phenotypic selection for improvement, it is necessary to wait until the corn grows to a certain stage before observation, while molecular assisted breeding can be used for detection in the corn seedling stage. However, the existing technology of molecular assisted breeding still requires backcrossing for more than 4-5 generations, and the large number of backcrossing generations leads to a relatively long breeding cycle. Summary of the invention

[0005] The purpose of the present invention is to provide a method for breeding corn germplasm with high resistance to southern rust, which can greatly shorten the backcrossing generation, accelerate breeding, greatly shorten the breeding cycle and improve efficiency.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for breeding corn germplasm with high resistance to southern rust disease comprises the following steps:

[0008] S1, using the recipient parent as the female parent and hybridizing it with the donor parent of the superior inbred line resistant to southern rust to obtain the corn hybrid F1 resistant to southern rust, and screening the F1 with heterozygous resistance to southern rust and a background recovery rate greater than or equal to 50% as the recurrent parent F1 by detecting the genotype of the F1;

[0009] S2. Backcrossing the recurrent parent F1 with the recipient parent to obtain BC1F1, and screening to obtain the recurrent parent BC1F1, the screening conditions include:

[0010] 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 85%; 3) The parent-offspring relationship is normal; 4) The flanking linkage markers are preferably broken on both sides, followed by the unilateral breaking distance ≤ 5M; 5) No national standard transgenic pollution;

[0011] S3, backcrossing the recurrent parent BC1F1 with the recipient parent to obtain BC2F1; and screening to obtain the selfing parent BC2F1, the screening conditions include:

[0012] 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 95%; 3) Parent-offspring relationship is normal; 4) Double-sided break of flanking linkage markers is preferred, followed by single-sided break distance ≤5M; 5) The difference site between the selfing parent BC2F1 and the recipient parent is ≤1; 6) No national standard transgenic pollution;

[0013] S4. BC2F2 is obtained by self-pollination of BC2F1, and the genotype of BC2F2 is tested. If BC2F2 has homozygous resistance to southern rust, BC2F2 is used as the selected germplasm. Otherwise, BC2F1 is backcrossed with the recipient parent to obtain BC3F1, and the materials are screened until the self-pollinated material has homozygous resistance to southern rust.

[0014] The present invention optimizes the screening conditions of each generation. First, the present invention adopts molecular assisted breeding. By detecting the genotype, it can be determined whether the sample has heterozygous resistance to southern rust. Compared with the existing technology that relies on experience and phenotypic selection for improvement, the breeding cycle can be shortened. Secondly, the background recovery rate, parent relationship, and flanking linkage marker breaking are introduced into the screening conditions of the invention, and the difference site ≤1 between the selfing parent BC2F1 and the recipient parent is introduced in the parental self-pollination generation, thereby greatly shortening the backcross generation. Compared with the existing technology, at least 4-5 generations of backcrossing are required. The present invention can breed stable germplasm with southern rust resistance at the earliest in BC2F2.

[0015] In a preferred embodiment, the background recovery rate is calculated as follows:

[0016] (1) Recode the genotype of each individual in the population according to the parental genotype, that is, convert the genotype file into an ABH format file;

[0017] (2) Calculate the background recovery rate based on the common allelic variation between the recurrent parent and a certain offspring.

[0018] In a preferred embodiment, the background recovery rate is calculated as follows:

[0019]

[0020] In the formula, Na represents the number of homozygous markers between the progeny and the recurrent parent; N hIt represents the number of markers that are homozygous in the recurrent parent and heterozygous in the offspring; N represents the total number of markers with genotypes (non-NA) existing in both the recurrent parent and the offspring.

[0021] In a preferred embodiment, the parent-offspring relationship reflects whether the parent-offspring relationship is normal by calculating the Mendel error rate.

[0022] In a preferred embodiment, the Mendelian error rate threshold is 5%, and errors exceeding 5% are considered to indicate an abnormal parent-child relationship.

[0023] In a preferred embodiment, the flanking linkage drag-breaking distance calculation process is:

[0024] Use a single-point product to measure the genotype of the progeny material; convert the genotype file into an ABH format file and find the breaking position.

[0025] In a preferred embodiment, the specific process of finding the breaking position is as follows:

[0026] 1) If the genotype of the flanking marker closest to the breakpoint is A, check the genotypes of the five flanking markers including the site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, then the marker position is the breaking position;

[0027] 2) If the genotype of the flanking marker next to the breakpoint is A, check the genotypes of the five flanking markers including this site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, and the next site is B or H, then the marker position is the breaking position;

[0028] 3) If the genotype of the first flanking marker is A, and the proportion of H or B in the genotypes of the next five markers is ≥ 80%, that is, at most one marker has a genotype of A, then the marker position is the breaking position;

[0029] 4) For the flanking markers at the remaining positions, loop through and judge: if the following conditions are met at the same time: the marker genotype is A, the 5 flanking marker genotypes including the site contain A proportion ≥ 80%, the next marker genotype is B or H, and the proportion of H or B in the last five marker genotypes is ≥ 80%, then the marker position is a breaking position.

[0030] In a preferred embodiment, a liquid phase chip is used to detect the genotype.

[0031] In a preferred embodiment, the differential loci are detected using multiple SSR detection products.

[0032] In a preferred embodiment, in step S1, quality control is performed on the parents: parents with lower deletion rate and heterozygosity rate are selected for hybridization.

[0033] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0034] The present invention adopts molecular assisted breeding. By detecting the genotype, it can determine whether the sample has heterozygous resistance to southern rust. Compared with the prior art that relies on experience and phenotypic selection for improvement, the breeding cycle can be shortened. Secondly, the screening conditions of the present invention introduce background recovery rate, parent-child relationship, and flanking linkage marker breaking, and introduce the difference site ≤1 between the selfing parent BC2F1 and the recipient parent in the parent self-pollination generation, thereby greatly shortening the backcrossing generation. Compared with the prior art that requires at least 4-5 backcrossing generations, the present invention can breed stable germplasm with southern rust resistance as early as BC2F2; the genetic background recovery rate of the recurrent parent of the technology at the BC2F1 stage can reach more than 95%, and the genetic background recovery rate of the recurrent parent of the seeds finally bred is more than 99%; the present invention can shorten the breeding cycle from two aspects of genotype detection time and shortening backcrossing generations. In particular, the present invention can greatly shorten the breeding cycle by shortening the backcrossing generation, and the screened germplasm has an extremely high genetic background recovery rate, indicating that the screened germplasm is of high quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0036] Figure 1 This is a flow chart for traditional backcross breeding;

[0037] Figure 2 This is a flow chart of the molecular-assisted backcross breeding of the present invention;

[0038] Figure 3 It is a schematic diagram of breaking the flanking linkage encumbrance of the present invention (left wing);

[0039] Figure 4 It is a schematic diagram of breaking the flank interlocking encumbrance of the present invention (right flank);

[0040] Figure 5 It is a flow chart of flanking linkage redundancy determination of the present invention;

[0041] Figure 6 It is a normal distribution diagram of the background recovery rate of BC1F1 of population Z-1808 of the present invention;

[0042] Figure 7 is the background recovery rate of the F1 generation of sample 1 in the case of the present invention;

[0043] Figure 8 This is the chromosome distribution map of the RppK introduced fragment of the F1 generation of sample 1 in the case of the present invention;

[0044] Fig. 9 This is the chromosome distribution map of the RppK introduced fragment in the BC2F2 generation in the case of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. The schematic implementation mode of the present invention and its description are only used to explain the present invention and are not intended to limit the present invention. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0046] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present invention. However, it is apparent to those of ordinary skill in the art that these specific details do not have to be employed to practice the present invention. In other embodiments, in order to avoid confusing the present invention, known structures, materials or methods are not specifically described. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.

[0047] Embodiment 1:

[0048] The existing technology uses empirical judgment to lead to a long breeding cycle, and uses molecular assisted breeding technology to shorten the genotype detection time. However, the number of backcross generations is large (more than 4-5 generations), which still leads to the problem of a long breeding cycle.

[0049] This embodiment provides a method for breeding corn germplasm with high resistance to southern rust. Figure 2 As shown, the following steps are included:

[0050] S1. Select a recipient parent with excellent agronomic traits (moderate plant height (the plant height of corn is between 200-250 cm), many kernels per ear (the number of kernels per ear of corn is between 400-600 kernels), high yield, and strong stress resistance) as the female parent, and hybridize it with an excellent inbred donor parent resistant to southern rust to obtain a corn hybrid F1 resistant to southern rust. Detect the genotype of F1 by liquid phase chip, screen out heterozygous resistance to southern rust, and select F1 with a background recovery rate greater than or equal to 50% as the recurrent parent F1; preferably, perform quality control on the parents: select parents with lower deletion rate and heterozygosity rate for hybridization. In the entire molecular marker-assisted backcross selection, all analyses are based on the markers of the parents. In order to avoid deviations caused by field management or testing, the parents are first quality controlled. The effect of backcross breeding depends to a large extent on whether the parent selection is appropriate. Parents with high deletion rate and high heterozygosity rate will cause abnormal results in the backcross introduction analysis.

[0051] S2. Backcrossing the recurrent parent F1 with the recipient parent to obtain BC1F1, and screening to obtain the recurrent parent BC1F1, the screening conditions include:

[0052] 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 85%; 3) The parent-offspring relationship is normal; 4) The flanking linkage markers are preferably broken on both sides, followed by the unilateral breaking distance ≤ 5M; 5) No national standard transgenic pollution;

[0053] S3, backcrossing the recurrent parent BC1F1 with the recipient parent to obtain BC2F1; and screening to obtain the selfing parent BC2F1, the screening conditions include:

[0054] 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 95%; 3) Parent-offspring relationship is normal; 4) Double-sided break of flanking linkage markers is preferred, followed by single-sided break distance ≤5M; 5) The difference site between the selfing parent BC2F1 and the recipient parent is ≤1; 6) No national standard transgenic pollution;

[0055] S4. BC2F2 is obtained through self-pollination of BC2F1, and the genotype of BC2F2 is tested. If BC2F2 has homozygous resistance to southern rust, BC2F2 is used as the selected germplasm. Otherwise, BC2F1 is backcrossed with the recipient parent to obtain BC3F1, and the materials are screened. The screening conditions of BC3F1 materials are the same as those of BC2F1, until the self-pollinated materials have homozygous resistance to southern rust.

[0056] Specifically: The calculation process of the background recovery rate is:

[0057] (1) Recode the genotype of each individual in the population according to the parental genotype, that is, convert the genotype file into an ABH format file; the genotype recoding rules are as follows:

[0058] A: genotype consistent with the defined parent A; H: heterozygous genotype of parent A and parent B; B: genotype consistent with the defined parent B; C: genotype of parent A is missing, but the offspring is a homozygous genotype of a non-parent B genotype; D: genotype of parent B is missing, but the offspring is a homozygous genotype of a non-parent A genotype; U: other conditions that do not meet the above conditions or the genotype is missing.

[0059] (2) Calculate the background recovery rate based on the common allelic variation between the recurrent parent and a certain offspring.

[0060] 3. A method for breeding corn germplasm with high resistance to southern rust according to claim 2, characterized in that the background recovery rate is calculated as follows:

[0061]

[0062] In the formula, Na represents the number of homozygous markers between the progeny and the recurrent parent; N h It represents the number of markers that are homozygous in the recurrent parent and heterozygous in the offspring; N represents the total number of markers with genotypes (non-NA) existing in both the recurrent parent and the offspring.

[0063] Calculation example:

[0064] 1) If the genotypes of the recurrent parent and offspring at a certain locus are C / C and C / C respectively, the "Na" of the molecule is increased by 1;

[0065] 2) If the genotypes of the recurrent parent and offspring at a certain locus are C / C and C / G respectively, the "Nh" of the numerator is increased by 1;

[0066] 3) If the genotypes of the recurrent parent and offspring at a certain locus are C / C and G / G respectively, the numerator is not calculated;

[0067] 4) If at a certain locus, the genotype exists in both the recurrent parent and the offspring, the denominator "N" is increased by 1.

[0068] Among them, the parent-child relationship reflects whether the parent-child relationship is normal by calculating the Mendel error rate.

[0069] 1) Theoretical background: Since corn is an open-pollinated crop, it is extremely susceptible to contamination by exogenous pollen during the breeding process. At the same time, the large-scale field management also inevitably results in a small number of errors. Therefore, Mendelian genetic error analysis based on molecular markers can effectively help determine the correspondence between parents and offspring, and thus determine the parent-child relationship between offspring and parents.

[0070] 2) Calculation principle and formula: The alleles of each genetic locus of the offspring are passed from parents to offspring in a Mendelian way. Therefore, the genetic locus of each allele between the parent-offspring pair consisting of the candidate parent and the offspring can be determined. If the two alleles at a certain locus of the offspring individual are inconsistent with the two alleles of the candidate parent, it can be regarded as a Mendelian error.

[0071] For example, if the offspring genotype is AA and the candidate parent genotype is CT; if one or two alleles at a certain locus of the offspring individual are the same as those of the candidate parent, and the candidate parent is a heterozygous genotype, then when the other parent is unknown, both alleles may be inherited from the parent, so Mendelian error calculation cannot be performed. If the offspring genotype is AA and the candidate parent genotype is AC, the number of such loci is recorded as {Nh}. Assuming that there are N loci that can be used for this test (excluding missing values), the number of Mendelian errors between suspected parent-offspring pairs is {Nme}, then the effective number of tests is {N-Nh}, and the Mendelian error rate is The Mendelian error rate threshold is 5%, above which the parent-offspring relationship is considered abnormal.

[0072] Among them, the flanking chain encumbrance:

[0073] 1) Theoretical background: Linkage drag refers to the phenomenon that when the target gene is transferred from the non-cyclic parent to the cyclic parent in the traditional backcross breeding process, chromosome fragments from the non-cyclic parent are often brought in. These chromosome fragments may carry genes with unfavorable traits. These unfavorable genes are linked to the target gene, resulting in a phenomenon where the breeding goal is inconsistent with the expected results. In short, in the breeding process, we hope to introduce a favorable gene, but at the same time we also have to introduce unfavorable genes linked to it. These unfavorable genes are like "burdens" that affect the effect of breeding. Avoiding or reducing linkage drag will help improve the efficiency of breeding.

[0074] 2) Definition of break: If the length of the transferred fragments other than the donor target gene is within 5M, it is considered a flanking linkage drag break. If one of the two sides exceeds 5M, it is considered a unilateral break.

[0075] 3) The calculation process of the flank chain encumbrance breaking distance (taking the left flank as an example) is as follows Figure 3-Figure 5 As shown:

[0076] According to the principle, the part of the target gene other than the transferred fragment should be consistent with the receptor, which is represented as genotype A in the ABH file. If genotype B or H appears, it means that other genes have been transferred. Based on this principle, the judgment and calculation process of breaking the flanking linkage drag of the present invention is designed:

[0077] A) Single-site products are designed with evenly distributed SNPs (hereinafter referred to as flanking markers) within 5M on both sides of the insertion breakpoint of the southern rust resistance RppK gene, and the genotype of the progeny material is measured using the single-site product.

[0078] B) Convert the genotype file into ABH format file and find the breaking position:

[0079] 1) If the genotype of the flanking marker closest to the breakpoint is A, check the genotypes of the five flanking markers including the site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, then the marker position is the breaking position (such as Figure 3 a);

[0080] 2) If the genotype of the flanking marker next to the breakpoint is A, check the genotypes of the five flanking markers including the site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, and the next site is B or H, then the marker position is the breaking position (such as Figure 3 b);

[0081] 3) If the genotype of the first flanking marker is A, and the proportion of H or B in the genotypes of the next five markers is ≥ 80%, that is, at most one marker has a genotype of A, then the marker position is the breaking position (e.g. Figure 3 c);

[0082] 4) For the flanking markers at the remaining positions, loop through and judge: if the following conditions are met at the same time: the marker genotype is A, the 5 flanking marker genotypes including the site contain A ratio ≥ 80%, the next marker genotype is B or H, and the 5 flanking marker genotypes contain H or B ratio ≥ 80%, then the marker position is a break position (such as Figure 3 d).

[0083] The liquid phase chip used in this example is the liquid phase chip sold by Boreidi 10K liquid phase chip for corn rust resistance backcross breeding; calculation of flanking linkage drag breaking distance is based on a product sold by Boredy The test results of the single-point detection products of the corn series; the single-point products can be used for RppK prospecting and flanking breakthrough; the multiple SSR detection products used are the liquid phase chips sold by Boredy Maize SSR fingerprinting kit.

[0084] This embodiment is achieved by The 10K liquid phase chip for backcross breeding of corn rust resistance was used to detect whether it contained national standard transgenic contamination. The specific detection method was based on the "GB / T 38570-2020 Target Sequence Sequencing Method for Determination of Plant Transgenic Components". This standard uses the target sequence sequencing method to identify exogenous genes and distinguish microbial contamination through exogenous gene characteristic fragments, and simultaneously detect multiple transgenic components through hybridization probes matching multiple transgenic elements. This example uses this highly accurate detection method to determine whether there is national standard transgenic contamination in the sample.

[0085] First, this embodiment adopts molecular assisted breeding. By detecting the genotype of the sample through a liquid phase chip, it can be determined whether the sample has heterozygous resistance to southern rust. Compared with the existing technology that relies on experience and phenotypic selection for improvement, it can shorten the breeding cycle.

[0086] The process of using the liquid phase chip to determine whether the material is resistant to southern rust in this embodiment is as follows: the planted material sprouts in about a week, and a puncher is used to punch 6 circular leaves with a radius of 6 mm on the material, and DNA can be extracted for experiments, which is faster and more accurate than manual identification.

[0087] Other non-artificial detection methods are also used in the prior art, such as PCR amplification to detect genes; non-specific amplification is prone to occur during PCR amplification, resulting in false positive and false negative results. Liquid chips can better avoid non-specific reactions during targeted capture due to their design advantages, such as specific binding on the microsphere surface and high sensitivity of fluorescence detection; and PCR amplification requires multiple temperature cycles, the operation is relatively complex, and each reaction takes a certain amount of time to complete. Liquid chips can achieve rapid and high-throughput detection, usually completing parallel processing of multiple samples in a short time.

[0088] Secondly, the screening conditions of the invention introduce background recovery rate, parent-child relationship, and breaking of flanking linkage markers, and in the parental self-pollination generation, the difference site ≤1 between the selfing parent BC2F1 and the recipient parent is introduced, thereby greatly shortening the backcrossing generations. Compared with the prior art, at least 4-5 backcrossing generations are required. The present invention can breed stable germplasm with resistance to southern rust from BC2F2 at the earliest.

[0089] The growing season of corn is 4-6 months. The prior art uses manual identification of whether a plant is resistant to southern rust. Starting from the F1 generation, if three backcrossings and four self-pollinations are performed, it will take at least about 2 years, including the waiting time for the plant to become diseased. In fact, the number of backcrossings in the prior art may be even greater. However, in this embodiment, starting from the F1 generation, two backcrossings and one self-pollination are performed, and the shortest time is only 1-1.5 years.

[0090] Currently, reverse backcrossing can be carried out through several backbone inbred lines, and the genetic background recovery rate of the recurrent parent can reach more than 90% at most, and an improved line with recessive disease resistance can be obtained; the breeding method of this embodiment can achieve a genetic background recovery rate of more than 95% for the recurrent parent at the BC2F1 stage, and the genetic background recovery rate of the recurrent parent of the seeds finally bred is more than 99%.

[0091] This embodiment introduces the genetic background recovery rate in the screening conditions of each generation of materials, among which the background recovery rate of F1 is greater than or equal to 50%; the background recovery rate of BC1F1 material is greater than or equal to 85%; the background recovery rate of BC2F1 material is greater than or equal to 95% (theoretical value is 87.5%), which can save at least one generation of backcrossing time and cost.

[0092] That is, this embodiment introduces the genetic background recovery rate into the screening conditions of each generation of materials, which can shorten the backcrossing generations and speed up breeding:

[0093] Example: The theoretical value of the background recovery rate of BC1F1 is 75%. 1808 BC1F1 materials of population Z are tested using a liquid phase chip, the background recovery rate is calculated and a distribution diagram is drawn, such as Figure 6 As shown, in the backcrossing process of this embodiment, materials with a background recovery rate greater than or equal to 85% can be directly selected for the next round of backcrossing, which can save at least one generation of backcrossing time and cost.

[0094] The consequences of not introducing SSR differential loci analysis:

[0095] Based on the national standard SSR provided in the national crop variety DNA fingerprint library, the multiple SSR products captured 5-30 highly polymorphic SNP markers on the left and right wings of the SSR respectively, selected the SNP markers that were inconsistent with the homozygous parents, and based on the principle of linkage drag, determined whether the SNP markers of the offspring were consistent with those of the recurrent parents to determine whether the SSR markers of the offspring were consistent with those of the recurrent parents.

[0096] Failure to identify SSR difference sites may lead to failure of variety approval.

[0097] If you do not introduce flanking the consequences of breaking the analysis:

[0098] If flanking break analysis is not performed, non-target gene fragments are likely to be introduced, which will reduce the quality of the variety or require more generations in the breeding process to remove these non-target genes, thereby reducing breeding efficiency.

[0099] Similarly, in the variety approval stage, the approval may fail due to impure materials.

[0100] In order to better illustrate the technology of this embodiment, the following specific case is used for illustration:

[0101] Case 1:

[0102] Dan340 modification (donor material: K22, acceptor material: Dan340) includes the following steps:

[0103] 1) Personal quality control

[0104] According to the background markers, the deletion rate and heterozygosity rate of the parents were statistically analyzed, as shown in Table 1:

[0105] Table 1

[0106]

[0107] In Table 1, NA indicates deletion and Het indicates heterozygous.

[0108] 2) Hybridization to obtain F1 generation

[0109] The parents hybridized to produce about 2,000 F1 plants, and 60 plants with consistent and good growth were selected for genetic testing, all of which had heterozygous resistance to southern rust. The background recovery rate of the 60 F1 generations was calculated, and the 60 F1 materials basically met the theoretical value of the background recovery rate of 50%. Figure 7 As shown; 60 F1 generations were used as recurrent parents.

[0110] 3) Backcrossing to obtain BC1F1 generation, and then using single point products and liquid phase chip testing

[0111] After pollination of the F1 plants, about 2400 BC1F1 grains were obtained and tested using a single-point product. Based on the results of the single-point product test, 10% (about 200) of the RppK heterozygous and bilaterally broken materials were selected for testing using a liquid phase chip;

[0112] According to the chip test results, the best individual plants (about 30 plants) with no transgenic contamination, RppK heterozygous, normal parent-offspring relationship (Mendel error rate is basically within 1%), short flank break distance (at least unilateral break is achieved), and high background recovery rate (basically 85%-90%) are selected and planted in the field to create the next generation.

[0113] The data of 30 strains of BC1F1 generation are shown in Table 2:

[0114] Table 2

[0115]

[0116]

[0117] In the table, "Left wing breaking position" and "Left wing breaking distance" are NA, which means that no flank breaking occurred on the left wing, that is, the sample is a unilateral break; "Right wing breaking position" and "Right wing breaking distance" are NA, which means that no flank breaking occurred on the right wing, that is, the sample is a unilateral break.

[0118] 4) Backcrossing to obtain BC2F1 generation, and then using single point products and liquid phase chip testing

[0119] After BC1F1 pollination, about 2,500 BC2F1 grains were obtained and tested using a single-point product. Based on the results of the single-point product test, 10% (about 200) of the RppK heterozygous materials with bilaterally broken encumbered were selected for testing using a liquid phase chip;

[0120] According to the chip test results, the best individual plants (about 30 plants) with no transgenic contamination, RppK heterozygous, normal parent-offspring relationship (Mendel error rate is basically within 0.4%), close flank break distance (most of them have achieved bilateral break), and high background recovery rate (greater than 95%) are selected and planted in the field to create the next generation.

[0121] The data of 30 BC2F1 strains are shown in Table 3:

[0122] Table 3

[0123]

[0124]

[0125] 5) BC2F1 self-pollination to obtain BC2F2 generation, and then use single-point products and liquid phase chip detection

[0126] About 2000 BC2F2 grains were obtained by self-pollination of BC2F1, and single-point product testing was used. According to the single-point product testing results, 10% (about 200) of the materials with the highest RppK homozygosity and the shortest bilateral breaking burden were selected for testing using liquid phase chips;

[0127] According to the chip test results, we selected the best individual plants (about 180 plants) with no transgenic contamination, homozygous RppK, normal parent-offspring relationship (Mendel error rate less than 0.4%), close flanking break distance (all achieved bilateral break), and high background recovery rate (greater than 98%), and used multiple SSR products for testing.

[0128] Based on the results of multiple product testing, materials (about 150 plants) with a difference site of ≤1 between the material and the recipient parent were screened out to obtain improved positive plants with homozygous disease-resistant chromosome segments.

[0129] Data display before and after improvement:

[0130] Figure 8 Chromosome distribution map of the RppK introduced fragment for the F1 generation; Fig. 9 This is the chromosome distribution map of the RppK introduced fragment in the BC2F2 generation; the RppK gene is located on chromosome 10 and is approximately 10Mbp in length.

[0131] Figure 8 , Fig. 9 In the middle, blue indicates heterozygous and red indicates homozygous; Figure 8 There is only blue, which means that the F1 generation is all heterozygous. Fig. 9 There is a red line on chromosome 10, indicating homozygosity; in the BC2F2 generation, which is the final germplasm material, only the RppK position shows homozygous introduction, and the remaining chromosomes are highly consistent with the recipient parent, the reaction background recovery rate is high, and the material is clean.

[0132] Part of the data of the BC2F2 generation is shown in Table 4:

[0133] Table 4

[0134]

[0135]

[0136] From Table 4, we can see that:

[0137] All samples in this case were introduced with RppK, and the background recovery rate was above 98%, the number of SSR differences was less than or equal to 1, and the Mendelian error rate was less than 0.5%.

[0138] Case 2:

[0139] This case is based on Case 1, and the difference from Case 1 is that the screening conditions of each generation do not use the background recovery rate of this embodiment.

[0140] According to the chip test results, the BC2F2 generation in this case does not have the RppK homozygous gene, but has the RppK heterozygous gene.

[0141] BC2F1 was backcrossed with the recurrent parent to obtain the BC3F1 generation. According to the chip test results, the BC3F1 generation had the homozygous RppK gene.

[0142] From the comparison between Case 2 and Case 1, we can see that:

[0143] If the background recovery rate of this embodiment is not used in the screening conditions of each generation, one more generation of backcrossing will be caused.

[0144] Case 3:

[0145] This case is based on Case 1, and the difference from Case 1 is that the screening conditions of each generation do not adopt the double-sided breaking priority of the flanking linkage markers of this embodiment, followed by the single-sided breaking distance ≤5M.

[0146] Starting from the BC1F1 generation, no flanking linkage drag breaking analysis was performed in each generation. Finally, there were about 2,600 BC2F2 germplasm materials, of which about 20% showed the expected southern rust resistance trait, and at the same time showed the undesirable trait of reduced stress resistance; about 30% of the materials showed the expected southern rust resistance trait, and at the same time showed the undesirable trait of decreased yield; the remaining about 50% of the materials showed the expected southern rust resistance trait, and no other good traits.

[0147] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for breeding corn germplasm with high resistance to southern rust, characterized in that: The following steps are involved: S1, using the recipient parent as the female parent and hybridizing it with the donor parent of the superior inbred line resistant to southern rust to obtain the corn hybrid F1 resistant to southern rust, and screening the F1 with heterozygous resistance to southern rust and a background recovery rate greater than or equal to 50% as the recurrent parent F1 by detecting the genotype of the F1; S2, backcrossing the recurrent parent F1 with the recipient parent to obtain BC1F1, and screening to obtain the recurrent parent BC1F1, the screening conditions including: 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 85%; 3) The parent-offspring relationship is normal; 4) The flanking linkage markers are preferably broken on both sides, followed by the unilateral breaking distance ≤ 5M; 5) No national standard transgenic pollution; S3, backcrossing the recurrent parent BC1F1 with the recipient parent to obtain BC2F1; and screening to obtain the selfing parent BC2F1, the screening conditions including: 1) Possessing heterozygous resistance to southern rust; 2) Background recovery rate is greater than or equal to 95%; 3) Parent-offspring relationship is normal; 4) Double-sided break of flanking linkage markers is preferred, followed by single-sided break distance ≤5M; 5) The difference site between the selfing parent BC2F1 and the recipient parent is ≤1; 6) No national standard transgenic pollution; S4. BC2F2 is obtained by self-pollination of BC2F1, and the genotype of BC2F2 is tested. If BC2F2 has homozygous resistance to southern rust, BC2F2 is used as the selected germplasm. Otherwise, BC2F1 is backcrossed with the recipient parent to obtain BC3F1, and the materials are screened until the self-pollinated material has homozygous resistance to southern rust.

2. The method for breeding corn germplasm with high resistance to southern rust according to claim 1, characterized in that: The calculation process of the background recovery rate is: (1) Recode the genotype of each individual in the population according to the parental genotype, that is, convert the genotype file into an ABH format file; (2) Calculate the background recovery rate based on the common allelic variation between the recurrent parent and a certain offspring.

3. The method for breeding corn germplasm with high resistance to southern rust according to claim 2, characterized in that: The calculation process of the background recovery rate is as follows: In the formula, Na represents the number of homozygous markers between the progeny and the recurrent parent; N h It represents the number of markers that are homozygous in the recurrent parent and heterozygous in the offspring; N represents the total number of markers with genotypes (non-NA) existing in both the recurrent parent and the offspring.

4. The method for breeding corn germplasm with high resistance to southern rust according to claim 1, characterized in that: The parent-offspring relationship reflects whether the parent-offspring relationship is normal by calculating the Mendel error rate.

5. The method for breeding corn germplasm with high resistance to southern rust according to claim 4, characterized in that: The Mendelian error rate threshold is 5%, above which the parent-offspring relationship is considered abnormal.

6. The method for breeding corn germplasm with high resistance to southern rust according to claim 1, characterized in that: in, The calculation process of the flanking chain encumbrance breaking distance is as follows: Use a single-point product to measure the genotype of the progeny material; convert the genotype file into an ABH format file and find the breaking position.

7. The method for breeding corn germplasm with high resistance to southern rust according to claim 6, characterized in that: The specific process of finding the breaking position is as follows: 1) If the genotype of the flanking marker closest to the breakpoint is A, check the genotypes of the five flanking markers including the site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, then the marker position is the breaking position; 2) If the genotype of the flanking marker next to the breakpoint is A, check the genotypes of the five flanking markers including this site. If the proportion of A is ≥ 80%, that is, at most one marker has a genotype of B or H, and the next site is B or H, then the marker position is the breaking position; 3) If the genotype of the first flanking marker is A, and the proportion of H or B in the genotypes of the next five markers is ≥ 80%, that is, at most one marker has a genotype of A, then the marker position is the breaking position; 4) For the flanking markers at the remaining positions, loop through and judge: if the following conditions are met at the same time: the marker genotype is A, the 5 flanking marker genotypes including the site contain A proportion ≥ 80%, the next marker genotype is B or H, and the proportion of H or B in the last five marker genotypes is ≥ 80%, then the marker position is a breaking position.

8. The method for breeding corn germplasm with high resistance to southern rust according to claim 1, characterized in that: Liquid phase chip was used to detect genotypes.

9. The method for breeding corn germplasm with high resistance to southern rust according to claim 1, characterized in that: The differential loci were detected using multiple SSR detection products.

10. A method for breeding corn germplasm with high resistance to southern rust according to any one of claims 1 to 9, characterized in that: In step S1, quality control is performed on the parents: parents with lower deletion rate and heterozygosity rate are selected for hybridization.

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