Molecular markers for detecting wheat Su1-Ph1 genes, preparation method of partially homologous chromosome recombinant wheat and its application

Abstract

The present invention provides a molecular marker for detecting wheat Su1-Ph1 gene, a preparation method of partially homologous chromosome recombinant wheat and its application. Among them, the molecular marker for detecting wheat Su1-Ph1 gene, the primers used to amplify the molecular marker include an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2. It can solve the problem of difficulty in detecting wheat Su1-Ph1 gene in the prior art, and is applicable to the field of molecular genetic breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetic breeding, and in particular to a molecular marker for detecting wheat Su1-Ph1 gene, a preparation method of partially homologous chromosome recombinant wheat and its application. Background Art

[0002] Long-term artificial directional selection has caused serious homogeneity of common wheat and tetraploid wheat germplasm resources, and the genetic basis is very narrow, which has become a bottleneck hindering further breakthroughs in wheat breeding. Wild wheat relatives have rich genetic variation and are an important gene pool for wheat variety improvement, containing a large number of excellent genes that wheat does not have. Using distant hybridization to integrate excellent genes from wheat relatives into tetraploid wheat or common wheat will greatly increase wheat genetic diversity.

[0003] However, in the process of allopolyploidization, wheat has formed a series of systems to inhibit the pairing of some homologous chromosomes in order to maintain the stability of polyploid subgenomic chromosomes. The genes that control this system are called Ph (Pairinghomoeologous) genes, including Ph1 gene (located on chromosome 5BL, with the strongest effect), Ph2 gene (located on chromosome 3DS, with a weaker effect than Ph1 gene) and Ph3 gene (located on chromosome 3AS, with the weakest effect). The existence of these Ph genes hinders the transfer of excellent genes from wheat related species to wheat and their use in breeding.

[0004] Manipulating the Ph gene to induce the pairing and recombination of some homologous chromosomes between wheat and closely related species to form chromosome translocation lines or introgression lines is an important way to utilize the excellent genes of closely related wheat species. Since the Ph1 gene has the strongest effect, most research revolves around this gene. Domestic and foreign scientists have successfully increased the recombination rate between some homologous chromosomes of wheat and its closely related species in three main ways: the deletion line of the Ph1 locus (ph1b), the Ph1 gene mutant and the Ph1 gene suppression system. Among them, the ph1b deletion line is the most widely used in wheat distant hybridization breeding, but the ph1b deletion line needs to be used under recessive homozygous conditions, and the breeding utilization cycle is long.

[0005] Compared with the recessive effect of the deletion line or mutant of the Ph1 locus, the Ph1 gene suppression system has a dominant effect, so it is easier to use and can greatly shorten the breeding cycle. The Su1-Ph1 gene from the 3S chromosome of Aegilops speltoides is one of the most effective genes. This gene has been successfully introgressed into the 3AL chromosomes of the tetraploid wheat variety Langdon and the hexaploid wheat Chinese Spring, and the obtained materials were named LDN-Su1-Ph1 and CS-Su1-Ph1, respectively. The Su1-Ph1 gene can induce the recombination between the 5S chromosome of Ae. searsii and the 5B chromosome of wheat, and can also promote the recombination between the 1E chromosome of Lophopyrum elongatum, which is more distantly related, and the 1A chromosome of wheat. The prior art believes that the Su1-Ph1 gene can efficiently induce the pairing and recombination of some homologous chromosomes in tetraploids, but it is speculated that there are inhibitory genes in the hexaploid Chinese Spring, which limits the induction of the Su1-Ph1 gene. Therefore, accurate molecular marker-assisted selection of the Su1-Ph1 gene, and then analyzing the molecular mechanism of its inhibition in hexaploid wheat materials, has important application value for the effective use of this gene in wheat breeding. The Su1-Ph1 gene is of great significance for promoting the transfer of excellent genes in wheat related species and the creation of chromosome translocation lines / introgression lines, and accelerating the breeding and utilization of exogenous excellent genes. At present, the identification of the genotype of the 3S translocation chromosome carrying the Su1-Ph1 gene mainly relies on linked dominant markers and TaqMan probes developed based on RFLP markers. These markers are physically far away from the gene, and the operation is cumbersome, the detection cycle is long, the cost is high, and it is not suitable for large-scale screening. Therefore, it is urgent to develop a new type of molecular marker that is fast, direct, high-throughput and tightly linked to the Su1-Ph1 gene locus. Summary of the invention

[0006] The main purpose of the present invention is to provide a molecular marker for detecting wheat Su1-Ph1 gene, a preparation method and application of partially homologous chromosome recombinant wheat, so as to solve the problem of difficulty in detecting wheat Su1-Ph1 gene in the prior art.

[0007] To achieve the above object, according to the first aspect of the present invention, a molecular marker for detecting wheat Su1-Ph1 gene is provided, wherein the primers for amplifying the molecular marker include an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2.

[0008] To achieve the above object, according to a second aspect of the present invention, a kit for detecting wheat Su1-Ph1 gene is provided, the kit comprising primers, wherein the primers include an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2.

[0009] To achieve the above object, according to the third aspect of the present invention, a method for detecting wheat Su1-Ph1 gene is provided, the detection method comprising: amplifying using the above molecular marker or the above kit, and detecting the amplified product.

[0010] Furthermore, the detection method includes: using the genomic DNA of the wheat to be tested as a template, performing PCR amplification using primers, and obtaining PCR amplification products; detecting the PCR amplification products, and judging the base sequence length of the PCR amplification products. If the base sequence length includes 1185bp, the wheat to be tested contains the Su1-Ph1 gene; if the base sequence length does not include 1185bp, the wheat to be tested does not contain the Su1-Ph1 gene; preferably, the PCR amplification procedure is: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30sec, annealing at 64°C for 30sec, extension at 72°C for 40sec, 35 cycles; extension at 72°C for 5min, and storage at 12°C.

[0011] Furthermore, if the base sequence length is 710 bp, the wheat to be tested does not contain the Su1-Ph1 gene.

[0012] Further, if the base sequence length includes 1185 bp and 710 bp, the wheat to be tested is a heterozygote containing the Su1-Ph1 gene; if the base sequence length includes 1185 bp but does not include 710 bp, the wheat to be tested is a homozygote containing the Su1-Ph1 gene.

[0013] In order to achieve the above-mentioned purpose, according to the fourth aspect of the present invention, there is provided a method for preparing a short-segment translocation line wheat containing the Su1-Ph1 gene, the preparation method comprising: a) hybridizing the translocation line wheat containing the Su1-Ph1 gene with wild-type wheat to obtain F1 plants; b) selfing the F1 plants to obtain F2 plants; c) using the above-mentioned molecular markers, the above-mentioned kit or the above-mentioned detection method to perform genotyping on the F2 plants, and screening to obtain the short-segment translocation line wheat containing the Su1-Ph1 gene.

[0014] Furthermore, the translocation line wheat containing the Su1-Ph1 gene includes the translocation line LDN-Su1-Ph1; preferably, the wild-type wheat is tetraploid wheat, more preferably tetraploid wheat Langdon.

[0015] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided a method for preparing wheat with partially homologous chromosomes, the preparation method comprising: a) hybridizing wheat containing Su1-Ph1 genes with translocation line wheat containing exogenous chromosomes to obtain F1 plants; b) detecting the F1 plants using the above-mentioned molecular markers, the above-mentioned kit or the above-mentioned detection method to screen and obtain a first plant containing the Su1-Ph1 gene; c) self-pollinating the first plant to obtain an F2 plant; d) screening and obtaining wheat with partially homologous chromosomes in the F2 plants.

[0016] Furthermore, wheat containing the Su1-Ph1 gene includes the translocation line LDN-Su1-Ph1; preferably, the translocation line wheat containing the exogenous chromosome is tetraploid wheat, more preferably the tetraploid wheat substitution line DS1E (LDN1A).

[0017] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, there is provided the application of the above-mentioned molecular marker, the above-mentioned kit, the above-mentioned detection method, the above-mentioned method for preparing the short segment translocation line wheat containing the Su1-Ph1 gene, or the above-mentioned method for preparing the partially homologous chromosome recombinant wheat in wheat breeding and / or wheat exogenous gene isolation and cloning.

[0018] By applying the technical scheme of the present invention, utilizing the molecular marker Pku8051 tightly linked to the Su1-Ph1 gene locus, and performing PCR amplification on wheat materials using the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2, it is possible to quickly, simply and accurately detect whether the wheat material contains the Su1-Ph1 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1The schematic diagram of Example 2 of the present invention is shown, which is a 3S exogenous chromosome fragment carried by the translocation line LDN-Su1-Ph1; wherein, number 1 represents the original donor of the Su1-Ph1 gene, Aegilops pseudo-speltii PI 369609; numbers 2 to 3 are Aegilops pseudo-speltii AE915 and AE159, respectively; number 4 is the translocation line LDN-Su1-Ph1; numbers 5 to 7 are tetraploid wheat Langdon, Svevo, and Zavitan, respectively; numbers 8 to 17 are hexaploid wheat ArinaLrFor, Jagger, Chinese Spring, CDC Landmark, Norin 61, SY Mattis, Julius, LongReach Lancer, Mace, and CDC Stanley, respectively; the blue line represents SNPs specific to Aegilops pseudo-speltii; the translocation line LDN-Su1-Ph1 carries a 22.8Mb exogenous chromosome, and the Su1-Ph1 gene is located at 689.3-712.1Mb (Svevo RefSeq v1.0).

[0021] Figure 2 A schematic diagram of wheat 3A chromosome according to Example 2 of the present invention is shown, wherein 3AL represents the long arm of the 3A chromosome, 3AS represents the short arm of the 3A chromosome, (a) is a schematic diagram of the 3A chromosome of the translocation line LDN-Su1-Ph1, which carries an exogenous 3S chromosome of about 22.8 Mb, as shown by the black rectangle in (a); (b) is a schematic diagram of the 3A chromosome of the recombinant 330-311 carrying an exogenous 3S chromosome, which carries an exogenous chromosome of about 3.6 Mb, as shown by the black rectangle in (b).

[0022] Figure 3 The agarose gel electrophoresis diagram for detecting the genotype of the individual plants of the self-pollinated progeny of the recombinant 330-311 in Example 3 of the present invention is shown, wherein lane 1 is the DL 2000 marker, lanes 2 to 14 are partially separated individual plants of the self-pollinated progeny of the recombinant 330-311, lane 15 is the tetraploid wheat Langdon, and lane 16 is the translocation line LDN-Su1-Ph1; M represents a DNA molecular weight standard, H represents a heterozygous plant, A represents a homozygous plant containing the Su1-Ph1 gene, and B represents a plant without the Su1-Ph1 gene.

[0023] Figure 4The agarose gel electrophoresis diagram of detecting Su1-Ph1 gene in different wheat species according to Example 4 of the present invention is shown, wherein lane 1 is DL 2000 marker, lanes 2 to 7 are tetraploid wheat varieties Langdon, Kronos, Zavitan, Svevo, Rusty, 8155-B1, lanes 8 to 14 are hexaploid wheat varieties China Spring, Jimai 22, Shannong 40, Ningchun No. 4, Fielder, Avocet-S, Cadenza, lane 15 is the translocation line LDN-Su1-Ph1, and M represents the DNA molecular weight standard. The positive control is LDN-Su1-Ph1, and the negative control is Langdon. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Terminology explanation:

[0026] Substitution line: a new type formed by replacing one or several pairs of chromosomes of a certain species with one or several pairs of chromosomes of other species.

[0027] Translocation line: a strain formed by the exchange of chromosome segments between non-homologous chromosomes or the exchange of chromosome segments between heterologous chromosomes of closely related species.

[0028] Homologous chromosomes: Chromosomes whose morphology, size and gene loci are not exactly the same. During meiotic pairing, their ability to pair with each other is not as good as that of homologous chromosomes.

[0029] Single nucleotide polymorphism (SNP) is a DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.

[0030] InDel marker: a marker designed based on the sequences on both sides of the insertion / deletion site in the genome for PCR amplification using specific primers. In essence, it is still a length polymorphism marker.

[0031] As mentioned in the background technology, the Su1-Ph1 gene can induce the pairing and recombination of some homologous chromosomes of wheat and closely related species to form chromosome translocation lines or introgression lines, thus playing an important role in wheat breeding. However, the methods used to detect the Su1-Ph1 gene in the prior art are cumbersome to operate, have a long detection cycle, high cost, and are not suitable for large-scale screening. Therefore, in the present application, the inventors attempt to develop a molecular marker that can quickly and easily detect the wheat Su1-Ph1 gene, and then propose a series of protection schemes of the present application.

[0032] In a first typical embodiment of the present application, a molecular marker for detecting wheat Su1-Ph1 gene is provided, which is Pku8051; the primers used to amplify the molecular marker include an upstream primer (Pku8051-F) shown in SEQ ID NO: 1 and a downstream primer (Pku8051-R) shown in SEQ ID NO: 2.

[0033] This application uses the published reference genome of Ae. speltoides TS01, as well as the annotation information of the reference genomes of tetraploid wheat Svevo and hexaploid wheat Chinese Spring, combined with the resequencing and transcriptome sequencing data of the translocation line LDN-Su1-Ph1, and obtains the potential candidate gene Ae. speltoides. TS01.3B01G0807800.1 related to chromosome recombination pairing in the 3.6Mb 3S genome through gene function prediction, polymorphism analysis, and transcriptome analysis. The gene is located at the position of 706.06Mb of the tetraploid wheat Svevo 3A chromosome (Svevo RefSeq v1.0). According to the annotation information of the reference genome, the gene encodes the recombinase MUS81, which is reported to play an important role in the DNA recombination repair process. Further analysis revealed that within the Ae.speltoides.TS01.3B01G0805100.1 gene, there was a 475 bp insertion in the homologous gene of the translocated 3S chromosome. Using this polymorphic site and Primer Premier 5.0 software, an InDel molecular marker Pku8051 tightly linked to the Su1-Ph1 gene was developed.

[0034] The molecular marker Pku8051 is an InDel molecular marker, and PCR amplification is performed using the upstream primer Pku8051-F and the downstream primer Pku8051-R. If the wheat contains the Su1-Ph1 gene of Aegilops pseudospeltii, a PCR product of 1185 bp can be amplified; if the wheat does not contain the Su1-Ph1 gene of Aegilops pseudospeltii, a PCR product of 1185 bp cannot be amplified, and a PCR product of 710 bp can be amplified with a high probability. Moreover, by judging whether the lengths of the PCR products are the same, it can be judged whether the chromosome containing the Su1-Ph1 gene in wheat is homozygous or heterozygous.

[0035] In a second typical embodiment of the present application, a kit for detecting wheat Su1-Ph1 gene is provided, the kit comprising the above-mentioned primers, wherein the primers comprise an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO: 2.

[0036] The above kit, i.e. the upstream primer and the downstream primer in the above kit, can be used to detect whether the wheat sample contains the Su1-Ph1 gene. In the prior art, when the wheat genome is complex and unclear, it is difficult to screen which molecular marker or which primer can be used to identify the wheat Su1-Ph1 gene in limited experiments.

[0037] In a third typical embodiment of the present application, a method for detecting wheat Su1-Ph1 gene is provided, the detection method comprising: performing amplification using the above-mentioned molecular marker or kit, and detecting the amplified product.

[0038] In a preferred embodiment, the detection method includes: using the genomic DNA of the wheat to be tested as a template, performing PCR amplification using primers to obtain PCR amplification products; detecting the PCR amplification products to determine the base sequence length of the PCR amplification products. If the base sequence length includes 1185bp, the wheat to be tested contains the Su1-Ph1 gene; if the base sequence length does not include 1185bp, the wheat to be tested does not contain the Su1-Ph1 gene.

[0039] By using existing technologies such as genome extraction kits, the genome of the wheat to be tested can be extracted and used as a template for PCR amplification. Alternatively, the wheat seedlings can be directly sampled and processed and used as a PCR template for PCR amplification. After PCR amplification, the PCR product is tested. If the base sequence length in the PCR product includes 1185 bp, the wheat to be tested contains the Su1-Ph1 gene; if the base sequence length in the PCR product does not include 1185 bp, the wheat to be tested does not contain the Su1-Ph1 gene.

[0040] In a preferred embodiment, if the base sequence length is 710 bp, the wheat to be tested does not contain the Su1-Ph1 gene.

[0041] In the present application, all existing wheat reference genomes and wheat materials of several hundred copies of exome sequencing that have been announced have been analyzed. The above-mentioned upstream primer Pku8051-F and downstream primer Pku8051-R and the genes at which the primers are located have very high conservatism in these materials and can all expand a PCR product of 710bp. Therefore, it is inferred that the wheat to be tested that does not contain the Su1-Ph1 gene of Aegilops pseudospeltii can theoretically expand a PCR product of 710bp.

[0042] In a preferred embodiment, if the base sequence length includes 1185bp and 710bp, the wheat to be tested is a heterozygote containing the Su1-Ph1 gene; if the base sequence length includes 1185bp but does not include 710bp, the wheat to be tested is a homozygote containing the Su1-Ph1 gene.

[0043] If the wheat to be tested is homozygous wheat containing the Su1-Ph1 gene, the base sequence length in the PCR product only includes 1185bp; if the wheat to be tested is heterozygous wheat containing the Su1-Ph1 gene, the base sequence length in the PCR product includes two fragment lengths of 1185bp and 710bp. The detection method of the above PCR product includes but is not limited to agarose gel electrophoresis. The above PCR amplification program can be flexibly adjusted according to factors such as the amount of template used in PCR and the type of DNA polymerase.

[0044] In a preferred embodiment, the program of PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 40 sec, 35 cycles; extension at 72°C for 5 min, and storage at 12°C.

[0045] In a fourth typical embodiment of the present application, a method for preparing a short-segment translocation line wheat containing the Su1-Ph1 gene is provided, and the preparation method comprises: a) hybridizing the translocation line wheat containing the Su1-Ph1 gene with wild-type wheat to obtain F1 plants; b) selfing the F1 plants to obtain F2 plants; c) using the above-mentioned molecular markers, the above-mentioned kit or the above-mentioned detection method to perform genotype identification on the F2 plants, and screen to obtain the short-segment translocation line wheat containing the Su1-Ph1 gene.

[0046] The present invention uses the obtained Aegilops pseudospeltii 3S chromosome-specific SNP to develop multiple pairs of specific molecular markers on the exogenous 3S chromosome of the translocation line LDN-Su1-Ph1, and uses the 3S chromosome-specific molecular markers to identify the genotype of the F2 plant to determine the size of the exogenous 3S chromosome fragment, and screen to obtain a short-segment translocation line wheat containing the Su1-Ph1 gene. In Example 2 of the present application, after obtaining the F2 plant, multiple molecular markers located on the 22.8-Mb 3S chromosome but not tightly linked to the Su1-Ph1 gene are used to determine the genotype of the F2 plant, detect whether the exogenous 3S chromosome has gene recombination, and then screen to obtain a short-segment translocation line wheat containing the Su1-Ph1 gene. However, except for Pku8051, the multiple 3S specific molecular markers developed are basically located on the recombined 3S chromosome. Once the above molecular markers are known, the identification of the Su1-Ph1 gene can be achieved by using the above molecular markers, kits or detection methods, thereby quickly and conveniently achieving the preparation of short-segment translocation wheat containing the Su1-Ph1 gene. The above-mentioned short-segment translocation wheat refers to wheat containing shortened chromosome fragments. In the prior art, the 3S chromosome fragment carried by the translocation wheat carrying the Su1-Ph1 gene is about 22.8Mb, that is, wheat containing the Su1-Ph1 gene and carrying a 3S chromosome fragment less than 22.8Mb all belong to the short-segment translocation wheat containing the Su1-Ph1 gene.

[0047] Preferably, the above preparation method also includes identifying whether the wheat is a short segment translocation line wheat, and the identification method includes: using multiple specific molecular markers distributed on the 3S chromosome to perform genotype identification on the F2 plants to screen and determine the short segment translocation line containing the Su1-Ph1 gene.

[0048] In a preferred embodiment, the translocation line wheat containing the Su1-Ph1 gene includes the translocation line LDN-Su1-Ph1; preferably, the wild-type wheat is tetraploid wheat, more preferably tetraploid wheat Langdon.

[0049] In a fifth typical embodiment of the present application, a method for preparing recombinant wheat with partial homoeologous chromosomes is provided, the method comprising: a) hybridizing wheat containing Su1-Ph1 gene with translocation wheat containing exogenous chromosomes to obtain F1 plants; b) using the above molecular markers, kits or detection methods to detect the F1 plants, and screening to obtain the first plants containing Su1-Ph1 genes; c) self-pollinating the first plants to obtain F2 plants; d) screening to obtain recombinant wheat with partial homoeologous chromosomes in the F2 plants. The above preparation method can achieve the purpose of using Su1-Ph1 gene to reduce the exogenous chromosome fragments, break linkage drag, and create short-segment translocation lines.

[0050] The above-mentioned partial homeologous chromosome recombination refers to the recombination between partial homeologous chromosomes (such as chromosomes 5S and 5B, or chromosomes 1E and 1A) that are not completely the same in shape, size and gene loci, and whose mutual pairing ability during meiosis is not as good as that of homologous chromosomes. Partial homeologous chromosome recombinant wheat is wheat containing the above-mentioned recombined partial homeologous chromosomes.

[0051] The above-mentioned translocation wheat containing exogenous chromosomes includes translocation wheat containing large fragments of exogenous chromosomes, wherein the large fragment refers to a larger fragment of the translocated exogenous chromosome, such as a whole chromosome replacement or a whole arm translocation, which may contain a large number of linked unfavorable genes.

[0052] In the above preparation method, wheat containing the Su1-Ph1 gene is first hybridized with translocation wheat containing a large exogenous chromosome to obtain a heterozygous F1 plant containing the Su1-Ph1 gene. The F1 plant is tested using the above molecular marker, kit or detection method to ensure that the F1 plant obtained is a successful hybrid. Since the F1 plant contains the Su1-Ph1 gene and the large translocated exogenous chromosome is a heterozygous type, it can induce recombination between some homologous chromosomes. Therefore, the genotype of the large translocated exogenous chromosome is identified in the F2 plant, and wheat with recombined homologous chromosomes can be screened. Using the above preparation method, the purpose of reducing the size of the exogenous translocated chromosome can be achieved.

[0053] In a preferred embodiment, the wheat containing the Su1-Ph1 gene includes the translocation line LDN-Su1-Ph1; preferably, the translocation line wheat containing the exogenous chromosome is tetraploid wheat, more preferably the tetraploid wheat substitution line DS1E (LDN1A).

[0054] In the sixth typical embodiment of the present application, there is provided a method for using the above-mentioned molecular marker, the above-mentioned kit, the above-mentioned detection method, the above-mentioned method for preparing the short segment translocation line wheat containing the Su1-Ph1 gene, or the above-mentioned method for preparing the partially homologous chromosome recombinant wheat in wheat breeding and / or wheat exogenous gene isolation and cloning.

[0055] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0056] Example 1: Aegilops spelta Su1-Ph1 gene induces the recombination of exogenous chromosome fragments

[0057] Lophopyrum elongatum has strong stems and a well-developed root system. It is salt-alkali tolerant, waterlogged tolerant, drought tolerant, and immune to wheat powdery mildew and three rusts (strip rust, leaf rust, and stem rust). It is also highly resistant to scab and other diseases, and is an important gene pool for wheat genetic improvement. However, due to the presence of the wheat Ph1 gene, the recombination exchange between it and common wheat chromosomes is limited, which is not conducive to the transfer of excellent genes to wheat. The Su1-Ph1 gene in the present invention can promote the recombination of exogenous chromosome fragments with wheat chromosomes.

[0058] LDN-Su1-Ph1 was hybridized with the substitution line DS1E (LDN1A) to obtain F1. The substitution line DS1E (LDN1A) is a tetraploid wheat, in which the 1E chromosome of Elymus longispinus replaced the 1A chromosome of the tetraploid wheat Langdon. The obtained F1 individual plant was self-pollinated to obtain F2. Since the Su1-Ph1 gene and the exogenous chromosome 1E will separate in the F2 generation plants, the genotype of the F2 population was identified with the help of molecular markers. The 1E / 1A heterozygous individual plant containing the Su1-Ph1 gene was selected for self-pollination, and the genotype of its offspring was identified with the help of molecular markers to detect whether the 1E / 1A partial homeologous chromosome recombination occurred; at the same time, the 1E / 1A heterozygous individual plant without the Su1-Ph1 gene was selected as a control to detect whether the 1E / 1A partial homeologous chromosome recombination occurred in its self-pollinated offspring. The results showed that no recombinants were detected in 64 plants without Su1-Ph1 gene, but 3 1E / 1A recombinants were detected in 63 plants with Su1-Ph1 gene (as shown in Table 1), indicating that the presence of Su1-Ph1 gene induced the recombination of 1E / 1A partial homologous chromosomes.

[0059] Table 1: Genotypes of F3 recombinants from the hybridization of DS1E (LDN1A) × LDN-Su1-Ph1

[0060]

[0061] Note: W and E represent 1A and 1E genotypes, respectively. 29, 38, and 46 are the numbers of the obtained recombinants.

[0062] Example 2 Creation of a short segment translocation line of Su1-Ph1 gene of Aegilops spelta

[0063] Aegilops spelta lacks a high-quality reference genome and the species has the characteristics of cross-pollination, so the sequence differences between different materials are large, which has caused certain difficulties in the development of specific molecular markers. The original donor of the Su1-Ph1 gene, Aegilops spelta PI 369609, two Aegilops spelta AE915 and AE159, the tetraploid background wheat Langdon and the translocation line LDN-Su1-Ph1 were resequenced, and the reference genome sequences of tetraploid and hexaploid wheat that had been sequenced were used, combined with bioinformatics analysis, to find the SNP specific to the 3S chromosome of Aegilops spelta. The data showed that the translocation line LDN-Su1-Ph1 carried a 3S exogenous chromosome fragment of about 22.8Mb, located between 689.3-712.1Mb of chromosome 3A of the tetraploid wheat SvevoRefSeq v1.0 reference genome (such as Figure 1 shown).

[0064] The present invention uses the obtained Aegilops pseudospeltii 3S chromosome-specific SNP to develop multiple pairs of specific molecular markers on the exogenous 3S chromosome of the translocation line LDN-Su1-Ph1 for subsequent large-scale recombinant screening. However, except for Pku8051, the multiple pairs of 3S chromosome-specific molecular markers developed are all located on the 3S chromosome that is recombined in subsequent experiments (the 3S chromosome fragment carried by the translocation line wheat carrying the Su1-Ph1 gene in the prior art is about 22.8Mb; the present application obtains a short-segment translocation line wheat carrying the Su1-Ph1 gene, which carries a 3S chromosome fragment of about 3.6Mb, and the length of the exogenous 3S chromosome is shortened by 19.2Mb).

[0065] Using the indoor plant growth room of the Modern Agricultural Research Institute of Peking University, LDN-Su1-Ph1 was hybridized with tetraploid wheat Langdon to obtain F1. The obtained F1 individual plant was self-pollinated to obtain F2. The presence of the Su1-Ph1 gene can induce the recombination of 3S and 3A partial homologous chromosomes, and there will be a short segment translocation line containing the Su1-Ph1 gene in the F2 population. Through molecular marker identification, heterozygous individual plants carrying shortened 3S / 3A chromosomes were selected in the F2 population and self-pollinated to obtain F3. Subsequently, with the help of molecular markers, it was detected whether the reduced 3S / 3A heterozygous chromosomes could re-occur with partial homologous chromosome recombination to determine whether the Su1-Ph1 gene existed. Through the above method, a key small segment recombinant was obtained, and the strain number was 330-311. Recombinant 330-311 is the recombinant with the smallest exogenous 3S chromosome segment carrying the Su1-Ph1 gene screened so far. Molecular marker identification showed that the exogenous 3S chromosome fragment contained in 330-311 was about 3.6 Mb in size, and the translocation fragment accounted for about 0.48% of the entire 3A chromosome (Svevo RefSeq v1.0). Figure 2 The genotype of the recombinant 330-311 is heterozygous, in which one sister chromatid does not carry the foreign 3S chromosome.

[0066] Example 3 Prediction of candidate genes for Su1-Ph1 gene of Aegilops speltatum and development of tightly linked molecular markers

[0067] Using the published reference genome of Ae. speltoides TS01, as well as the annotation information of the reference genomes of tetraploid wheat Svevo and hexaploid wheat Chinese Spring, combined with the resequencing and transcriptome sequencing data of the translocation line LDN-Su1-Ph1, through gene function prediction, polymorphism analysis, and transcriptome analysis, we obtained a potential candidate gene Ae. speltoides. TS01.3B01G0807800.1 related to chromosome recombination pairing in the 3.6Mb 3S genome. This gene is located at the 706.06Mb position of the tetraploid wheat Svevo3A chromosome (Svevo RefSeq v1.0). According to the annotation information of the reference genome, this gene encodes the recombinase MUS81, which is reported to play an important role in the process of DNA recombination repair. We found a 475bp insertion in the homologous gene of the translocation 3S chromosome in the Ae.speltoides.TS01.3B01G0805100.1 gene. Using this polymorphic site and Primer Premier 5.0 software, we developed the InDel molecular marker Pku8051 that is tightly linked to the Su1-Ph1 gene. This marker is located at 705.67Mb (SvevoRefSeq v1.0) of the tetraploid wheat Svevo 3A chromosome, and the physical distance to our inferred candidate gene is only 0.39Mb (Svevo RefSeq v1.0). In theory, this marker is tightly linked to our inferred candidate gene and can be used as a tightly linked marker of the gene.

[0068] The sequences of the amplification primers used to amplify the molecular marker Pku8051 are as follows:

[0069] Pku8051-F: 5'-ACCCTAGAGTCGCAATGGAC-3', SEQ ID NO: 1;

[0070] Pku8051-R: 5'-TTTCAGTATGTCGTCCGCTC-3', SEQ ID NO: 2.

[0071] Specifically, the recombinant 330-311 is a heterozygote, and its self-pollination produces segregating offspring. The molecular marker Pku8051 is used to screen homozygous recombinant plants in the offspring. The molecular marker Pku8051 is used to perform PCR amplification on the genomic DNA of the individual plants in the self-pollination offspring population of the recombinant 330-311, and the reaction system is as follows:

[0072] DNA template (100ng / μL) 0.5μL,

[0073] 2×Taq Plus PCR MasterMix (RB-PCRD01) 6.25 μL,

[0074] 0.5 μL of each 10 μM primer,

[0075] Add ddH2O to make up to 12.5 μL.

[0076] PCR amplification reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 40 sec, 35 cycles; extension at 72°C for 10 min, and storage at 12°C.

[0077] The above products were analyzed by 1% agarose gel electrophoresis, the electrophoresis buffer was 1×TAE, and the constant voltage of the electrophoresis instrument was 150 V. The gel was stained with EB and photographed using a BIO-RAD imager. Some of the results are shown in Figure 3 As shown, lanes 2, 3, and 13 amplified the same bands as LDN-Su1-Ph1 (lane 16), indicating that it contained a homozygous 3S short-fragment chromosome; the bands in lanes 4, 7, and 8 were the same as Langdon (lane 15), indicating that it did not contain a 3S chromosome; and lanes 5, 6, 9, 10, 11, 12, and 14 were heterozygous band types, indicating that they were heterozygous individual plants.

[0078] Example 4 Verification of the tightly linked molecular marker Pku8051 of the Su1-Ph1 gene of Aegilops spelta

[0079] The molecular marker Pku8051 was used to identify the genotypes of 82 tetraploid wheat (T. turgidum) and 68 hexaploid wheat (T. aestivum) materials from home and abroad.

[0080] Specifically, the molecular marker Pku8051 was used to perform PCR amplification on the genomic DNA of different wheat materials, and the reaction system was as follows:

[0081] DNA template (100 ng / μL) 0.5 μL;

[0082] 2×Taq Plus PCR Master Mix (RB-PCRD01, Ruibo Xingke) 6.25 μL;

[0083] 0.5 μL each of 10 μM primers;

[0084] Add ddH2O to make up to 12.5 μL.

[0085] PCR amplification reaction program: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 40 sec, 35 cycles; extension at 72°C for 10 min, and storage at 12°C.

[0086] The above products were analyzed by 1% agarose gel electrophoresis, the electrophoresis buffer was 1×TAE, and the constant voltage of the electrophoresis instrument was 150 V. The gel was stained with EB and photographed using a BIO-RAD imager. Some of the results are shown in Figure 4 The results show that none of the tetraploid and hexaploid wheat materials amplified bands consistent with the donor parent PI 369609 and the translocation line LDN-Su1-Ph1, indicating that these materials do not contain the Su1-Ph1 gene. These results show that the molecular marker Pku8051 provided by the present invention can efficiently and accurately identify whether the wheat material contains the Aegilops pseudospeltii Su1-Ph1 gene, and the statistical results are shown in Table 2.

[0087] Table 2: Identification of different tetraploid and hexaploid wheat materials by molecular marker Pku8051

[0088]

[0089]

[0090] In addition, we analyzed all published wheat reference genomes and several hundred wheat materials with exome sequencing. The upstream primer Pku8051-F and downstream primer Pku8051-R designed by us, as well as the genes where the primers are located, are highly conserved in these materials and can amplify a 710bp PCR product. Therefore, it is speculated that the wheat to be tested that does not contain the Su1-Ph1 gene of Aegilops pseudospeltii can theoretically amplify a 710bp PCR product.

[0091] It can be seen from the above description that the above embodiments of the present invention achieve the following technical effects: the present invention provides a molecular marker Pku8051 for detecting the wheat Su1-Ph1 gene. By using the upstream primer Pku8051-F and the downstream primer Pku8051-R, the molecular marker can be amplified using the genome of the wheat sample to be detected as a template, thereby detecting whether the wheat sample contains the Aegilops pseudospeltii Su1-Ph1 gene, and the detection difficulty is low.

[0092] From the above description, it can be seen that the above embodiment of the present invention achieves the following technical effects: the molecular marker Pku8051 is very closely linked to the wheat Su1-Ph1 gene, and the detection result is more accurate; the length of the fragment after PCR amplification of the two wheat materials with and without the Su1-Ph1 site is very different, so the detection difficulty is low. Using the molecular marker Pku8051 that is closely linked to the Su1-Ph1 gene site, by using the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2 to perform PCR amplification on the wheat material, it is possible to quickly, simply and accurately detect whether the wheat material contains the Su1-Ph1 gene. Using this molecular marker, it can play an important role in wheat breeding. By detecting whether the wheat progeny produced by hybridization contains the Su1-Ph1 gene, the progeny without the Su1-Ph1 gene can be screened out, reducing unnecessary breeding and gene detection workload. This molecular marker can also provide convenience and material basis for subsequent research such as cloning of the gene.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting wheat Su1-Ph1 A gene kit, characterized in that The kit comprises primers, wherein the primers comprise an upstream primer shown in SEQ ID NO: 1 and a downstream primer shown in SEQ ID NO:

2.

2. A kind of wheat Su1-Ph1 A method for detecting a gene, characterized in that: The detection method comprises: Amplifying using the kit according to claim 1, and detecting the amplified product; The detection method comprises: Using the genomic DNA of the wheat to be tested as a template, performing PCR amplification using the primers to obtain a PCR amplification product; Detecting the PCR amplification product to determine the base sequence length of the PCR amplification product, If the base sequence length includes 1185 bp, the wheat to be tested contains Su1-Ph1 Gene; If the base sequence length does not include 1185 bp, the wheat to be tested does not contain the Su1-Ph1 Gene.

3. The detection method according to claim 2, characterized in that: The procedure of the PCR amplification is: Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 40 sec, 35 cycles; extension at 72°C for 5 min, and storage at 12°C.

4. The detection method according to claim 2 or 3, characterized in that: If the base sequence length is 710 bp, the wheat to be tested does not contain Su1-Ph1 Gene.

5. The detection method according to claim 2 or 3, characterized in that: If the base sequence lengths include 1185 bp and 710 bp, the wheat to be tested contains the Su1-Ph1 Heterozygous for a gene; If the base sequence length includes 1185 bp but does not include 710 bp, the wheat to be tested contains the Su1- Ph1 Homozygous for the gene.

6. A Su1-Ph1 A method for preparing a short gene translocation line wheat, characterized in that: The preparation method comprises: a) will contain Su1-Ph1 The translocation line wheat of the gene is crossed with wild-type wheat to obtain F1 plants; b) selfing the F1 plants to obtain F2 plants; c) using the kit according to claim 1 or the detection method according to any one of claims 2 to 5 to identify the genotype of the F2 plants, and screen for the plants containing Su1-Ph1 Short fragment translocation lines of genes in wheat.

7. The preparation method according to claim 6, characterized in that: Said containing Su1-Ph1 Gene translocation lines of wheat include the translocation line LDN-Su1-Ph1.

8. The preparation method according to claim 6, characterized in that: The wild type wheat is tetraploid wheat.

9. The preparation method according to claim 8, characterized in that: The wild type wheat is tetraploid wheat Langdon.

10. A method for preparing wheat with partially homologous chromosomes, characterized in that: The preparation method comprises: a) will contain Su1-Ph1 The wheat with the gene was crossed with the translocation line wheat containing the foreign chromosome to obtain F1 plants; b) using the kit according to claim 1 or the detection method according to any one of claims 2 to 5 to detect the F1 plants, and screen for the plants containing Su1-Ph1 The first plant of genes; c) selfing the first plant to obtain an F2 plant; d) Screening and obtaining the recombinant wheat with the partially homologous chromosomes in the F2 plants.

11. The preparation method according to claim 10, characterized in that: Said containing Su1-Ph1 The wheat gene includes the translocation line LDN-Su1-Ph1.

12. The preparation method according to claim 10, characterized in that: The translocation wheat containing exogenous chromosomes is tetraploid wheat.

13. The preparation method according to claim 12, characterized in that: The translocation wheat line containing exogenous chromosomes is a tetraploid wheat substitution line DS1E (LDN1A).

14. The kit according to claim 1, the detection method according to any one of claims 2 to 5, the composition containing any one of claims 6 to 9 Su1-Ph1 The method for preparing a wheat line with a short gene translocation or the method for preparing a wheat with partially homologous chromosomes as described in any one of claims 10 to 13 are used in wheat breeding and / or wheat exogenous gene isolation and cloning.

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