Wheat crossbreeding method

Abstract

The invention relates to the technical field of wheat breeding, in particular to a wheat crossbreeding method which comprises the following steps: hybridizing a variety carrying a NeI1 gene as a male parent with a non-Ne gene carrying variety or a Ne1 carrying variety, and selecting a hybrid of a progeny carrying the NeI1 gene and the Ne1 gene as a parent 1; selecting a parent 2 carrying the Ne2 gene through wide test cross; hybridizing the parent 1 and the parent 2 to complete seed production. According to the wheat breeding method provided by the invention, the problem that gene exchange among regional varieties is hindered due to the fact that varieties in different regions carry Ne genes is solved by virtue of the suppressor genes, and the development of wheat breeding is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of wheat breeding, and particularly relates to a wheat cross-breeding method utilizing gene interaction. Background Art

[0002] In the current breeding research field, aggregating excellent genes is the basis for cultivating high-yield, high-quality, disease-resistant, and widely adaptable excellent wheat varieties. In wheat breeding, the uncoordinated development and severe decline of hybrids severely restrict the utilization of alien germplasm and excellent interspecific genes by breeders. This phenomenon was discovered as early as 1942. In actual breeding practice, hybrid decline is simply considered by breeders as an interspecific gene exchange barrier or physiological disharmony; in addition, due to the extremely poor agronomic phenotypes of the declining individuals, they are prematurely eliminated by breeders.

[0003] From a pathological perspective, hybrid decline belongs to a special case of senescence, and senescence is a typical programmed cell death process. Research on plant-pathogen interactions shows that the plant system relies on two immune resistance methods to resist pathogen infection, namely pathogen-associated molecular pattern-triggered immunity (PTI) and effector protein-triggered immunity (ETI). The typical symptom of HR in wheat is the programmed rapid death of plant cells at the infection site, triggering the expression of locally acquired resistance (LAR) and systemically acquired resistance (SAR), inducing pathogenesis-related genes and proteins, etc., as well as the coupling and following of long non-coding RNAs, thereby inhibiting the growth of pathogens. Therefore, the research on this type of decline that is similar to hypersensitive necrosis and has a stronger phenotypic response than the HR response is also helpful for strengthening the utilization of disease resistance. However, unfortunately, the necrosis of F1 hybrids seriously affects gene transfer, so it has not received the attention of scientists from various countries and in-depth research has not been carried out.

[0004] The above research on wheat hybrid decline lacks genetic evidence. In spring wheat, it has been confirmed that the hybrid chlorosis decline trait is regulated by the complementary action of two pairs of dominant genes Ne1 and Ne2 , which are located on chromosomes 5BL and 2BS respectively. Among them, Ne2 gene encodes the leaf rust resistance gene Lr13 , and both of them have bilateral genetic markers with a genetic distance of about 0.2 cM. It is worth noting that genetic analysis of wheat crosses in different wheat regions has found that the main types of chlorosis genes ( Ne1 / Ne2 ) are different in different regions, but their frequencies in the released varieties all show an increase rather than a decrease. Thus, it can be seen that the region where the single Ne gene is located must carry many excellent trait genes, that is, it has the potential to be an important genetic region, and has been selectively retained by breeders during empirical breeding.

[0005] Backbone parents generally have excellent agronomic traits, good disease resistance, many excellent genes, high compatibility, and strong genetic transmission. In China's wheat variety improvement, 14 farmer varieties and introduced germplasms have played a core role in hybrid breeding, such as Grasshopper Wheat, Piedmont Wheat, Afu, Niu Zhute, Zhengyin No. 4, Nonglin No. 10, Lovelin 13 and its derivatives Xiaoyan No. 6, Yumai No. 2, Aymeng Niu, Zhou 8425B, Lumai 13, Lumai 14, etc., which have laid the foundation for grain production and variety improvement in the Huanghuai winter wheat region and achieved multiple variety changes in the wheat region. The latter has also become the most important backbone parent in the southern part of the Huanghuai wheat region. Research on these backbone parents will undoubtedly further promote the utilization efficiency of backbone parents and tap the greater potential of their genes in breeding production.

[0006] In the early stage of the polygenetic disease-resistant breeding, the team of the present invention accidentally discovered that the self-bred wild two-grain wheat AS846 derived family N0439 was hybridized with Zhou 8425B derived variety Zhoumai 22, and the F1 showed hybrid decay phenomenon in the seedling stage. But interestingly, the yellowed plants can complete the life cycle and produce healthy offspring plant individuals, which show similar phenotypes to the decay genes carried by Pan 555 and Zheng 891 in the winter wheat area of ​​the northern part of the Huanghuai region. Then, with N0439 as the core parent, the genetic distribution of hybrid decay regulatory genes in the winter wheat area of ​​the southern part of the Huanghuai region was preliminarily randomly predicted, and it was found that the occurrence frequency of the yellowed gene carried by Zhoumai 22 was as high as 38%. In connection with the core backbone parent position of Zhou 8425B in the Huanghuai wheat area, it is evaluated that the genetic segment where the gene it carries is located has a high genetic contribution.

[0007] However, 30% to 50% of these materials are Ne gene carriers, especially materials from different ecological zones often carry a certain Ne gene, that is, they contain Ne1 or Ne2 For example, about 30% of the varieties in Shaanxi contain Ne1 Gene, more than 50% of varieties in Henan contain Ne2 Individuals with these two genes alone show good growth ability, but when germplasm materials or varieties from different regions are hybridized to utilize hybrid advantages or further breed varieties, F1 shows a decline phenomenon, that is, hybrid disadvantage, which seriously hinders the progress of wheat breeding. Summary of the invention

[0008] In order to solve the problem that varieties from different regions carry one Ne gene, which hinders gene exchange between regional varieties, the present invention provides a wheat hybrid breeding method targeting quality trait genes controlled by complementary effects with the help of inhibitory genes. This method is used in combination with molecular markers to promote the development of wheat breeding.

[0009] To achieve the above object, the technical solution adopted by the present invention is: A wheat hybrid breeding method comprises the following steps: Using the variety carrying NeI1 gene as the male parent, cross it with a non-Ne gene-carrying variety or Ne1 a carrying variety, and select the offspring carrying NeI1 gene and Ne1 gene as Parent 1; Through extensive test crosses, select Parent 2 carrying Ne2 gene; Cross the said Parent 1 and the said Parent 2 to complete seed production. Among them, Parent 1 and Parent 2 are preferably hybrids with excellent agronomic traits and high combining ability.

[0010] The F1 generation of the hybrid grows completely normally and exhibits the heterosis of both parents; in the F2 generation, due to the inhibitory gene NeI1 / Ne1 gene, and Ne2 the separation of the triple heterozygous locus, 3 / 16 of the single plant individuals showing the hybrid decline phenotype are produced.

[0011] Preferably, the offspring carrying NeI1 gene and Ne1 gene are selected by molecular marker-assisted selection.

[0012] More preferably, the specific process of molecular marker-assisted selection is as follows: Extract the genomic DNA of the wheat to be detected; Synthesize molecular markers linked to NeI1 gene and Ne1 gene respectively; Using the genomic DNA of the wheat to be detected as a template, perform PCR amplification respectively with the molecular markers linked to NeI1 and Ne1 gene as primers; Detect the amplified products by electrophoresis. The electrophoresis bands of the offspring containing NeI1 gene are consistent with the bands of the normal phenotype single plants, or contain this dominant band; the electrophoresis bands of the offspring containing Ne1 gene are consistent with the control of the Ne1 carrying variety.

[0013] Even more preferably, each 10ul PCR system contains: 1ul of 10×PCR buffer, 0.8ul of 2mM dNTP MIX, 0.5ul each of 10uM primers F and R, 0.1ul of 5U / ul Taq enzyme, 100 - 200ng of template DNA, and ddH2O is added to make up to 10ul; The PCR reaction procedure is as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 98°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 1 min; (5) starting from step (2), a total of 35 cycles of PCR amplification are carried out; (6) extension at 72°C for 10 min.

[0014] Preferably, the hybrid carrying NeI1 gene and Ne1 gene is obtained by introgressing NeI1 and Ne1 genes using the backcross breeding method.

[0015] Preferably, the variety carrying NeI1 gene is selected from any one of wheat N15025N, Jimai 22, Bainong 207, Changwu 134, Jinmai 47 or Luyuan 502.

[0016] Preferably, the parent 1 and the parent 2 are crossed, and in the F2 generation of the cross, the double co-dominant molecular markers of the hybrid necrosis gene are used to continue selecting Ne1 and Ne2 gene double homozygous normal single plants to form an F3 family and enter conventional breeding. This method utilizes the self-pollination and breeding characteristics of wheat to transform the F1 hybrid disadvantage into hybrid advantage and transfer it into conventional breeding, further making full use of the role of the hybrid combination, promoting the development of wheat breeding, and providing more opportunities for breeders to hybridize between varieties.

[0017] Preferably, the double co-dominant molecular markers of the hybrid necrosis gene Ne1 are Xbarc89 or Xbarc74, and the double co-dominant molecular markers of the hybrid necrosis gene Ne2 are TC67744 or Xgwm374; The sequence of Xbarc89 is shown in SEQ ID NO.1, the sequence of Xbarc74 is shown in SEQ ID NO.4, the sequence of TC67744 is shown in SEQ ID NO.13, and the sequence of Xgwm374 is shown in SEQ ID NO.14.

[0018] The present invention has the following beneficial effects: Aiming at the problem that the Ne gene carried by varieties in different regions hinders the gene exchange between regional varieties, and the problem of commercial protection of hybrids caused by the self-pollination characteristics of wheat, the present invention provides a wheat hybrid breeding method for qualitative trait genes controlled by complementary action with the help of inhibitory genes. Based on the death of some plants caused by gene segregation in the next generation, commercial protection of hybrids is achieved, that is, the interests of hybrid producers are indirectly protected.

[0019] The present invention combines the hybrid necrosis of dominant complementary inheritance Ne1and Ne2 The genetic characteristics of the gene, when used in cooperation with the inhibitory gene discovered in the present invention NeI1 overcome the difficulty that gene exchange between varieties cannot be achieved among gene carriers of Ne1 and Ne2 and realize the utilization of heterosis between varieties. The introduction of the inhibitory gene NeI1 utilizes the scientific law of genetic segregation of wheat self-pollination, which not only protects the rights and interests of commercial hybrid producers in the utilization of heterosis, but also can use the inhibitory gene to cultivate Ne1 and Ne2 gene pyramiding varieties in conventional wheat breeding, while breaking the genetic barriers of Ne1 and Ne2 gene combinations and realizing the accumulation of excellent gene genetic effects in their respective genetic segments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a diagram showing an example of hybrid necrosis; after crossing Parent 1 carrying Ne1 gene and Parent 2 carrying Ne2 gene, the hybrid F1 plants died; Figure 2 is a diagram showing an example of introducing a hybrid necrosis inhibitory gene into commercial breeding; after crossing Parent 1 carrying the hybrid necrosis inhibitory genes NeI1 and Ne1 gene and Parent 2 carrying Ne2 gene, the hybrid F1 plants grew normally and showed heterosis; trait segregation occurred in F2, with both necrotic individuals and normal-growing individuals.

[0021] Figure 3 is Ne1 and NeI1 gene-linked marker PCR electrophoresis gel diagram; A: N34: Hybrid necrosis gene Ne1 control N9134; Ne1: Hybrid necrosis gene Ne1 carrier; ne: Non-hybrid necrosis gene Ne1 carrier; B: M: marker; H: Necrotic individual; N: Normal individual, i.e., NeI1 carrier; Figure 4 is Ne2 gene-linked marker PCR electrophoresis gel diagram; Z22, Hybrid necrosis gene Ne2 control Zhoumai 22; Ne2, Hybrid necrosis gene Ne2 carrier; ne, Non-hybrid necrosis gene Ne2 carrier. DETAILED DESCRIPTION OF THE INVENTION

[0022] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] In wheat breeding, the uncoordinated development and senescence of hybrids severely restrict breeders' utilization of alien germplasm and excellent interspecific genes in wheat. Studying the senescence related to hypersensitive necrosis, which is similar but has a stronger phenotypic response than the HR response, helps to enhance the utilization of disease resistance. However, unfortunately, the necrosis of F1 hybrids seriously affects gene transfer, so it has not been taken seriously by scientists in various countries and relevant research has not been carried out in depth.

[0024] It has been confirmed in spring wheat that the trait of hybrid chlorosis and senescence is regulated by two pairs of dominant genes Ne1 and Ne2 with complementary effects, which are located on chromosomes 5BL and 2BS respectively. Among them, Ne2 gene encodes the leaf rust resistance gene Lr13 , and both of them have bilateral genetic markers with a genetic distance of about 0.2 cM. The research team of the present invention accidentally found that the F1 of the hybrid between the self-bred wild emmer wheat AS846-derived line N0439 and the Zhou 8425B-derived variety Zhoumai 22 showed the phenomenon of hybrid senescence at the seedling stage during the previous polymerization disease resistance breeding. Interestingly, however, the chlorotic plants can complete their life cycles and produce healthy offspring plant individuals. Furthermore, taking N0439 as the core parent, the genetic distribution of the hybrid senescence regulatory gene in the southern winter wheat area of the Huanghuai region was preliminarily predicted randomly, and it was found that the occurrence frequency of the chlorosis gene carried by Zhoumai 22 was as high as 38%. Considering the status of Zhou 8425B as a core backbone parent in the Huanghuai wheat area, it is evaluated that the genetic segment where the gene is located has a high genetic contribution.

[0025] However, 30% - 50% of these materials are Ne gene carriers. Especially, materials from different ecological regions often carry a certain Ne gene, that is, contain Ne1 or Ne2 . Individuals with these two genes alone show good growth ability. However, when germplasm materials or varieties from different regions are hybridized with each other to utilize heterosis or further breed varieties, the F1 shows the phenomenon of senescence, that is, hybrid disadvantage, which seriously hinders the progress of wheat breeding.

[0026] Based on this, the present invention provides a wheat hybridization breeding method, including the following steps: Using the variety carrying the NeI1 gene as the male parent, hybridizing with non-Ne gene-carrying varieties or Ne1 carrying varieties, and selecting the offspring carryingNeI1 Gene and Ne1 the hybrid of the gene as Parent 1; Through extensive test crosses, select Parent 2 carrying Ne2 the gene; Hybridize the said Parent 1 and the said Parent 2 to complete seed production.

[0027] The present invention provides a wheat cross-breeding method for qualitative trait genes controlled by complementary action by means of a suppressor gene NeI1 Based on the death of some plants caused by gene segregation in the next generation, commercial protection of hybrids is achieved, that is, the interests of hybrid producers are indirectly protected.

[0028] Some varieties involved in the present invention carrying NeI1 , Ne1 , Ne2 genes are shown in Table 1. However, it should be clear that this is not a special limitation on the technical solution of the present invention.

[0029] Table 1 Some variety names carrying NeI1 , Ne1 , Ne2 genes The reagents used in the present invention can be purchased from the market without special instructions; primers can obtain sequence information from public databases such as NCBI (https: / / www.ncbi.nlm.nih.gov / ), WheatOmics (http: / / wheatomics.sdau.edu.cn / ), GrainGenes (https: / / wheat.pw.usda.gov / GG3 / ) and be synthesized by biological companies.

[0030] Example 1 A wheat cross-breeding method, comprising the following steps: S1. Using the wheat variety N15025N carrying NeI1 gene as the male parent and the commercial wheat variety Chinese Spring as the female parent, and using the method of recurrent breeding to select in the offspring wheat materials with high combining ability, excellent agronomic traits and containing both NeI1 gene and Ne1 gene as the heterosis utilization Parent 1.

[0031] With the aid of molecular marker-assisted breeding method, screen materials containing NeI1 gene and Ne1 gene, specifically including the following steps: (1) Extract the genomic DNA of the wheat to be detected; (2) Synthesize withNeI1 The molecular marker BARC4 linked to the gene, and Ne1 the molecular marker Xbarc89 linked to the gene; BARC4: >chrUn chrUn:65957614..65957771, as shown in SEQ ID NO.1: CACCACACATGCCACCTTCTTTAAAGGGCCACCTCAACATGTATGTTAATTATTATTATTATTATTATTATTATTATTATTATTATTATTATACCATGCTACTTCTTTTTTCTTTCTTTCTAGAAACACCGATTAGAACGCAGACACAAACACGC Xbarc89: >chr5B chr5B:331475051..331475185, as shown in SEQ ID NO.3: GGGCGCGGCACCAGCACCACCGGGGCTTCATCATCATCATCATCATCATCATCATCATCGGGAAAGTCGCAGAGGATCTCCCTCTGACGCGGGGCAGAGGATTCCTCCACCATCTTGTCTTCGGTGGCCTCGGAG (3)Using the genomic DNA of the wheat to be detected as a template, and using NeI1 and Ne1 the linked molecular markers of the gene as primers respectively for PCR amplification; The primer sequences are as follows: BARC4-F: 5’-GCGTGTTTGTGTCTGCGTTCTA-3’, SEQ ID NO.5; BARC4-R: 5’-CACCACACATGCCACCTTCTTT-3’, SEQ ID NO.6; Xbarc89-F: 5’-GGGCGCGGCACCAGCACTACC-3’, SEQ ID NO.9; Xbarc89-R: 5’-CTCCGAGGCCACCGAAGACAAGATG-3’, SEQ ID NO.10.

[0032] The PCR system is: 1ul of 10×PCR buffer, 0.8ul of 2mM dNTP MIX, 0.5ul each of 10uM primer F and R, 0.1ul of 5U / ul Taq enzyme, 100~200ng of template DNA, and sterilized ddH2O is added to make up to 10ul in volume.

[0033] The PCR reaction procedure is as follows: (1) pre-denaturation at 94°C for 3 min; (2) denaturation at 98°C for 30 s; (3) annealing at 58°C for 30 s; (4) extension at 72°C for 1 min; (5) starting from step (2), perform 35 cycles of PCR amplification in total; (6) extension at 72°C for 10 min and store at 12°C.

[0034] (4) Detection by polyacrylamide gel electrophoresis, the electrophoretic band of the material containing NeI1 the gene is consistent with the electrophoretic band of the normal phenotype single plant, or contains this dominant band (such as Figure 3 B); the electrophoretic band of the material containing Ne1 the gene is consistent with that of the Ne1 carrier material (such as N9134) control (such as Figure 3 A).

[0035] After polyacrylamide gel electrophoresis, stain the PAGE gel using the silver staining method.

[0036] S2. Through extensive test crosses, select Zhoumai 22 that carries Ne2 the gene and has high combining ability as the parent 2; Ne2 There are many excellent agronomic trait genes in the 2BS segment where the gene is located. Therefore, the gene carriers usually show high combining ability and good yield. More than 50% of the varieties in the Huang-Huai wheat region, especially the main Henan varieties with the blood relationship of Zhoumai, are all gene carriers. Use molecular markers to detect and select the materials carrying Ne2 the gene, and then use them as parents to perform test crosses with the materials developed from S1 to identify the specific combining ability of the two parents and determine the materials to be paired with the S1 materials.

[0037] With the help of molecular marker-assisted breeding technology, screen the materials containing Ne2 the gene, which specifically includes the following steps: (1) Extract the genomic DNA of the wheat to be detected; (2) Synthesize the molecular marker TC67744 linked to the Ne2 gene; TC67744: >chr2B chr2B:156591333..156591577, SEQ ID NO.13: TGTTGACCTCGTCAGCAGAGCCATAGGTGACGAAACCGAAACCCCTCGACCTGCCGCTGTCACGGTCATAGATGACCTTGGCATCAAGCACCTTTCCTTGTTCGCTGAACAGGTTCTCGAGCGTTGAGTTGTCGACGCCCCAGGATAGGTTTCCGACGTAGAGCTTGTTGGCTGAATCAAAGCTGCCGCCGCCGCCGCCACCACCCATGGTCCTCGGTGTTCTGGGTGCGAATTCATCTCTGGGT (3)Using the genomic DNA of the wheat to be detected as a template, and using the Ne2 linked molecular marker of the gene as a primer for PCR amplification; TC67744-F: 5'-ACCCAGAGATGAATTCGCAC-3', SEQ ID NO.15; TC67744-R: 5'-TGTTGACCTCGTCAGCAGAG-3', SEQ ID NO.16.

[0038] Both the PCR system and the PCR reaction program are the same as those in S1.

[0039] (4)Detecting by polyacrylamide gel electrophoresis, the main target band of the electrophoresis of the material containing the Ne2 gene is consistent with the dominant band of the Ne2 carrier control (Zhoumai 22) ( Figure 4 ); After the polyacrylamide gel electrophoresis, the PAGE gel is stained using the silver staining method.

[0040] S3. Crossing the above-mentioned parent 1 and parent 2 to complete seed production.

[0041] Example 2 A wheat cross-breeding method, comprising the following steps: S1. Using the Luyuan 502 variety carrying the NeI1 gene as the male parent, and using the commercial wheat variety Chuanmai 41 as the female parent, and using the method of recurrent breeding to select wheat materials with high combining ability, excellent agronomic traits and containing both the NeI1 gene and the Ne1 gene in the offspring as the heterosis utilization parent 1.

[0042] Screening the materials containing the NeI1 gene and the Ne1 gene by means of molecular marker-assisted breeding method, specifically comprising the following steps: (1)Extracting the genomic DNA of the wheat to be detected; (2) Synthesis and NeI1 molecular marker WMC73 linked to the Ne1 gene, and molecular marker Xbarc74 linked to the WMC73: >chr5B chr5B:119164656..119164848, as shown in SEQ ID NO.2: TTGTGCACCGCACTTACGTCTCGATGGCTCGAGGTTGTGCAACGCGCACACACACACACACACACACACACACACGGCGTTGGCGGCTCGAGGTTGTCCGCCGCACACACACACGGCGCCGGTGGCTTCGAGCAGCAGCTCGAGGTTGTGTGTGGCAAGCACGCACGGCGCCTAAGGATCGGAGACCGGGTGT Xbarc74: >chr5B chr5B:402787119..402787293, as shown in SEQ ID NO.4: CGCGGGAGAACCACCAGTGACAGAGCCACCCACCTCTGTTTCTCTCTCTCCCTCTCTTAAACCTAACAGCCGGCCCCACCTATGAGTCTGTCTCTCTCTCTCTCTCTCTATCTATCTATCTATCTATCTATCTATCTCTCTCTCTCTCTCTCTCTCTCTCGCCTGAAGGGGCAAG (3) Using the genomic DNA of the wheat to be detected as a template, and using NeI1 and Ne1 the linked molecular markers of the gene as primers respectively for PCR amplification; The primer sequences are as follows: WMC73-F: 5’-TTGTGCACCGCACTTACGTCTC-3’, SEQ ID NO.7; WMC73-R: 5’-ACACCCGGTCTCCGATCCTTAG-3’, SEQ ID NO.8; Xbarc74-F: 5’-GCGCTTGCCCCTTCAGGCGAG-3’, SEQ ID NO.11; Xbarc74-R: 5’-CGCGGGAGAACCACCAGTGACAGAGC-3’, SEQ ID NO.12.

[0043] The PCR system is as follows: 1 μl of 10×PCR buffer, 0.8 μl of 2 mM dNTP MIX, 0.5 μl each of 10 μM primers F and R, 0.1 μl of 5 U / μl Taq enzyme, 100 - 200 ng of template DNA, and sterilized ddH2O is added to make up a volume of 10 μl.

[0044] The PCR reaction program is as follows: (1) Pre-denaturation at 94°C for 3 min; (2) Denaturation at 98°C for 30 s; (3) Annealing at 58°C for 30 s; (4) Extension at 72°C for 1 min; (5) Starting from step (2), a total of 35 cycles of PCR amplification are carried out; (6) Extension at 72°C for 10 min and preservation at 12°C.

[0045] (4) Detection by polyacrylamide gel electrophoresis. The electrophoresis band of the material containing NeI1 the gene is consistent with the band of the normal phenotype single plant, or contains this dominant band; the electrophoresis band of the material containing Ne1 the gene is consistent with that of the Ne1 carrier material (such as N9134) control.

[0046] After polyacrylamide gel electrophoresis, the PAGE gel is stained using the silver staining method.

[0047] S2. Through extensive test crosses, select Yangmai 11 carrying Ne2 the gene and having high combining ability as parent 2; Ne2 There are many excellent agronomic trait genes in the 2BS section where the gene is located. Therefore, the gene carriers usually show high combining ability and good yield. More than 50% of the varieties in the Huang-Huai wheat region, especially the major Henan varieties with Zhoumai blood relationship, are carriers of this gene. Use molecular markers to detect and select materials carrying Ne2 the gene, and then use them as parents to test cross with the materials developed from S1 to identify the specific combining ability of the two parents and determine the materials to be paired with the S1 materials.

[0048] With the help of molecular marker-assisted breeding technology, screen materials containing Ne2 the gene, which specifically includes the following steps: (1) Extract the genomic DNA of the wheat to be detected; (2) Synthesize the molecular marker Xgwm374 linked to the Ne2 gene; Xgwm374: >chr2B chr2B:165578329..165578541, SEQ ID NO.14: TCTAATTAGCGTTGGCTGCCCACAGTCATATGCATGTTAGTCTTAGTAATTAAACTAACCTAAACAACGGGGCCACATGGCAGTGTGCCACTACTAACCTAACACACACACACACACACACACACACACACACACTAATTACACCGGGCCGGCCACACACAGTCGTGGCCAAGCCGGCCACACACACACCACACACAGCATGCAACACACTAT (2)Using the genomic DNA of the wheat to be detected as a template, Ne2 perform PCR amplification with the linked molecular markers of the Xgwm374-F: 5’-ATAGTGTGTTGCATGCTGTGTG-3’, SEQ ID NO.17 Xgwm374-R: 5’-TCTAATTAGCGTTGGCTGCC-3’, SEQ ID NO.18 Both the PCR system and the PCR reaction program are the same as S1.

[0049] (3)Detect by polyacrylamide gel electrophoresis. The main target band of the electrophoresis of the material containing the Ne2 gene is consistent with the dominant band of the Ne2 carrier control (Zhoumai 22).;

[0050] After the polyacrylamide gel electrophoresis, stain the PAGE gel with silver staining method.

[0051] S3. Cross the above-mentioned parent 1 and parent 2 to complete seed production.

[0052] Example 3 A wheat cross-breeding method, comprising the following steps: S1. Using the Jimai 22 variety carrying the NeI1 gene as the male parent, and using the commercial wheat variety Lunxuan 169 as the female parent, select the wheat materials with high combining ability, excellent agronomic traits and containing both the NeI1 gene and the Ne1 gene in the offspring by the method of recurrent breeding as the heterosis utilization parent 1.

[0053] Screen the materials containing the NeI1 gene and the Ne1 gene by means of molecular marker-assisted breeding method. The specific steps are as follows: (1)Extract the genomic DNA of the wheat to be detected; (2)Synthesize the molecular marker WMC73 linked to the NeI1 gene, and theNe1 Molecular marker Xbarc89 linked to genes WMC73: >chr5B chr5B:119164656..119164848, as shown in SEQ ID NO.2: TTGTGCACCGCACTTACGTCTCGATGGCTCGAGGTTGTGCAACGCGCACACACACACACACACACACACACACACGGCGTTGGCGGCTCGAGGTTGTCCGCCGCACACACACACGGCGCCGGTGGCTTCGAGCAGCAGCTCGAGGTTGTGTGTGGCAAGCACGCACGGCGCCTAAGGATCGGAGACCGGGTGT Xbarc89: >chr5B chr5B:331475051..331475185, as shown in SEQ ID NO.3:GGGCGCGGCACCAGCACCACCGGGGCTTCATCATCATCATCATCATCATCATCATCATCGGGAAAGTCGCAGAGGATCTCCCTCTGACGCGGGGCAGAGGATTCCTCCACCATCTTGTCTTCGGTGGCCTCGGAG (3)Using the genomic DNA of the wheat to be detected as a template, and using NeI1 and Ne1 the linked molecular markers of the genes as primers respectively for PCR amplification; The primer sequences are as follows: WMC73-F: 5’-TTGTGCACCGCACTTACGTCTC-3’, SEQ ID NO.7; WMC73-R: 5’-ACACCCGGTCTCCGATCCTTAG-3’, SEQ ID NO.8; Xbarc89-F: 5’-GGGCGCGGCACCAGCACTACC-3’, SEQ ID NO.9; Xbarc89-R: 5’-CTCCGAGGCCACCGAAGACAAGATG-3’, SEQ ID NO.10.

[0054] (4)Detect by polyacrylamide gel electrophoresis. The electrophoresis band of the material containing NeI1 the gene is consistent with the band of the normal phenotype single plant, or contains the dominant band; the electrophoresis band of the material containing Ne1 the gene is consistent with the control of the Ne1 carrying material (such as N9134).

[0055] Among them, the PCR system is as follows: 1 μl of 10×PCR buffer, 0.8 μl of 2 mM dNTP MIX, 0.5 μl each of 10 μM primers F and R, 0.1 μl of 5 U / μl Taq enzyme, 100 - 200 ng of template DNA, and sterilized ddH2O is added to make up a volume of 10 μl.

[0056] The PCR reaction procedure is as follows: (1) Pre-denaturation at 94°C for 3 min; (2) Denaturation at 98°C for 30 s; (3) Annealing at 58°C for 30 s; (4) Extension at 72°C for 1 min; (5) Starting from step (2), a total of 35 cycles of PCR amplification are carried out; (6) Extension at 72°C for 10 min, and stored at 12°C; After the polyacrylamide gel electrophoresis, the PAGE gel is stained using the silver staining method.

[0057] S2. Through extensive test crosses, Xinnong 938 carrying Ne2 gene and having high combining ability is selected as parent 2; Ne2 There are many excellent agronomic trait genes in the 2BS section where the Ne2 gene is located. Therefore, the carriers of this gene usually show high combining ability and good yield. More than 50% of the varieties in the Huang-Huai wheat region, especially the leading varieties in Henan with Zhoumai blood relationship, are carriers of this gene. Molecular markers are used to detect and select materials carrying the

[0058] gene, and then they are used as parents to test cross with the materials developed from S1 to identify the specific combining ability of the two parents and determine the materials to be paired with the S1 materials. Ne2 With the help of molecular marker-assisted breeding technology, materials containing the gene are screened, which specifically includes the following steps: (1) Extract the genomic DNA of the wheat to be detected; Ne2 (2) Synthesize the molecular marker TC67744 linked to the TC67744: >chr2B chr2B:156591333..156591577, SEQ ID NO.13: TGTTGACCTCGTCAGCAGAGCCATAGGTGACGAAACCGAAACCCCTCGACCTGCCGCTGTCACGGTCATAGATGACCTTGGCATCAAGCACCTTTCCTTGTTCGCTGAACAGGTTCTCGAGCGTTGAGTTGTCGACGCCCCAGGATAGGTTTCCGACGTAGAGCTTGTTGGCTGAATCAAAGCTGCCGCCGCCGCCGCCACCACCCATGGTCCTCGGTGTTCTGGGTGCGAATTCATCTCTGGGT (3)Using the genomic DNA of the wheat to be detected as a template, and using the Ne2 linked molecular marker of the gene as a primer for PCR amplification; TC67744-F: 5’-ACCCAGAGATGAATTCGCAC-3’, SEQ ID NO.15; TC67744-R: 5’-TGTTGACCTCGTCAGCAGAG-3’, SEQ ID NO.16.

[0059] Both the PCR system and the PCR reaction program are the same as those in S1.

[0060] (4)Detecting by polyacrylamide gel electrophoresis, the main target band of the electrophoresis of the material containing the Ne2 gene is consistent with the dominant band of the Ne2 carrier control (Zhoumai 22).

[0061] After the polyacrylamide gel electrophoresis, the PAGE gel is stained by the silver staining method.

[0062] S3. Completing the seed production by crossing the above-mentioned Parent 1 and Parent 2.

[0063] Example 4 A wheat cross-breeding method, comprising the following steps: S1. Using the Jinmai 47 variety carrying the NeI1 gene as the male parent, and using the commercial wheat variety Shanmai 159 as the female parent, and selecting, by the method of recurrent breeding, the wheat materials with high combining ability, excellent agronomic traits and containing both the NeI1 gene and the Ne1 gene as the heterosis utilization parent 1.

[0064] Screening the materials containing the NeI1 gene and the Ne1 gene by means of molecular marker-assisted breeding method, specifically including the following steps: (1)Extracting the genomic DNA of the wheat to be detected; (2) Synthesis of molecular marker BARC4 linked to the gene; synthesis of NeI1 molecular marker Xbarc74 linked to the gene; Ne1 Xbarc74: >chr5B chr5B:402787119..402787293, as shown in SEQ ID NO.4: BARC4: >chrUn chrUn:65957614..65957771, as shown in SEQ ID NO.1: CACCACACATGCCACCTTCTTTAAAGGGCCACCTCAACATGTATGTTAATTATTATTATTATTATTATTATTATTATTATTATTATTATTATACCATGCTACTTCTTTTTTCTTTCTTTCTAGAAACACCGATTAGAACGCAGACACAAACACGC Xbarc74: >chr5B chr5B:402787119..402787293, as shown in SEQ ID NO.4: CGCGGGAGAACCACCAGTGACAGAGCCACCCACCTCTGTTTCTCTCTCTCCCTCTCTTAAACCTAACAGCCGGCCCCACCTATGAGTCTGTCTCTCTCTCTCTCTCTCTATCTATCTATCTATCTATCTATCTATCTCTCTCTCTCTCTCTCTCTCTCTCGCCTGAAGGGGCAAG (3) Using the genomic DNA of the wheat to be detected as a template, and using NeI1 and Ne1 the linked molecular markers of the gene as primers to perform PCR amplification respectively; The primer sequences are as follows: BARC4-F: 5'-GCGTGTTTGTGTCTGCGTTCTA-3', SEQ ID NO.5; BARC4-R: 5'-CACCACACATGCCACCTTCTTT-3', SEQ ID NO.6; Xbarc74-F: 5'-GCGCTTGCCCCTTCAGGCGAG-3', SEQ ID NO.11; Xbarc74-R: 5'-CGCGGGAGAACCACCAGTGACAGAGC-3', SEQ ID NO.12.

[0065] (4) Detection by polyacrylamide gel electrophoresis, containing NeI1The electrophoretic band of the material with the Ne1 gene is consistent with that of the single plant with normal phenotype, or contains this dominant band; the material containing Ne1 gene has the same electrophoretic band as the control of the carrier material (such as N9134).

[0066] Among them, the PCR system is as follows: 1 μl of 10×PCR buffer, 0.8 μl of 2 mM dNTP MIX, 0.5 μl each of 10 μM primers F and R, 0.1 μl of 5 U / μl Taq enzyme, 100 - 200 ng of template DNA, and sterilized ddH2O is added to make up a volume of 10 μl; The PCR reaction program is as follows: (1) Pre-denaturation at 94°C for 3 min; (2) Denaturation at 98°C for 30 s; (3) Annealing at 58°C for 30 s; (4) Extension at 72°C for 1 min; (5) Starting from step (2), a total of 35 cycles of PCR amplification are carried out; (6) Extension at 72°C for 10 min and preservation at 12°C; After the polyacrylamide gel electrophoresis, the PAGE gel is stained using the silver staining method.

[0067] S2. Through extensive test crosses, select Yannong 0428 with high combining ability and carrying the Ne2 gene as parent 2; Ne2 There are many excellent agronomic trait genes in the 2BS section where the Ne2 gene is located. Therefore, the carriers of this gene usually show high combining ability and good yield. More than 50% of the varieties in the Huang-Huai wheat region, especially the leading varieties in Henan with Zhoumai blood relationship, are carriers of this gene. Use molecular markers to detect and select materials carrying the

[0068] gene, and then use it as a parent to test cross with the breeding materials of S1 to identify the specific combining ability of the two parents and determine the materials to be paired with the S1 materials. Ne2 With the help of molecular marker-assisted breeding technology, screen materials containing the (1) Extract the genomic DNA of the wheat to be detected; (2) Synthesize the molecular marker Xgwm374 linked to the Ne2 gene; Xgwm374: >chr2B chr2B:165578329..165578541, SEQ ID NO.14: TCTAATTAGCGTTGGCTGCCCACAGTCATATGCATGTTAGTCTTAGTAATTAAACTAACCTAAACAACGGGGCCACATGGCAGTGTGCCACTACTAACCTAACACACACACACACACACACACACACACACACACTAATTACACCGGGCCGGCCACACACAGTCGTGGCCAAGCCGGCCACACACACACCACACACAGCATGCAACACACTAT (3)Using the genomic DNA of the wheat to be detected as a template, and using the Ne2 linked molecular marker of the gene as a primer for PCR amplification; Xgwm374-F: 5'-ATAGTGTGTTGCATGCTGTGTG-3', SEQ ID NO.17 Xgwm374-R: 5'-TCTAATTAGCGTTGGCTGCC-3', SEQ ID NO.18 Both the PCR system and the PCR reaction program are the same as those in S1.

[0069] (4)Detect by polyacrylamide gel electrophoresis. The main target band of the electrophoresis of the material containing the Ne2 gene is consistent with the dominant band of the carrier control (Zhoumai 22); Ne2 After the polyacrylamide gel electrophoresis, use the silver staining method to stain the PAGE gel.

[0070] S3. Cross the above-mentioned parent 1 and parent 2 to complete seed production.

[0071] Since the breeding effects obtained by the methods provided in Examples 1 to 4 are basically the same, only Example 1 is taken here to illustrate the effect.

[0072] Figure 1 In the parent 1 carrying Ne1 and the parent 2 carrying Ne2 , very obvious hybrid necrosis phenomenon occurred in the F1 generation of the hybrid offspring, while Figure 2 after introducing the hybrid necrosis suppressor gene NeI1 , the hybrid F1 grew normally and showed strong heterosis. And the genes of the self-pollinated F2 generation segregated. 3 / 16 of the individuals in the F2 population showed hybrid necrosis due to Ne1 and Ne2 gene complementation. The hybrid seeds are sold to growers through commercial sales, and the heterosis is converted into economic yield in field planting.

[0073] ​Since the above methods (except S2) are consistent with conventional wheat breeding, it is also possible to continue selection in the F2 generation using the co-dominant molecular markers (Ne1: Xbarc89, Xbarc74; Ne2: TC67744, Xgwm374) on both sides of the developed hybrid necrosis genes Ne1 and Ne2 homozygous normal single plants with double genes to form F3 families and enter conventional pedigree breeding. This method can provide breeders with more opportunities for inter-varietal hybridization combinations, convert the F1 hybrid disadvantage into hybrid advantage and transfer it into conventional breeding.

[0074] The present invention combines the genetic characteristics of hybrid necrosis Ne1 and Ne2 genes ([[]] Figure 1 ), and is used in coordination with the NeI1 gene (inhibitor gene) discovered by the present invention to overcome the difficulty that gene exchange between varieties cannot be achieved between carriers of Ne1 and Ne2 genes and realize the utilization of inter-varietal heterosis. The introduction of the inhibitor gene NeI1 utilizes the scientific law of genetic segregation of wheat self-pollination, which not only protects the rights and interests of commercial hybrid producers in the utilization of heterosis, but also can use the inhibitor gene to cultivate Ne1 and Ne2 gene pyramiding varieties ([[]] Figure 2 ) in conventional wheat breeding, and realizes the accumulation of excellent gene genetic effects in their respective genetic segments while breaking the genetic barrier of the hybrid necrosis Ne1 and Ne2 gene combinations.

[0075] Use molecular marker-assisted breeding technology to select Ne1 and Ne2 homozygous normal single plants with double genes. The specific steps are the same as S1 and S2.

[0076] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have

[0077] various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A wheat cross-breeding method, characterized in that, It includes the following steps: Using the variety carrying NeI1 gene as the male parent, cross it with a non-Ne gene-carrying variety or Ne1 carrying variety, and select the hybrid offspring carrying NeI1 gene and Ne1 gene as Parent 1; Through extensive test crosses, select the parent 2 carrying Ne2 gene; Hybridize the said Parent 1 and the said Parent 2 to complete seed production.

2. The wheat cross-breeding method according to claim 1, characterized in that, Carry NeI1 gene and Ne1 The hybrid offspring of the gene are selected by molecular marker-assisted selection.

3. The wheat cross-breeding method according to claim 2, characterized in that, The specific process of molecular marker-assisted selection is as follows: Extract the genomic DNA of the wheat to be detected; Synthesize respectively with NeI1 gene and Ne1 molecular markers linked to the gene; Using the genomic DNA of wheat to be detected as a template, and using the primers of the linked molecular markers of the NeI1 and Ne1 genes to perform PCR amplification; The amplified products were detected by electrophoresis. The offspring containing NeI1 gene had the same electrophoresis bands as those of the single plants with normal phenotypes, or contained the dominant bands; the offspring containing Ne1 gene had electrophoresis bands consistent with those of the Ne1 carrying variety control.

4. The wheat cross-breeding method according to claim 3, characterized in that, Each 10ul PCR system contains: 1ul of 10×PCR buffer, 0.8ul of 2mM dNTP MIX, 0.5ul each of 10uM primers F and R, 0.1ul of 5U / ul Taq enzyme, 100 - 200ng of template DNA, and ddH2O is added to make up to 10ul.

5. The wheat cross-breeding method according to claim 3, characterized in that, The PCR reaction program is: (1) Pre-denaturation at 94°C for 3min; (2) Denaturation at 98°C for 30s; (3) Annealing at 58°C for 30s; (4) Extension at 72°C for 1min; (5) Starting from step (2), a total of 35 cycles of PCR amplification are carried out; (6) Extension at 72°C for 10min.

6. The wheat cross-breeding method according to claim 1, characterized in that, Progeny carry NeI1 gene and Ne1 gene hybrids are developed by backcross breeding methods after transferring NeI1 and Ne1 gene.

7. The wheat cross-breeding method according to claim 1, characterized in that, Carry NeI1 The variety carrying the gene is selected from any one of wheat N15025N, Jimai 22, Bainong 207, Changwu 134, Jinmai 47 or Luyuan 502.

8. The wheat cross-breeding method according to claim 1, characterized in that, Hybridize the parental line 1 and the parental line 2, and utilize the bilateral codominant molecular markers of the hybrid necrosis gene in the F2 generation of the hybridization to continue selecting Ne1 and Ne2 normal single plants that are double homozygous for the gene, and form F3 families to enter conventional breeding. Ne1 and Ne2 ​ 9. The wheat cross-breeding method according to claim 8, characterized in that Hybrid necrosis gene Ne1 The co-dominant molecular markers on both sides are Xbarc89 or Xbarc74, and the hybrid necrosis gene Ne2 The co-dominant molecular markers on both sides are TC67744 or Xgwm374; The sequence of Xbarc89 is as shown in SEQ ID NO.1, the sequence of Xbarc74 is as shown in SEQ ID NO.4, the sequence of TC67744 is as shown in SEQ ID NO.13, and the sequence of Xgwm374 is as shown in SEQ ID NO.14.

Images