Molecular marker of sorghum plant height related gene SbPH11 and application thereof
By developing a molecular marker for the sorghum plant height-related gene SbPH11 and utilizing the polymorphism detection of SNP1 and SNP2, the problem of plant height localization in sorghum breeding was solved, enabling efficient sorghum plant height identification and breeding, and improving breeding efficiency.
Patent Information
- Application Number
- CN202211390692.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-07
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2042-11-07
AI Technical Summary
The lack of effective molecular markers in existing technologies for the location and detection of sorghum plant height leads to low sorghum breeding efficiency and makes it difficult to breed new high-yielding sorghum varieties.
A molecular marker for the gene SbPH11, which is associated with sorghum plant height, was developed. By detecting the polymorphism or genotype of SNP1 and SNP2, KASP technology was used to identify sorghum plant height and assist in breeding, providing a method and product for detecting and identifying sorghum plant height.
It has enabled efficient and accurate identification and breeding of sorghum plant height, improved the efficiency of sorghum breeding, and enabled early prediction and screening of tall sorghum varieties, thus promoting the development of the sorghum industry.
Smart Images
Figure BDA0003929278610000121 
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Figure BDA0003929278610000141
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of molecular biology, and particularly relates to a molecular marker of a sorghum height-related gene SbPH11 and application thereof. BACKGROUND
[0002] Sorghum bicolor, also known as sorghum, is an annual herbaceous plant of the Poaceae family. Sorghum is one of the world's five major crops, and belongs to C4 crops, with high photosynthetic efficiency and high biological yield. Sorghum has a wide range of uses, such as food, feed, brewing, bioenergy, and chemical materials. Sorghum is the world's most important food and feed crop due to its characteristics of drought and flood tolerance, salt and alkali tolerance, and easy cultivation.
[0003] Soil salinization is an important problem affecting agricultural production and ecological environment. About 20% of the available arable land has been or is being salinized to varying degrees, and the loss of land available for crop cultivation has become a serious problem for agricultural sustainability. Planting salt-tolerant crops is one of the most effective and economical methods for utilizing and improving saline-alkali soils. Therefore, studying the salt-tolerance of sorghum and breeding new sweet sorghum varieties with high yield and salt-tolerance has broad prospects for utilizing and improving soil resources in saline-alkali areas.
[0004] For a long time, the breeding of new sorghum varieties in China has mainly focused on high-stalk and large-eared types, with most varieties having a plant height of 2.5-3 m. Plant height is a basic index for morphological investigation of crops and an important factor affecting the biological yield of sorghum. Generally, the higher the plant height of sorghum, the higher its biological yield. The yield of most high-stalk varieties is higher than that of short-stalk varieties. Therefore, breeding sorghum parent lines and hybrids containing high-stalk haplotypes is crucial for promoting the development of the sorghum industry.
[0005] Molecular marker-assisted selection breeding can select target traits at the DNA level, resulting in stable results and allowing selection at the seedling stage to reduce the cost of phenotypic evaluation and improve the efficiency of sorghum breeding. Single nucleotide polymorphism (SNP) markers have characteristics such as genetic stability, large number, wide distribution, and ease of detection, making them suitable for large-scale detection and analysis. After finding key marker sites through genetic analysis, it is necessary to convert them into easy-to-use molecular markers. KASP (Kompetitive allele-specific PCR), a competitive allele-specific PCR technology, has been widely used in high-throughput SNP genotyping due to its high stability, accuracy, and low cost.
[0006] Therefore, it is of great significance to study the genes regulating plant height, obtain KASP molecular markers closely linked to plant height genes, locate and detect major gene loci of plant height in sorghum, effectively regulate the plant height type of sorghum, and breed new sorghum varieties with expected plant height type to improve the yield of sorghum. At present, there are few reports on the development of molecular markers closely linked to target QTL and the application of patent in sorghum, and there is no patent report related to plant height. SUMMARY
[0007] The technical problem to be solved by the present application is to provide a molecular marker of a sorghum plant height related gene SbPH11.
[0008] To solve the above technical problems, the present application provides the following applications:
[0009] The application is P1 or P2.
[0010] The P1 is the application of a substance for detecting the polymorphism or genotype of SNP1 and SNP2 in identifying or assisting in identifying the plant height of sorghum, or preparing products for identifying or assisting in identifying the plant height of sorghum, or sorghum breeding or preparing sorghum breeding products, wherein the SNP1 is a SNP in the genome of sorghum, which is the 425th nucleotide of SEQ ID No. 1 in the sequence listing, and is A or C; and the SNP2 is a SNP in the genome of sorghum, which is the 401st nucleotide of SEQ ID No. 1 in the sequence listing, and is C or T.
[0011] The P2 is the application of a substance for detecting a haplotype in identifying or assisting in identifying the plant height of sorghum, or preparing products for identifying or assisting in identifying the plant height of sorghum, or sorghum breeding or preparing sorghum breeding products, wherein the haplotype is a combination of the polymorphism of SNP1 and SNP2 on a chromosome of sorghum.
[0012] The nucleotide sequence of Sequence 1 is as follows:
[0013] CTCCAGTGTGCACCGGCCGCGGGGGTTGCAGGGTGAGGGTCCGTTGGAAGGTGGAACATAGAAGAAAGGGATGGGAAAGGGAAAAAAGAGACTGACGAGTGGGCCCCACGTGGTAAGGTGCCTCATCCAACTTTCTAGTGTCCCTCAACCAAACACAGAGGAGAGATGCTCCCATCCCTCAAAACTGGGATGGGACCGTCCCATCCCACCATGTCCCAAACCAAACACAAACTTAACTAGTTTGTATCGAGTCACATTAGGTTTGTTAAGCGCCTAAATATAAGTGTAACCATCAAAGAAATAAACTCCCGTTCATTGGCGTGAGTTGTCTTAGACCGTGGTGTGAAATCCTTCCTCATTCGATGACAACTGCCTATAGTCGTGCCTACCTCGCTACCCTCTTTGTTCTCTTTCTGACACACATATACTACTCTACTCTCCCTAGCGTTCTGTTTGTCGTGCATGCCATAACAAGTTGGCATCATAGATCGACCAATGGGCTTTGACTATGTTGGAATCAACATTACGAAGCTAAACCATATCATCGCCCAGCTCGTGACGATG.
[0014] The substance can be a product. The detection substance can include reagents, kits and instruments for detecting the above-mentioned single nucleotide polymorphism. Specifically, primers and other reagents and instruments required for in vitro amplification of nucleic acids for detecting the above-mentioned single nucleotide polymorphism.
[0015] The SEQ ID No. 1 is the genomic sequence of the gene of SbPH11, which includes an intron sequence. In actual detection, the polymorphism of SNP1 and SNP2 can be detected and analyzed by detecting the nucleotide polymorphism of the mRNA transcribed from the SbPH11 gene, the cDNA reverse transcribed from the SbPH11 mRNA, or the amino acid polymorphism of the SbPH11 protein caused by the polymorphism of SNP1 and SNP2.
[0016] In this application, the genotype of SNP1 can be genotype AA or genotype CC, genotype AA is homozygous type of SNP1 being A; and genotype CC is homozygous type of SNP1 being C.
[0017] The genotype of the SNP2 can be genotype CC or genotype TT, the genotype CC is homozygous type of C of the SNP2, and the genotype TT is homozygous type of T of the SNP2.
[0018] The genotype CA is a heterozygous type of C and A of the SNP1, and the genotype TA is a heterozygous type of T and A of the SNP2.
[0019] The SNP1 is located at 53908336 of chromosome 4 of the sorghum inbred line BTx623, with the sorghum genome (BTx623 (v3.1) as a reference genome.
[0020] The SNP2 is located at 53908312 of chromosome 4 of the sorghum inbred line BTx623, with the sorghum genome (BTx623 (v3.1) as a reference genome.
[0021] To solve the above technical problems, the application further provides a product;
[0022] The product provided in the application contains the product containing the substance for detecting the polymorphism or genotype of the two SNPs of the SNP1 and the SNP2 of the sorghum genome or the product containing the substance for detecting the haplotype in claim 1, and can be any one of the following G1) to G3):
[0023] G1) a product for detecting a single nucleotide polymorphism or genotype related to the plant height of sorghum;
[0024] G2) a product for identifying or assisting in identifying the plant height of sorghum;
[0025] G3) a product for sorghum breeding.
[0026] To solve the above technical problems, the application further provides a method for identifying or assisting in identifying the plant height of sorghum;
[0027] The method for identifying or assisting in identifying the plant height of sorghum provided in the application is method A or method B:
[0028] The method A is a method for identifying or assisting in identifying the plant height of sorghum, comprising detecting the genotype of the two SNPs of the SNP1 and the SNP2 in claim 1 in the to-be-tested sorghum, and identifying or assisting in identifying the plant height of sorghum according to the genotype of the to-be-tested sorghum:
[0029] The height of the sorghum with the genotype CCCC is higher or is a candidate for being higher than the height of the sorghum with the genotype CCTT and AACC, the height of the sorghum with the genotype CCTT is higher or is a candidate for being higher than the height of the sorghum with the genotype AACC; the CCCC is a two-SNP combined genotype of CC of the SNP1 and CC of the SNP2, the CCTT is a two-SNP combined genotype of CC of the SNP1 and TT of the SNP2, and the AACC is a two-SNP combined genotype of AA of the SNP1 and CC of the SNP2; the CC genotype of the SNP1 is a homozygous genotype in which the 425th nucleotide of SEQ ID No. 1 in the sequence listing is C; the AA genotype of the SNP1 is a homozygous genotype in which the 425th nucleotide of SEQ ID No. 1 in the sequence listing is A; the CC genotype of the SNP2 is a homozygous genotype in which the 401st nucleotide of SEQ ID No. 1 in the sequence listing is C; and the TT genotype of the SNP2 is a homozygous genotype in which the 401st nucleotide of SEQ ID No. 1 in the sequence listing is T;
[0030] The method B is a method for identifying or assisting in identifying the height of sorghum, comprising detecting the haplotype in claim 1 in the sorghum to be tested, and identifying or assisting in identifying the height of sorghum according to the haplotype of the sorghum to be tested:
[0031] The height of the sorghum with the homozygous genotype corresponding to the haplotype SbPH11-Hap2 is higher or is a candidate for being higher than the height of the sorghum with the homozygous genotype corresponding to the haplotype SbPH11-Hap1 or SbPH11-Hap3, and the height of the sorghum with the homozygous genotype corresponding to the haplotype SbPH11-Hap1 is higher or is a candidate for being higher than the height of the sorghum with the homozygous genotype corresponding to the haplotype SbPH11-Hap3; the haplotype SbPH11-Hap2 is a haplotype in which the SNP1 is C and the SNP2 is C, the haplotype SbPH11-Hap1 is a haplotype in which the SNP1 is C and the SNP2 is T, and the haplotype SbPH11-Hap3 is a haplotype in which the SNP1 is A and the SNP2 is C.
[0032] As an embodiment, the method for identifying or assisting in identifying the height of sorghum can comprise the following steps:
[0033] (1) using the genomic DNA of the sorghum to be tested as a template, and performing KASP using primer set F1 and primer set F2;
[0034] The primer set F1 can comprise primer F1-A, primer F1-B, and primer F1-C; and the combined primer set F2 can comprise primer F2-A, primer F2-B, and primer F2-C;
[0035] The primer F1-A can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 2 in the sequence listing;
[0036] The primer F1-B can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 3 in the sequence listing;
[0037] The primer F1-C can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 4 in the sequence listing;
[0038] The primer F2-A can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 5 in the sequence listing;
[0039] The primer F2-A can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 6 in the sequence listing;
[0040] The primer F2-A can be a single-stranded DNA molecule with a nucleotide sequence of SEQ ID NO: 7 in the sequence listing.
[0041] (2) After step (1) is completed, fluorescence detection is performed to determine the genotypes of the SNP1 and SNP2 sites of the to-be-tested sorghum;
[0042] (3) According to the genotype results, the height of the to-be-tested sorghum is identified or assisted to identify: the height of the to-be-tested sorghum (such as a sorghum inbred line) with the genotype CCCC of the two SNP sites of SNP1 and SNP2 is higher than or is expected to be higher than the height of the to-be-tested sorghum (such as a sorghum inbred line) with the genotype CCTT or AACC; the height of the to-be-tested sorghum (such as a sorghum inbred line) with the genotype CCTT is higher than or is expected to be higher than the height of the to-be-tested sorghum (such as a sorghum inbred line) with the genotype AACC.
[0043] In the above-mentioned application, product, or method, the sorghum is a sorghum pure line or inbred line.
[0044] The above-mentioned method is applied in sorghum breeding.
[0045] In the above-mentioned application, product, method, or the above-mentioned application, product, or method, the purpose of the breeding includes breeding or selecting a sorghum with low height or high height.
[0046] In the above-mentioned application, product, method, or the above-mentioned application, product, or method, the substance for detecting the polymorphism or genotype of the two SNPs of SNP1 and SNP2, or the substance for detecting the haplotype, is as follows: D1), D2), D3), or D4):
[0047] D1) contains in vitro nucleic acid amplification primers for specifically amplifying the SNP1 and SNP2 sites;
[0048] D2) contains in vitro nucleic acid amplification reagents containing the in vitro nucleic acid amplification primers of D1);
[0049] D3) a kit containing the in vitro nucleic acid amplification primer of D1) or the in vitro nucleic acid amplification reagent of D2);
[0050] D4) a detection instrument containing the in vitro nucleic acid amplification primer of D1), the in vitro nucleic acid amplification reagent of D2), or the kit of D3).
[0051] In the present application, the in vitro nucleic acid amplification technique can be polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), and nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification technique (HDA), or Qβ replication technique.
[0052] In the present application, the in vitro nucleic acid amplification technique can be polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), and nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification technique (HDA), or Qβ replication technique.
[0053] The specific amplification in D1) can detect the nucleotide sequence of the SNP1 and SNP2 polymorphic sites by the presence or absence of the amplification product or by the presence or absence of the amplification product in combination with a probe or other auxiliary reagent.
[0054] In the above applications, methods, and products, the in vitro nucleic acid amplification primer can or can not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radioactive labels such as 32P; binding moieties such as biotin; hapten such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The label can include or be combined with a nucleic acid or protein sequence, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label.
[0055] In the above applications, products, or methods, the in vitro nucleic acid amplification primer includes primer set F1-1, primer set F1-2, primer set F2-1, and / or primer set F2-2:
[0056] The primer set F1-1 is F1-1-1 or F1-1-2:
[0057] F1-1-1, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is from 22nd to 44th of SEQ ID No. 2 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence table;
[0058] F1-1-2, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 2 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence table;
[0059] The primer set F1-2 is F1-2-1 or F1-2-2:
[0060] F1-2-1, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is from 22nd to 46th of SEQ ID No. 3 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence table;
[0061] F1-2-2, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 3 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 4 in the sequence table;
[0062] The primer set F2-1 is F2-1-1 or F2-1-2:
[0063] F1-2-1, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is from 22nd to 40th of SEQ ID No. 5 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence table;
[0064] F1-2-2, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 5 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence table;
[0065] The primer set F2-2 is F2-2-1 or F2-2-2:
[0066] F1-2-1, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is from 22nd to 41st of SEQ ID No. 6 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence table;
[0067] F1-2-2, a primer combination consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 6 in the sequence table and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence table.
[0068] In the present application, the primer F1-1 group is an in vitro nucleic acid amplification primer for detecting SNP1 polymorphism site A, which can be F1-1-1 or F1-1-2: F1-1-1 is a primer composition consisting of a single-stranded DNA with nucleotide sequence of 22-44 of SEQ ID No. 2 in the sequence listing and a single-stranded DNA with nucleotide sequence of SEQ ID No. 4 in the sequence listing (named as universal primer F1-C);
[0069] F1-1-2, a primer composition consisting of a single-stranded DNA with nucleotide sequence of SEQ ID No. 2 in the sequence listing (1-21 is a FAM tag sequence, named as F1-A) and a single-stranded DNA with nucleotide sequence of SEQ ID No. 4 in the sequence listing;
[0070] The primer F1-2 group is an in vitro nucleic acid amplification primer for detecting SNP1 polymorphism site C, which can be F1-2-1 or F1-2-2: F1-2-1 is a primer composition consisting of a single-stranded DNA with nucleotide sequence of 22-46 of SEQ ID No. 3 in the sequence listing and a single-stranded DNA with nucleotide sequence of SEQ ID No. 4 in the sequence listing (named as universal primer F1-C);
[0071] F1-1-2, a primer composition consisting of a single-stranded DNA with nucleotide sequence of SEQ ID No. 3 in the sequence listing (1-21 is a FAM tag sequence, named as F1-B) and a single-stranded DNA with nucleotide sequence of SEQ ID No. 4 in the sequence listing;
[0072] The primer F2-1 group is an in vitro nucleic acid amplification primer for detecting SNP2 polymorphism site T, which can be F2-1-1 or F2-1-2: F2-1-1 is a primer composition consisting of a single-stranded DNA with nucleotide sequence of 22-40 of SEQ ID No. 5 in the sequence listing and a single-stranded DNA with nucleotide sequence of SEQ ID No. 7 in the sequence listing (named as universal primer F2-C);
[0073] F2-1-2, a primer composition consisting of a single-stranded DNA with nucleotide sequence of SEQ ID No. 5 in the sequence listing (1-21 is a FAM tag sequence, named as F2-A) and a single-stranded DNA with nucleotide sequence of SEQ ID No. 7 in the sequence listing;
[0074] The primer F2-2 group is an in vitro nucleic acid amplification primer for detecting the SNP2 polymorphic site C, which can be F2-2-1 or F2-2-2: F2-2-1 is a primer composition consisting of a single-stranded DNA with the nucleotide sequence of SEQ ID No. 6 in the sequence listing (positions 22-41) and a single-stranded DNA with the nucleotide sequence of SEQ ID No. 7 in the sequence listing (named universal primer F2-C);
[0075] F2-1-2, a primer composition consisting of a single-stranded DNA with the nucleotide sequence of SEQ ID No. 6 in the sequence listing (positions 1-21 are FAM label sequences, named F2-B) and a single-stranded DNA with the nucleotide sequence of SEQ ID No. 7 in the sequence listing.
[0076] The SNP1 can be at position 53908336 on chromosome 4 of sorghum inbred line BTx623, and the SNP2 can be at position 53908312 on chromosome 4 of sorghum inbred line BTx623.
[0077] In the above applications, products and methods, the sorghum can be a pure line or a sorghum inbred line.
[0078] In the above applications and methods, the product can be a reagent or a kit or a system, which can include a combination of reagents or kits, instruments and analysis software, such as a product composed of PCR primers, PARMS master mix reagents, an enzyme marker and online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), a combination product composed of PCR primers, PARMS master mix reagents, online software SNP decoder and a fluorescence quantitative PCR instrument. The product can include the above-mentioned substances for detecting the polymorphism or genotype of SNP1 and SNP2 sites in the sorghum genome.
[0079] In order to solve the above problems, the present application provides a plant breeding method.
[0080] The plant breeding method comprises replacing the 425th nucleotide and the 401st nucleotide in the sequence 1 region in the genome of the plant of interest with the 425th nucleotide being C and the 401st nucleotide being T, the 425th nucleotide being C and the 401st nucleotide being C, or the 425th nucleotide being A and the 401st nucleotide being C, to obtain a plant with high or low height.
[0081] The plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are C has a higher plant height than or is a candidate for having a higher plant height than the plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are C and T, or the plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are A and C.
[0082] In the present application, the plant is a homozygous plant.
[0083] The method for breeding the plant can be to replace the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 on all chromosomes to obtain a homozygous plant. Or, the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 on one chromosome are replaced first, and then a homozygous plant is obtained through sexual reproduction and homozygous screening.
[0084] To solve the above problems, the present application also provides the following applications.
[0085] The application of the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome being C, C, or C corresponding to different plant heights in any of the following:
[0086] (1) Regulating the plant height of sorghum;
[0087] (2) Preparing a product for regulating the plant height of sorghum;
[0088] (3) Breeding sorghum;
[0089] (4) Preparing a product for breeding the plant height of sorghum;
[0090] (5) A product for detecting single nucleotide polymorphism or genotype related to the plant height of sorghum;
[0091] (6) A product for identifying or assisting in identifying the plant height of sorghum.
[0092] In the present application, the plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are C has a higher plant height than or is a candidate for having a higher plant height than the plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are C and T, or the plant in which the nucleotide at position 425 of SEQ ID NO: 1 and the nucleotide at position 401 of SEQ ID NO: 1 in the plant genome are A and C.
[0093] The plant is a homozygous plant.
[0094] The sorghum can be at least one of:
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KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL, KORAIL,Kaffir corn 374, Kaffir corn 382, Kaffir corn 384, Kaffir corn 395, Kaffir corn 48, Kaffir corn 4, Kaffir corn 61, Kaffir corn 62, Kaffir corn 67, Kaffir corn 70, Kaffir corn 86, Kaffir corn GW102, Kaffir corn GW59, Kaffir corn J136, Kaffir corn J146, Kaffir corn J14, Kaffir corn J150, Kaffir corn J158, Kaffir corn J20, Kaffir corn J22, Kaffir corn J25, Kaffir corn J26, Kaffir corn J27, Kaffir corn J28, Kaffir corn J2, Kaffir corn J30, Kaffir corn J40, Kaffir corn J42, Kaffir corn J44, Kaffir corn J49, Kaffir corn J50, Kaffir corn J53, Kaffir corn J54, Kaffir corn J55, Kaffir corn J57, Kaffir corn J5, Kaffir corn J66, Kaffir corn J67, Kaffir corn J69, Kaffir corn J77, Kaffir corn J81, Kaffir corn J85, Kaffir corn J93, Kaffir corn J95, Kaffir corn J97, Kaffir corn J99, Kaffir corn J9, Kaffir corn SL129, Kaffir corn SL136, Kaffir corn SL37, Kaffir corn SL43, Kaffir corn SL47, Kaffir corn SL62, Kaffir corn Tu14, Kaffir corn Tu17, Kaffir corn WSC10, Kaffir corn WSC16, Kaffir corn WSC19, Kaffir corn WSC24, Kaffir corn WSC25, Kaffir corn WSC2, Kaffir corn WSC32, Kaffir corn WSC38, Kaffir corn WSC56, Kaffir corn WSC5, Kaffir corn WSC66, Kaffir corn WSC6, Kaffir corn 128, Kaffir corn 131, Kaffir corn 136, Kaffir corn 137, Kaffir corn 139, Kaffir corn 157, Kaffir corn 175, Kaffir corn 176, Kaffir corn 177, Kaffir corn 2031, Kaffir corn 2050, Kaffir corn 2084, Kaffir corn 2085, Kaffir corn 264, Kaffir corn 268, Kaffir corn 269, Kaffir corn 372, Kaffir corn 383, Kaffir corn 386, Kaffir corn 400, Kaffir corn 8, Kaffir corn J101, Kaffir corn SL105, Kaffir corn SL12, Kaffir corn WSC105, Kaffir corn WSC18, Kaffir corn WSC68, Kaffir corn WSC8.
[0096] In the above-mentioned uses, methods and products, the substance can be a reagent and / or a kit and / or an instrument required for determining the polymorphism or genotype of the SNP by at least one of the following methods: in vitro nucleic acid amplification, DNA sequencing, restriction enzyme digestion fragment length polymorphism, single strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. The SNP chip includes chip based on nucleic acid hybridization reaction, chip based on single base extension reaction, chip based on allele specific primer extension reaction, chip based on "one-step" reaction, chip based on primer ligation reaction, chip based on restriction enzyme reaction, chip based on protein DNA binding reaction, and chip based on fluorescence molecule DNA binding reaction.
[0097] In the above-mentioned uses and methods, the Kaffir corn inbred can be selected as the parent for breeding.
[0098] The Kaffir corn plant height can be the plant height of Kaffir corn at the mature stage.
[0099] The breeding aims include breeding or selecting low or tall statured sorghum.
[0100] In the above applications and methods, the PCR primers can or can not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. Labels include, but are not limited to, dyes; radioactive labels such as32P; binding moieties such as biotin; hapten such as digoxigenin (DIG); luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The label can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The label can include or be combined with nucleic acid or protein sequences, as long as the sequence comprising the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., the sequence is directly read).
[0101] In the above applications and methods, the product can be a reagent or a kit or a system, which can include a combination of reagents or kits, instruments and analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, a microplate reader and online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), a combination product consisting of PCR primers, PARMS master mix reagents, online software SNP decoder and a fluorescent quantitative PCR instrument. The product can include the above-mentioned substances for detecting the polymorphism or genotype of SNP1 and / or SNP2 sites in the sorghum genome.
[0102] In the embodiments of the present application, through genetic variation analysis of the SbPH11 gene in a sorghum inbred line association population, two SNPs, SNP1 and SNP2, are found in the SbPH11 gene related to plant height in the sorghum genome, i.e., at positions 425 and 401 of the sequence table SEQ ID No. 1. The two SNPs exist in three haplotypes in combination: haplotype SbPH11-Hap1 (CT), haplotype SbPH11-Hap2 (CC), and haplotype SbPH11-Hap3 (AC). Experiments prove that the plant height of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap2 (CC) is significantly higher than that of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap1 (CT) or that of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap3 (AC), and the plant height of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap1 (CT) is significantly higher than that of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap3 (AC). The plant height of 77.69% of sorghum corresponding to the homozygous genotype of haplotype SbPH11-Hap2 (CC) is higher than 250 cm. The molecular marker of haplotype SbPH11-Hap2 (CC) can be used for early prediction and screening of sorghum plant height, and can also be used for molecular marker assisted selection breeding of sorghum and breeding of high-stalk sorghum varieties. DETAILED DESCRIPTION
[0103] The present application will be further described in conjunction with the specific embodiments. The embodiments provided below serve only to illustrate the present application and are not intended to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.
[0104] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.
[0105] In the following examples, the data are processed using GraphPad Prism v8.0 statistical software, and the experimental results are expressed as mean ± standard deviation. One-way ANOVA test is used, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, and P<0.001 (***) indicates extremely significant difference.
[0106] The 254 sorghum inbred line association population in the following examples: provided by the Key Laboratory of Northern Resources and Plants, Institute of Botany, Chinese Academy of Sciences, Jing Haichun Laboratory, and recorded in the following literature and its annex materials: Xiaoyuan Wu, Yuanming Liu, Hong Luo, Li Shang, Chuanyuan Leng, Zhiquan Liu, Zhigang Li, Xiaochun Lu, Hongwei Cai, Huaiqin g Hao, Hai-Chun Jing, (2022, 5), Genomic footprints of sorghum domestication and breeding selection for multiple end uses, Molecular Plant, Volume 15, Issue 3, 7 March 2022, Pages 537-551. The variety details are shown in Table 1. The public can obtain this biological material from the applicant, which is only used for repeating the experiments of the invention and cannot be used for other purposes.
[0107] Example 1, haplotype combination and related haplotype molecular marker associated with sorghum plant height
[0108] I. Plant height statistics of test materials
[0109] 1. Planting of test materials
[0110] In 2021, 254 sorghum inbred line association population germplasm resources were planted in the farmland soil of light saline-alkali land in Dongying City, Shandong Province, China, using a randomized complete block design, with a test plot length of 3 m and a width of 2 m, planting 5 rows, 10 plants per row, with a plant spacing of 0.3 m and a row spacing of 0.5 m, and normal irrigation.
[0111] 2. Plant height statistics of test materials
[0112] After the 254 sorghum (as shown in Table 1) inbred line association population was fully matured, 3 plants were selected from each material, and the plant height of each main stem was counted, and the average value of three repetitions was taken as the final result of the plant height of the sample, as shown in Table 1.
[0113] II. Discovery of haplotype combination and haplotype molecular marker related to gene SbPH11
[0114] 1. Whole genome sequencing of 254 sorghum inbred line association population
[0115] The whole genome sequencing data of the 254 sorghum inbred line association population was provided by the Key Laboratory of Northern Resources and Plants, Institute of Botany, Chinese Academy of Sciences, Jing Haichun Laboratory.
[0116] 2. A haplotype combination associated with the gene SbPH11 and the discovery of a haplotype molecular marker associated therewith
[0117] According to the plant height of the sorghum inbred lines and the sequencing results of the genomes thereof, a haplotype combination associated with the gene SbPH11 and the plant height of the sorghum is screened. The haplotype combination comprises two SNP sites, namely SNP1 and SNP2. The SNP1 corresponds to the 53908336th position on the 4th chromosome of the sorghum inbred line BTx623 (BTx623(v3.1) sorghum genome sequence information), the nucleotide of which is A or C (A / C binary polymorphism), which corresponds to the 425th position of SEQ ID No. 1 in the sequence listing, and the 21st amino acid thereof is tyrosine or aspartic acid (i.e., the SAP1 site, tyrosine / aspartic acid binary polymorphism, which corresponds to the 425th nucleotide of SEQ ID No. 1 being A or C); and the SNP2 corresponds to the 53908312th position on the 4th chromosome of the sorghum inbred line BTx623, the nucleotide of which is C or T, which corresponds to the 401st position of SEQ ID No. 1 in the sequence listing, and the 29th amino acid thereof is glutamic acid mutation or lysine (i.e., the SAP2 site, glutamic acid mutation / lysine binary polymorphism, which corresponds to the 401st nucleotide of SEQ ID No. 1 being C or T). In the sequence listing, m of SEQ ID No. 1 represents a or c, and y represents t or c.
[0118] The genotype detection results of each sorghum variety are shown in Table 1. The results show that the SNP1 site has two genotypes (referred to as SNP1 genotype) AA or CC, the genotype AA is the homozygous type of SNP1 being A, and the genotype CC is the homozygous type of SNP1 being C; the SNP2 site has two genotypes (referred to as SNP2 genotype) CC or TT, the genotype CC is the homozygous type of SNP2 being C, and the genotype TT is the homozygous type of SNP2 being T. In the test population, the two SNP combinations, i.e., the haplotype combination, exist in three haplotypes: haplotype SbPH11-Hap1(CT) (referred to as SbPH11-Hap1), haplotype SbPH11-Hap2(CC) (referred to as SbPH11-Hap2), and haplotype SbPH11-Hap3(AC) (referred to as SbPH11-Hap3). The haplotype SbPH11-Hap1(CT) is the combination of SNP1 being C and SNP2 being T, the haplotype SbPH11-Hap2(CC) is the combination of SNP1 being C and SNP2 being C, and the haplotype SbPH11-Hap3(AC) is the combination of SNP1 being A and SNP2 being C.
[0119] The plant height of sorghum with the homozygous genotype SbPH11-Hap2(CC) was significantly higher than that of sorghum with the homozygous genotype SbPH11-Hap1(CT) or SbPH11-Hap3(AC). The plant height of sorghum with the homozygous genotype SbPH11-Hap1(CT) was significantly higher than that of sorghum with the homozygous genotype SbPH11-Hap3(AC). The genotype of sorghum corresponding to the haplotype SbPH11-Hap2(CC) is CCCC, which is a combination of two SNPs: SNP1 genotype CC and SNP2 genotype CC. The genotype of sorghum corresponding to the haplotype SbPH11-Hap1(CT) is CCTT, which is a combination of two SNPs: SNP1 genotype CC and SNP2 genotype TT. The genotype of sorghum corresponding to the haplotype SbPH11-Hap3(AC) is AACC, which is a combination of two SNPs: SNP1 genotype AA and SNP2 genotype CC.
[0120] Therefore, the haplotype combination related to the SbPH11 gene obtained above was selected for identification or auxiliary identification of the plant height of different sorghum varieties; the SbPH11-Hap2(CC) haplotype in the haplotype combination was used as a molecular marker for identification or auxiliary identification of the plant height of different sorghum varieties, and the plant height of sorghum varieties containing the SbPH11-Hap2(CC) haplotype molecular marker may be higher than 250 cm.
[0121] III. Design and establishment of specific primers for SbPH11-related haplotype combinations and SbPH11-Hap2(CC) haplotype molecular markers.
[0122] 1. Design of genome-specific primers for haplotype combination-related SNP sites
[0123] The specific primer sequences for SNP1 (SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4 in the sequence listing) and the specific primer sequences for SNP2 (SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7 in the sequence listing) were designed and synthesized by Zhongyu Gold Label (Beijing) Biotechnology Co., Ltd.
[0124] The primer set F1 for identifying SNP1 site polymorphism is as follows:
[0125] Specific primer F1-A (SEQ ID No. 2):
[0126] 5'- GAGGTGCACCAAGAACCATGCTTTGTTCTCTTTCTGACACACATC-3'
[0127] Specific primer F1-B (SEQ ID No. 3):
[0128] 5'-TGCCTACCTCGCTACCCTT-3' GAGGTGCACCAAGAACCATGCT CTTTGTTCTCTTTCTGACACACATC-3'
[0129] Universal primer F1-C (SEQ ID No. 4):
[0130] 5'-GAACGCTAGGGAGAGTAGAGTAGTA-3'
[0131] The primer set F2 for identifying polymorphism of SNP2 site is as follows:
[0132] Specific primer F2-A (SEQ ID No. 5):
[0133] 5'-TGCCTACCTCGCTACCCTT-3' GAGGTGCACCAAGAACCATGCT TGCCTACCTCGCTACCCTC-3'
[0134] Specific primer F2-B (SEQ ID No. 6):
[0135] 5'-TGCCTACCTCGCTACCCTT-3' GAGGTGCACCAAGAACCATGCT GTGCCTACCTCGCTACCCTT-3'
[0136] Universal primer F2-C (SEQ ID No. 7):
[0137] 5'-GAACGCTAGGGAGAGTAGAGTAGTA-3'
[0138] The primer set F1 for identifying polymorphism of SNP1 site is designed according to the sequence SEQ ID No. 1 (sense strand), and the primer set F2 for identifying polymorphism of SNP2 site is designed according to the sequence SEQ ID No. 1 (sense strand).
[0139] The underlined sequence in the above primers F1-A and F2-A is FAM sequence; the underlined sequence in F1-B and F2-B is HEX sequence.
[0140] The fragment of SNP1 site with A in the single-stranded DNA molecule amplification sequence list SEQ ID No. 1 shown in the above sequences SEQ ID No. 2 and SEQ ID No. 4 can be read by the fluorescence signal of the fluorescent group combined with FAM sequence in the template by using an enzyme marker or a fluorescent quantitative PCR instrument;
[0141] The fragment of SNP1 site with C in the single-stranded DNA molecule amplification sequence list SEQ ID No. 1 shown by the above sequence SEQ ID No. 3 and SEQ ID No. 4 can be read to the fluorescence signal of the fluorescence group combined with the HEX sequence in the template by using the enzyme marker or the fluorescence quantitative PCR instrument.
[0142] The fragment of SNP2 site with C in the single-stranded DNA molecule amplification sequence list SEQ ID No. 1 shown by the above sequence SEQ ID No. 5 and SEQ ID No. 7 can be read to the fluorescence signal of the fluorescence group combined with the FAM sequence in the template by using the enzyme marker or the fluorescence quantitative PCR instrument.
[0143] The fragment of SNP2 site with T in the single-stranded DNA molecule amplification sequence list SEQ ID No. 1 shown by the above sequence SEQ ID No. 6 and SEQ ID No. 7 can be read to the fluorescence signal of the fluorescence group combined with the HEX sequence in the template by using the enzyme marker or the fluorescence quantitative PCR instrument.
[0144] 2. Establishment of detection method
[0145] 2.1 DNA extraction
[0146] The test sample variety (shown in Table 1) sorghum leaf genomic DNA was extracted, dissolved with ddH2O as a template for PCR amplification.
[0147] 2.2 PCR amplification and fluorescence signal detection
[0148] The template obtained in 2.1 was subjected to PCR amplification using primer set F1 and primer set F2 in step 1 respectively, and the polymorphism (nucleotide type) and genotype of the SNP site were detected; the PCR products of primer set F1 and primer set F2 were subjected to fluorescence data reading by using Douglas-Araya high-throughput flow line type fluorescence signal scanner, and the fluorescence signal was processed by using Douglas special software-Kraken.
[0149] Preparation of primer mixture: first dilute the three primers primer F1-A, primer F1-B and primer F1-C into 100 mmol·L-1 with ddH2O respectively, then mix them to prepare the primer mixture. -1, respectively, to obtain primer F1-A solution, primer F1-B solution, and primer F1-C solution. Take 60 μL of primer F1-A solution, 60 μL of primer F1-B solution, and 150 μL of primer F1-C solution, and add 230 μL of 10 mM Tris-HCL to obtain primer mixture F1. Use the above method to obtain primer F2-A solution, primer F2-B solution, and primer F2-C solution, take 60 μL of primer F2-A solution, 60 μL of primer F2-B solution, and 150 μL of primer F2-C solution, and add 230 μL of 10 mM Tris-HCL to obtain primer mixture F2.
[0150] 2 μL of the PCR fluorescent quantitative detection reaction system comprises: 50 ng of test sample genomic DNA, 0.02 μL of primer mixture F1 or F2, 0.6 μL of 1x KASP Mix (Low Rox) from LGC, and the rest is ddH2O. According to the operation manual of Douglas-Nexar and Soellex water bath system, edit the program and run, and save the data.
[0151] If the F1 primer group PCR product shows only the fluorescence signal of the fluorescent group combined with the FAM sequence, the genotype of the SNP1 site of the to-be-tested sorghum is AA (i.e., the SNP1 site in the sorghum genome is homozygous for A); if only the fluorescence signal of the fluorescent group combined with the HEX sequence is shown, the genotype of the SNP1 site of the to-be-tested sorghum is CC (i.e., the SNP1 site in the sorghum genome is homozygous for C); if both the fluorescence signal of the fluorescent group combined with the FAM sequence and the fluorescence signal of the fluorescent group combined with the HEX sequence are shown, the genotype of the SNP1 site of the to-be-tested sorghum is CA (i.e., the SNP1 site in the sorghum genome is heterozygous for C and A). If the F2 primer group PCR product shows only the fluorescence signal of the fluorescent group combined with the FAM sequence, the genotype of the SNP2 site of the to-be-tested sorghum is CC (i.e., the SNP2 site in the sorghum genome is homozygous for C); if only the fluorescence signal of the fluorescent group combined with the HEX sequence is shown, the genotype of the SNP2 site of the to-be-tested sorghum is TT (i.e., the SNP2 site in the sorghum genome is homozygous for T); if both the fluorescence signal of the fluorescent group combined with the FAM sequence and the fluorescence signal of the fluorescent group combined with the HEX sequence are shown, the genotype of the SNP2 site of the to-be-tested sorghum is TC (i.e., the SNP2 site in the sorghum genome is heterozygous for T and C).
[0152] Determination of haplotype and genotype of the haplotype combination related to gene SbPH11 to identify or assist in identifying the plant height of the tested sorghum variety: the plant height of sorghum (e.g., sorghum inbred line) with genotype CCC at SNP1 and SNP2 is higher than or candidate higher than that of sorghum (e.g., sorghum inbred line) with genotype CCTT or genotype AACC at SNP1 and SNP2. The plant height of sorghum (e.g., sorghum inbred line) with homozygous genotype corresponding to haplotype SbPH11-Hap2 (CC) is higher than or candidate higher than that of sorghum (e.g., sorghum inbred line) with homozygous genotype corresponding to haplotype SbPH11-Hap1 or haplotype SbPH11-Hap3.
[0153] Example 2, Application of the significantly associated haplotype combination of sorghum plant height and the molecular marker of haplotype SbPH11-Hap2 (CC) to the tested sorghum: 254 sorghum inbred lines of the association population
[0154] I. Determination of sorghum plant height
[0155] The method is the same as that in Example 1. The results show that the plant heights of different sorghum varieties are significantly different when 254 sorghum inbred lines are planted in the farmland soil of the light saline-alkali land in Nonggao New District, Dongying City, Shandong Province, China. The plant height of sorghum ranges from 111 to 382 cm, and the plant height of 153 sorghum inbred lines exceeds 250 cm, accounting for about 60.24% of the association population.
[0156] II. Molecular identification or assisted identification of the plant height of sorghum inbred lines
[0157] The genome DNA of the to-be-tested sorghum is extracted and dissolved with ddH2O as a template. The SNP1 primer group F1 and the SNP2 primer group F2 of the haplotype combination related SNP site in Example 1 are used for PCR amplification respectively to obtain the polymorphism information of the two SNP sites in the haplotype combination related to the SbPH11 gene, so as to determine the haplotype and genotype of the haplotype combination related to the SbPH11 gene of the to-be-tested sorghum, thereby identifying or assisting in identifying the plant height of the sorghum variety under test: the plant height of the sorghum (such as a sorghum inbred line) with the genotype CCCC of the two SNPs of SNP1 and SNP2 is higher than or is expected to be higher than that of the sorghum (such as a sorghum inbred line) with the genotype CCTT of the two SNPs of SNP1 and SNP2 or the sorghum (such as a sorghum inbred line) with the genotype AACC of the two SNPs of SNP1 and SNP2, and the plant height of the sorghum (such as a sorghum inbred line) with the genotype CCTT of the two SNPs of SNP1 and SNP2 is higher than or is expected to be higher than that of the sorghum (such as a sorghum inbred line) with the genotype AACC of the two SNPs of SNP1 and SNP2. The plant height of the sorghum (such as a sorghum inbred line) with the homozygous genotype corresponding to the haplotype SbPH11-Hap2 (CC) is higher than or is expected to be higher than that of the sorghum (such as a sorghum inbred line) with the homozygous genotype corresponding to the haplotype SbPH11-Hap1 (CT) or the sorghum (such as a sorghum inbred line) with the homozygous genotype corresponding to the haplotype SbPH11-Hap3 (AC), and the plant height of the sorghum (such as a sorghum inbred line) with the homozygous genotype corresponding to the haplotype SbPH11-Hap1 (CT) is higher than or is expected to be higher than that of the sorghum (such as a sorghum inbred line) with the homozygous genotype corresponding to the haplotype SbPH11-Hap3 (AC).
[0158] The genotypes of the two SNP sites and the plant height of the sorghum in 254 to-be-tested sorghums are shown in Tables 1 and 2. The SNP1 site of the to-be-tested sorghum contains two genotypes CC and AA (shown in the SNP1 genotype column); the SNP2 site contains two genotypes TT and CC (shown in the SNP2 genotype column). The two SNP sites in the genome of the sorghum under test exist in three haplotype combinations in the order of the genome, i.e. the haplotype SbPH11-Hap1 (CT), SbPH11-Hap2 (CC) and SbPH11-Hap3 (AC). The detection results show that among the 254 sorghum varieties, the plant height of 101 sorghum varieties with the haplotype SbPH11-Hap2 (CC) among the 130 sorghum varieties with the haplotype SbPH11-Hap2 (CC) is higher than 250 cm, i.e. the plant height of 77.69% of the sorghum varieties with the haplotype SbPH11-Hap2 (CC) is higher than 250 cm.
[0159] Among the 96 sorghum varieties of haplotype SbPH11-Hap1(CT) type, the plant height of 45 sorghum varieties is higher than 250 cm, i.e. the plant height of 46.87% of the sorghum varieties of haplotype SbPH11-Hap1(CT) is higher than 250 cm.
[0160] Among the 28 sorghum varieties of haplotype SbPH11-Hap3(AC) type, the plant height of 21 sorghum varieties is lower than 250 cm, i.e. the plant height of 75% of the sorghum varieties of haplotype SbPH11-Hap3(AC) is lower than 250 cm.
[0161] This indicates that the low plant height sorghum varieties can be eliminated by using haplotype SbPH11-Hap2(CC), the high plant height sorghum varieties of haplotype SbPH11-Hap2(CC) can be selected, and the haplotype molecular marker can be used for the assisted selection of sorghum plant height.
[0162] Table 1, plant height of 254 sorghum inbred lines and genotypes of two SNP loci
[0163]
[0164]
[0165]
[0166]
[0167]
[0168]
[0169]
[0170] Note:
[0171] IS: Sweet Sorghum; IG: Grain Sorghum; LG: Grain Sorghum; AL: unknown; LB: Broom Sorghum
[0172] The following examples use SPSS 11.5 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, One-way ANOVA test is used, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, and P<0.001 (***) indicates extremely significant difference.
[0173] The difference significant analysis of three haplotypes and plant height shows that the plant height of the homozygous genotype of haplotype SbPH11-Hap1 is extremely significantly different from the plant height of the homozygous genotype of haplotype SbPH11-Hap2 (P<0.0001), the plant height of the homozygous genotype of haplotype SbPH11-Hap1 is significantly different from the plant height of the homozygous genotype of haplotype SbPH11-Hap3 (P<0.05), the plant height of the homozygous genotype of haplotype SbPH11-Hap2 is extremely significantly different from the plant height of the homozygous genotype of haplotype SbPH11-Hap3 (P<0.0001), the plant height of the homozygous genotype of haplotype SbPH11-Hap2 (CC) is higher than or candidate higher than the plant height of the homozygous genotype of haplotype SbPH11-Hap1 (CT) and haplotype SbPH11-Hap3 (AC). The plant height of the homozygous genotype of haplotype SbPH11-Hap1 (CT) is higher than the plant height of the homozygous genotype of haplotype SbPH11-Hap3 (AC) (as shown in Table 2).
[0174] Table 2 The plant height of 254 sorghum inbred lines and difference analysis according to the genotype and haplotype combination of gene SbPH11
[0175] genotype haplotype type haplotype combination number of varieties / individual plant height (cm) CCTT SbPH11-Hap1 CT 96 242.2 ± 58.39 a ]] CCCC SbPH11-Hap2 CC 130 281.6 ± 43.16 b ]] AACC SbPH11-Hap3 AC 28 206.2 ± 44.71 c ]]
[0176] The difference significant analysis of the genotype of SNP1 or the genotype of SNP2 and plant height shows that the plant height of the homozygous genotype (CC) of SNP1 is significantly different from the plant height of the homozygous genotype (AA) of SNP1 (P<0.05), the plant height of the genotype SNP1-CC is higher than or candidate higher than the plant height of the genotype SNP1-AA. The plant height of the homozygous genotype (TT) of SNP2 is significantly different from the plant height of the homozygous genotype (CC) of SNP2 (P<0.05), the plant height of the genotype SNP2-CC is higher than or candidate higher than the plant height of the genotype SNP2-TT.
[0177] Table 3 The plant height of 254 sorghum inbred lines and difference analysis according to the different genotypes of two SNP sites of gene SbPH11
[0178]
[0179] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.
Claims
1. Use, characterized in that, The application is P1 or P2; The P1 is the use of a substance for detecting the polymorphism or genotype of the two SNPs of SNP1 and SNP2 in identifying or assisting in identifying the plant height of sorghum, or, in preparing a product for identifying or assisting in identifying the plant height of sorghum, or, in sorghum breeding or preparing a sorghum breeding product; The P2 is the use of a substance for detecting a haplotype in identifying or assisting in identifying the plant height of sorghum, or, in preparing a product for identifying or assisting in identifying the plant height of sorghum, or, in sorghum breeding or preparing a sorghum breeding product, wherein the haplotype is the polymorphism combination of SNP1 and SNP2 on a chromosome of sorghum; The SNP1 is a SNP in the genome of sorghum, which is the 425th nucleotide of SEQ ID No. 1 in the sequence listing, and is A or C; and the SNP2 is a SNP in the genome of sorghum, which is the 401st nucleotide of SEQ ID No. 1 in the sequence listing, and is C or T; The purpose of the breeding includes cultivating or selecting sorghum with high plant height.
2. Use according to claim 1, characterized in that, The sorghum is a pure line or inbred line of sorghum.
3. Use according to claim 1 or 2, characterized in that, The substance is D1), D2), D3) or D4) as follows: D1) contains specific in vitro nucleic acid amplification primers for amplifying SNP1 and SNP2 sites; D2) contains in vitro nucleic acid amplification reagents containing D1) in vitro nucleic acid amplification primers; D3) contains a kit containing D1) in vitro nucleic acid amplification primers or D2) in vitro nucleic acid amplification reagents; D4) contains a detection instrument containing D1) in vitro nucleic acid amplification primers, D2) in vitro nucleic acid amplification reagents or D3) kits.
4. Use according to claim 3, characterized in that, The in vitro nucleic acid amplification primers include primer group F1-1, primer group F1-2, primer group F2-1 and / or primer group F2-2: The primer group F1-1 is F1-1-1 or F1-1-2: F1-1-1: a primer composition consisting of a single-stranded DNA with a nucleotide sequence of 22-44 of SEQ ID No. 2 in the sequence listing and a single-stranded DNA with a nucleotide sequence of SEQ ID No. 4 in the sequence listing; F1-1-2: a primer composition consisting of a single-stranded DNA with a nucleotide sequence of SEQ ID No. 2 in the sequence listing and a single-stranded DNA with a nucleotide sequence of SEQ ID No. 4 in the sequence listing; The primer group F1-2 is F1-2-1 or F1-2-2: F1-1-1: a primer composition consisting of a single-stranded DNA with a nucleotide sequence of 22-46 of SEQ ID No. 3 in the sequence listing and a single-stranded DNA with a nucleotide sequence of SEQ ID No. 4 in the sequence listing; F1-1-2: a primer composition consisting of a single-stranded DNA with a nucleotide sequence of SEQ ID No. 3 in the sequence listing and a single-stranded DNA with a nucleotide sequence of SEQ ID No. 4 in the sequence listing; The primer group F2-1 is F2-1-1 or F2-1-2: F1-2-1: a primer composition consisting of a single-stranded DNA with a nucleotide sequence of 22-40 of SEQ ID No. 5 in the sequence listing and a single-stranded DNA with a nucleotide sequence of SEQ ID No. 7 in the sequence listing; F1-2-2: a primer composition consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 5 in the sequence listing and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence listing; The primer set F2-2 is F2-2-1 or F2-2-2: F1-2-1: a primer composition consisting of a single-stranded DNA whose nucleotide sequence is positions 22-41 of SEQ ID No. 6 in the sequence listing and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence listing; F1-2-2: a primer composition consisting of a single-stranded DNA whose nucleotide sequence is SEQ ID No. 6 in the sequence listing and a single-stranded DNA whose nucleotide sequence is SEQ ID No. 7 in the sequence listing.
5. A method for identifying or assisting in identifying the plant height of sorghum, which is method A or method B: The method A is a method for identifying or assisting in identifying the plant height of sorghum, comprising detecting the genotypes of the two SNPs of SNP1 and SNP2 in claim 1 in the sorghum to be tested, and identifying or assisting in identifying the plant height of sorghum according to the genotypes of the sorghum to be tested: The plant height of the sorghum with the genotype of CCCC is higher or is a candidate for being higher than the plant height of the sorghum with the genotype of CCTT, and the plant height of the sorghum with the genotype of CCTT is higher or is a candidate for being higher than the plant height of the sorghum with the genotype of AACC; the CCCC is a combined genotype of the two SNPs of the genotype CC of SNP1 and the genotype CC of SNP2, the CCTT is a combined genotype of the two SNPs of the genotype CC of SNP1 and the genotype TT of SNP2, and the AACC is a combined genotype of the two SNPs of the genotype AA of SNP1 and the genotype CC of SNP2; the genotype CC of SNP1 is a homozygous type of the 425th nucleotide in SEQ ID No. 1 in the sequence listing being C; the genotype AA of SNP1 is a homozygous type of the 425th nucleotide in SEQ ID No. 1 in the sequence listing being A; the genotype CC of SNP2 is a homozygous type of the 401st nucleotide in SEQ ID No. 1 in the sequence listing being C; and the genotype TT of SNP2 is a homozygous type of the 401st nucleotide in SEQ ID No. 1 in the sequence listing being T; The method B is a method for identifying or assisting in identifying the plant height of sorghum, comprising detecting the haplotype in claim 1 in the sorghum to be tested, and identifying or assisting in identifying the plant height of sorghum according to the haplotype of the sorghum to be tested: The plant height of the homozygous genotype of haplotype SbPH11-Hap2 is higher than or higher than the plant height of the homozygous genotype of haplotype SbPH11-Hap1, and the plant height of the homozygous genotype of haplotype SbPH11-Hap1 is higher than or higher than the plant height of the homozygous genotype of haplotype SbPH11-Hap3; the haplotype SbPH11-Hap2 is a haplotype of SNP1 being C and SNP2 being C, the haplotype SbPH11-Hap1 is a haplotype of SNP1 being C and SNP2 being T, and the haplotype SbPH11-Hap3 is a haplotype of SNP1 being A and SNP2 being C.
6. The method of claim 5, wherein, The sorghum is a sorghum pure line or inbred line.
7. The use of the method of claim 5 in sorghum breeding, and the purpose of the breeding includes breeding or selecting sorghum with high plant height.