InDel molecular markers for selecting or assisting in the selection of stem nodule sesbania and their application

Abstract

The present invention belongs to the technical field of sesbania genetic breeding, and specifically relates to molecular markers for breeding or assisting in breeding of stem nodule sesbania and their application in hybrid breeding and haploid breeding. The molecular markers provided by the present invention are a set of InDel molecular markers for detecting the genetic similarity between sesbania varieties and germplasm and stem nodule sesbania, which are distributed on chromosomes 1, 3, 4, 5, and 6 of sesbania. The haplotypes of InDel molecular markers differ greatly between stem nodule sesbania and sesbania core germplasm and varieties, and are stably inherited. According to the difference between molecular markers in stem nodule sesbania and root nodule sesbania, the present invention develops primer pairs for specifically amplifying the molecular markers. By using the primer pairs provided by the present invention, it is only necessary to extract sesbania genomic DNA and then perform PCR amplification and agarose gel electrophoresis to quickly and accurately identify or assist in identifying whether the genetic source of sesbania is stem nodule sesbania.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to InDel molecular markers for breeding or auxiliary breeding of stem tumor sesbania and applications thereof. Background Art

[0002] At present, the breeding of sesbania is still mainly based on conventional breeding technology, which has a long breeding cycle and low efficiency. However, by inducing haploids and doubling the genome of hybrid offspring with excellent agronomic traits, the aggregation and rapid homozygosity of excellent alleles can be achieved, the breeding efficiency of sesbania can be improved, and the breeding process of sesbania can be accelerated. Stem nodule sesbania has the excellent trait of stem nodulation, and can be hybridized with sesbania varieties or germplasm with excellent traits such as large biomass, strong tolerance to environmental stress, and root nodulation to produce a genome heterozygous F1 generation with multiple excellent traits. Using haploid technology to induce F 1 The new varieties with homozygous genome and stable phenotype were produced. 1 The authenticity of hybrid genetic combinations and high-throughput and accurate identification of induced haploid progeny are of great significance for sesbania breeding. Molecular markers are basic tools for genetic research and are widely used in the fields of genetic diversity research, germplasm identification, genetic map construction and gene positioning. The identification of target varieties using molecular marker-assisted selection technology is more accurate, simple, and not affected by the environment, and has high efficiency. Molecular markers have the advantages of genetic stability and are not easily affected by the environment and materials. The method of identifying sesbania varieties and purity in the prior art is mainly field plant phenotypic identification (morphological markers). Due to the slow growth of sesbania seedlings and the instability of the phenotypes between individuals with large genetic background differences, it is difficult to use morphological markers to quickly identify the authenticity and purity of the variety.

[0003] DNA molecular markers have been widely used in the identification of crop varieties and germplasm authenticity. In the evaluation of genetic diversity and identification of kinship of Sesbania sesbania germplasm resources, only SNP and SSR molecular markers are used, while the widely studied and applied InDel markers have not been developed and used in Sesbania sesbania. Compared with SSR molecular markers, Indel markers have higher resolution and accuracy and shorter identification cycle. With the development of sequencing technology, the development of InDel markers using high-throughput genomic data has received great attention. InDel molecular markers are base insertion / deletion phenomena, which have the advantages of wide distribution in the genome, high density, stable variation, strong polymorphism, and easy detection. At present, they have been widely used in major food crops and cash crops, especially in variety and germplasm identification, germplasm purity identification and molecular identity card construction. However, there are no relevant reports on the use of InDel molecular markers in the identification of Sesbania sesbania varieties and germplasm, purity detection and molecular identity card construction.

[0004] Therefore, providing an Indel molecular marker primer set and application for identifying stem tumor sesbania varieties is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an Indel molecular marker primer set and application for breeding or assisting in breeding of tuber sesbania varieties and germplasms. The InDel molecular marker provided by the present invention can distinguish tuber sesbania from other tuber-free sesbania phenotypes in sesbania approved varieties and core germplasms. The molecular marker can be used to quickly and accurately screen sesbania with tuber sesbania genetic background, and has broad application prospects in breeding forage and greening sesbania.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides an application of InDel molecular markers for breeding or assisting in breeding of stem tumor sesbania in identifying sesbania varieties and germplasm, wherein the InDel molecular markers are 5 in total, which are located on chromosomes 1, 3, 4, 5, and 6 of the sesbania genome, respectively, and the primer set for amplifying the InDel molecular markers includes the following primer pairs:

[0008] The primer pair IDE3 includes an upstream primer IDE3-F and a downstream primer IDE3-R, the sequence of IDE3-F is shown in SEQ ID NO. 1, and the sequence of IDE3-R is shown in SEQ ID NO. 2;

[0009] The primer pair IDE15 includes an upstream primer IDE15-F and a downstream primer IDE15-R, the sequence of IDE15-F is shown in SEQ ID NO. 3, and the sequence of IDE15-R is shown in SEQ ID NO. 4;

[0010] The primer pair IDE20 includes IDE20-F and IDE20-R, the sequence of IDE20-F is shown in SEQ ID NO.5, and the sequence of IDE20-R is shown in SEQ ID NO.6;

[0011] The primer pair IDE25 includes an upstream primer IDE25-F and a downstream primer IDE25-R, the sequence of IDE25-F is shown in SEQ ID NO.7, and the sequence of IDE25-R is shown in SEQ ID NO.8;

[0012] The primer pair IDE30 includes an upstream primer IDE30-F and a downstream primer IDE30-R, wherein the sequence of IDE30-F is shown in SEQ ID NO. 9, and the sequence of IDE30-R is shown in SEQ ID NO. 10

[0013] The second aspect of the present invention provides the use of the above-mentioned InDel molecular marker for selecting or assisting in selecting nodular sesbania in hybrid breeding of nodular sesbania.

[0014] The third aspect of the present invention provides the use of the above-mentioned InDel molecular markers for selecting or assisting in the selection of stem nodule sesbania in identifying the genetic origin of hybrid sesbania.

[0015] The fourth aspect of the present invention provides the use of the above-mentioned InDel molecular marker for selecting or assisting in the selection of nodular sesbania in haploid breeding of nodular sesbania.

[0016] The fifth aspect of the present invention provides the use of the above-mentioned InDel molecular markers for selecting or assisting in the selection of stem tumor sesbania in identifying the genetic origin of the offspring sesbania produced by haploid induction.

[0017] A sixth aspect of the present invention provides a method for identifying or assisting in identifying whether the genetic source of Sesbania sesbania is stem tumor Sesbania sesbania, comprising the following steps:

[0018] a. Extracting genomic DNA from Sesbania odorata materials;

[0019] b. Using the DNA obtained in step a as a template, PCR amplification was performed using primer pair IDE3, primer pair IDE15, primer pair IDE20, primer pair IDE25, and primer pair IDE30, and the amplified fragments were analyzed by 1.5% agarose gel electrophoresis;

[0020] c. When primer pair IDE3 was used, a band appeared at the 759 bp position of the electrophoresis graph for Sesbania tuberulata, while the electrophoresis showed 1204 bp for non-sebasica tuberulata; when primer pair IDE15 was used, a band appeared at the 994 bp position of the electrophoresis graph for Sesbania tuberulata, while the electrophoresis showed 691 bp for non-sebasica tuberulata; when primer pair IDE20 was used, a band appeared at the 1000 bp position of the electrophoresis graph for Sesbania tuberulata, while the electrophoresis showed 593 bp for non-sebasica tuberulata; when primer pair IDE25 was used, a band appeared at the 1390 bp position of the electrophoresis graph for Sesbania tuberulata, while the electrophoresis showed 1000 bp for non-sebasica tuberulata; when primer pair IDE30 was used, a band appeared at the 1248 bp position of the electrophoresis graph for Sesbania tuberulata, while the electrophoresis showed 818 bp for non-sebasica tuberulata.

[0021] Furthermore, in step b, the PCR amplification system is: 1.0 μL genomic DNA, 15.0 μL 2×PCRMix, 1 μL each of upstream and downstream primers, and ddH 2 O to 30μL; the PCR amplification program was: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 40s, 35 cycles; 72℃ extension for 5min.

[0022] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention analyzes the high-quality reference genome results of tuber sesbania and approved varieties, uses bioinformatics methods to screen out InDel sites with high polymorphism, and develops InDel markers suitable for electrophoresis detection, and performs molecular marker validity identification on sesbania varieties and germplasms of different chromosome ploidy, different geographical origins and different tuber phenotypes, and finally screens out 5 pairs of InDel markers with polymorphism and clear bands distributed on chromosomes 1, 3, 4, 5 and 6, which can completely distinguish tuber sesbania from 16 sesbania varieties and germplasms. The sesbania Indel molecular marker primer set for identifying sesbania varieties and germplasms disclosed in the present invention can be used for tuber sesbania varieties and germplasm identification, purity detection and molecular identity card construction, thereby improving the inspection accuracy and efficiency of germplasm, and laying a certain foundation for the subsequent development of sesbania breeding work. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 The accompanying drawing shows the genetic relationship analysis of 1 stem nodule sesbania and 16 root nodule sesbania varieties and germplasms of the present invention;

[0026] Figure 2 The attached figure shows the detection results of 17 sesbania varieties and germplasms using the InDel molecular marker provided by the present invention; wherein, M is 2K Marker, No. 1 is stem tumor sesbania, No. 2 to 17 are sesbania core germplasms and varieties, and No. 18 is a blank control. DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0028] In the present invention, the genetic distance between the stem nodule sesbania and other sesbania varieties and core germplasm is relatively far, and the genetic variation is relatively large ( Figure 1). The nodule phenotype is a unique excellent trait of nodule sesbania, and there is a significant phenotypic difference between it and root nodule sesbania. The InDel molecular marker of the present invention is designed based on the structural variation site between nodule sesbania and the Zhongkejing 1 reference genome. The variation site is stable and effective in sesbania with multiple genetic diversities, and has significant selectivity for distinguishing nodule sesbania in the population. The present invention provides an important reference basis for the breeding of nodule sesbania and molecular marker-assisted breeding, and has broad application prospects in the molecular breeding of new varieties and new lines of nodule sesbania.

[0029] Example 1: Development, screening and validation of InDel molecular markers

[0030] 1. Use the Zhongkejing 1 reference genome (A and B subgenomes) to compare with the stem tumor sesbania reference genome, screen the InDel site information, and design primers: use Primer3 and BLAST to design InDel marker primers.

[0031] 2. Based on the InDel sites identified from the reference genome data, we screened sites with insertion and deletion bases greater than or equal to 300 and less than 450, a sequencing depth greater than 10, and sites that could be identified by comparing the A and B subgenomes of Zhongkejing 1 with those of Sesbania tuberulata. We randomly screened out 67 InDel sites that could be designed into primers and were evenly distributed on the 6 chromosomes of the Sesbania tuberulata reference genome.

[0032] 3. Extract 500 bp of reference genome base sequence upstream and downstream of the mutation site on the reference genome of Sesbania scaly. Design primers according to the primer design standard, annealing temperature of 58-60°C, primer length of (20±5) bp, and PCR product size of 750-1500 bp.

[0033] 4. The varieties and germplasms of Sesbania sesbania have a wide geographical distribution, including Zhongkejing No. 1 and 16 core germplasms, a total of 17 varieties and germplasms (Table 1); among them, there is 1 forage variety (No. 16) and 16 core germplasms (No. 1-15, 17); they were collected from 7 regions, 2 in Beijing (No. 2-3), 2 in Shandong (No. 4 and 17), 3 in Hainan (No. 5-7), 1 in Guangxi (No. 8), 2 in Fujian (No. 9-10), 3 in Guangdong (No. 11-13), 1 in Yunnan (No. 15), and 1 in Pakistan (No. 14).

[0034] Table 1: 17 Sesbania cultivars and germplasms

[0035]

[0036] 5. Extract leaf DNA by CTAB method.

[0037] 6. InDel marker screening and verification: Using the genomic DNA of the Zhongkejing No. 1 sesbania variety and the tumor sesbania germplasm as templates, PCR amplification and 1.5% agarose gel electrophoresis analysis were performed, and 11 pairs of InDel markers with stable amplified bands and obvious polymorphism were initially screened out. Then, 17 sesbania varieties and germplasms were used for rescreening, and finally 5 pairs of InDel markers with clear and stable bands, good repeatability and high polymorphism were obtained.

[0038] 7. The PCR amplification system is 30 μL, including 1.0 μL genomic DNA (100 ng·μL -1 ), 15.0 μL 2×PCRMix, forward and downstream primers (10 μmol·L -1 ) 1 μL each, add ddHO 2 O to 30 μL. The amplification program was: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 40 s, 35 cycles; 72°C extension for 5 min. PCR amplification products were detected by 1.5% agarose gel electrophoresis (1.0×TAE buffer) and photographed with nucleic acid dye.

[0039] 8.5 pairs of Indel primer information sequences

[0040] Using the genomic DNA of the Zhongkejing No. 1 sesbania variety and the stem tumor sesbania germplasm with large differences in genetic background as templates, PCR amplification and 1.5% agarose gel electrophoresis analysis were performed, and 11 pairs of InDel markers with stable amplified bands and obvious polymorphism were initially screened. Then 17 sesbania varieties and germplasms were used for rescreening, and finally 5 pairs of InDel markers with clear and stable bands, good repeatability and high polymorphism were obtained. The size of the amplified fragment is between 750 and 1500bp, distributed on 5 of the 6 chromosomes in the sesbania genome. The specific information is as follows:

[0041] The primer pair IDE3 for amplifying the InDel molecular marker of chromosome 1 of stem tumor sesbania, the primer pair IDE3 comprises an upstream primer IDE3-F and a downstream primer IDE3-R; the nucleotide sequence of the upstream primer IDE3-F is shown in SEQ ID NO. 1, the nucleotide sequence of the downstream primer IDE3-R is shown in SEQ ID NO. 2, and the specific primer sequences are as follows:

[0042] SEQ ID NO.1: 5'-TGAGCCCGTTTGGATACACT-3';

[0043] SEQ ID NO.2: 5'-ACGAGCCAAGCCCATAACTA-3'.

[0044] The primer pair IDE15 for amplifying the InDel molecular marker of chromosome 3 of stem tumor sesbania, the primer pair IDE3 comprises an upstream primer IDE15-F and a downstream primer IDE15-R; the nucleotide sequence of the upstream primer IDE15-F is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer IDE15-R is shown in SEQ ID NO.4, and the specific primer sequences are as follows:

[0045] SEQ ID NO.3: 5'-ATGTCCTTGCACACTCATGTC-3';

[0046] SEQ ID NO.4: 5'-AATTCCAGCCTCCCTTGCTTT-3'.

[0047] The primer pair IDE20 for amplifying the InDel molecular marker of chromosome 4 of stem tumor sesbania, the primer pair IDE20 includes an upstream primer IDE20-F and a downstream primer IDE20-R; the nucleotide sequence of the upstream primer IDE20-F is shown in SEQ ID NO.5, the nucleotide sequence of the downstream primer IDE20-R is shown in SEQ ID NO.6, and the specific primer sequences are as follows:

[0048] SEQ ID NO.5: 5'-AGGTGGGGCATCTTTTCAC-3';

[0049] SEQ ID NO.6: 5'-GCCGCCATAAACAAACCTGT-3'.

[0050] The primer pair IDE25 for amplifying the InDel molecular marker of chromosome 5 of stem tumor sesbania, the primer pair IDE25 comprises an upstream primer IDE25-F and a downstream primer IDE25-R; the nucleotide sequence of the upstream primer IDE25-F is shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer IDE25-R is shown in SEQ ID NO.8, and the specific primer sequences are as follows:

[0051] SEQ ID NO.7: 5'-CTCAAACCACACAGAAGGGC-3';

[0052] SEQ ID NO.8: 5'-GCAGTGGGGAGTTAGCAGAT-3'.

[0053] The primer pair IDE30 for amplifying the InDel molecular marker of chromosome 6 of stem tumor sesbania, the primer pair IDE30 includes an upstream primer IDE30-F and a downstream primer IDE30-R; the nucleotide sequence of the upstream primer IDE30-F is shown in SEQ ID NO. 9, the nucleotide sequence of the downstream primer IDE30-R is shown in SEQ ID NO. 10, and the specific primer sequences are as follows:

[0054] SEQ ID NO.9: 5'-AGCCAAAAGAAAAGAACATTCAACA-3';

[0055] SEQ ID NO.10: 5'-ACATGGAGCCGTCAAGAGAT-3'.

[0056] Example 2 Differentiation of Nodular Sesbania and 16 Sesbania Varieties and Germplasms Based on Five Pairs of InDel Markers

[0057] The 5 pairs of primers for InDel molecular markers provided in Example 1 were used to perform genotyping on the 17 sesbania varieties and germplasms in Table 1. Genomic DNA of the 17 sesbania varieties and germplasms was extracted, and the identification results were analyzed using PCR and agarose gel electrophoresis.

[0058] The genotype identification results of the above 17 sesbania varieties and germplasms are as follows Figure 2 As shown, using primer pair IDE3, a band appeared at the 759 bp position of the electrophoresis graph for Sesbania tuberculosa, while the electrophoresis showed 1204 bp for non-sebasica tuberculosa; using primer pair IDE15, a band appeared at the 994 bp position of the electrophoresis graph for Sesbania tuberculosa, while the electrophoresis showed 691 bp for non-sebasica tuberculosa; using primer pair IDE20, a band appeared at the 1000 bp position of the electrophoresis graph for Sesbania tuberculosa, while the electrophoresis showed 593 bp for non-sebasica tuberculosa; using primer pair IDE25, a band appeared at the 1390 bp position of the electrophoresis graph for Sesbania tuberculosa, while the electrophoresis showed 1000 bp for non-sebasica tuberculosa; using primer pair IDE30, a band appeared at the 1248 bp position of the electrophoresis graph for Sesbania tuberculosa, while the electrophoresis showed 818 bp for non-sebasica tuberculosa.

[0059] The results show that the verification results of the five pairs of primers are consistent, No. 1 is the tuber sesbania, No. 2 to 17 are the core germplasms and varieties of sesbania, and the InDel molecular markers developed in this application have a 100% match rate between the genotypes and the actual genetic backgrounds of these sesbania materials, and the genetic backgrounds of tuber sesbania and other sesbania varieties and germplasms can be completely distinguished according to the band size of the amplified fragment. Therefore, the present invention can be applied to the identification of tuber sesbania varieties and germplasms, purity detection and molecular ID card construction, thereby improving the accuracy and efficiency of germplasm testing, and laying a certain foundation for the subsequent development of sesbania breeding.

[0060] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. 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. Application of an InDel molecular marker primer set in breeding or auxiliary breeding of stem tumor Sesbania sesbania, characterized in that: The InDel molecular marker primer set includes the following primer pairs: The primer pair IDE3 includes an upstream primer IDE3-F and a downstream primer IDE3-R, the sequence of IDE3-F is shown in SEQ ID NO. 1, and the sequence of IDE3-R is shown in SEQ ID NO. 2; The primer pair IDE15 includes an upstream primer IDE15-F and a downstream primer IDE15-R, the sequence of IDE15-F is shown in SEQ ID NO. 3, and the sequence of IDE15-R is shown in SEQ ID NO. 4; The primer pair IDE20 includes IDE20-F and IDE20-R, the sequence of IDE20-F is shown in SEQ ID NO.5, and the sequence of IDE20-R is shown in SEQ ID NO.6; The primer pair IDE25 includes an upstream primer IDE25-F and a downstream primer IDE25-R, the sequence of IDE25-F is shown in SEQ ID NO. 7, and the sequence of IDE25-R is shown in SEQ ID NO. 8; The primer pair IDE30 includes an upstream primer IDE30-F and a downstream primer IDE30-R, the sequence of IDE30-F is shown in SEQ ID NO.9, and the sequence of IDE30-R is shown in SEQ ID NO.

10.

2. A method for identifying or assisting in identifying whether the genetic origin of Sesbania sesbania is stem tumor Sesbania sesbania, characterized in that: The following steps are included: a. Extracting genomic DNA from Sesbania sesbania materials; b. Using the DNA obtained in step a as a template, PCR amplification is performed using the primer pair IDE3, primer pair IDE15, primer pair IDE20, primer pair IDE25, and primer pair IDE30 described in claim 1, and the amplified fragments are analyzed by 1.5% agarose gel electrophoresis; c. When primer pair IDE3 was used, a band appeared at the 759 bp position of the electrophoresis graph, which was Sesbania tuberulata, while the electrophoresis showed 1204 bp for non-Sesbania tuberulata; when primer pair IDE15 was used, a band appeared at the 994 bp position of the electrophoresis graph, which was Sesbania tuberulata, while the electrophoresis showed 691 bp for non-Sesbania tuberulata; when primer pair IDE20 was used, a band appeared at the 1000 bp position of the electrophoresis graph, which was Sesbania tuberulata, while the electrophoresis showed 593 bp for non-Sesbania tuberulata; when primer pair IDE25 was used, a band appeared at the 1390 bp position of the electrophoresis graph, which was Sesbania tuberulata, while the electrophoresis showed 1000 bp for non-Sesbania tuberulata; when primer pair IDE30 was used, a band appeared at the 1248 bp position of the electrophoresis graph, which was Sesbania tuberulata, while the electrophoresis showed 818 bp for non-Sesbania tuberulata.

3. The method for identifying or assisting in identifying whether the genetic origin of Sesbania sesbania is stem tumor Sesbania according to claim 2, characterized in that: In the step b, the PCR amplification system is: 1.0 μL genomic DNA, 15.0 μL 2×PCR Mix, 1 μL each of upstream and downstream primers, and ddH2O is added to 30 μL; the PCR amplification program is: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 40 s, 35 cycles; 72°C extension for 5 min.

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