Codominant molecular marker method developed based on broom corn millet regulatory gene Hd1

Abstract

The invention belongs to the technical field of crop genetic breeding, and particularly relates to a broomcorn millet regulatory gene Hd1 development-based codominant molecular marking method, which comprises the following specific steps: acquiring an Hd1 gene sequence of a paddy rice related species from an NCBI database, comparing BLAST with a broomcorn millet reference genome, positioning a broomcorn millet Hd1 homologous region, and identifying the broomcorn millet regulatory gene Hd1 homologous region. By sequencing Hd1 genes of 20-30 parts of broom corn millet varieties, combining with generic genome data, identifying low-frequency PAVs and TE derivative variation, supplementing the defects of traditional SNP / InDel, preferentially developing markers of a coding region and a promoter region, and directly influencing gene functions by variation of the regions, the polymorphism detection efficiency is improved; cross-species verification is carried out by using Hd1 markers of related species such as rice and millet, a broom corn millet marker is rapidly developed by adjusting primers and amplification conditions, a differential expression region of Hd1 is identified through transcriptome sequencing, and an EST-SSR or InDel marker is designed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop genetic breeding, in particular to a co-dominant molecular marker method developed based on proso millet regulatory gene Hd1. BACKGROUND

[0002] The proso millet is called yellow rice after shelling, and is divided into two types of glutinous and non-glutinous, the former is used to make traditional food and yellow rice, and the latter is used as staple food. The whole plant has forage value, and the stems and leaves can be used as high-quality forage grass. The nutritional components contain 13.6%-17.9% of protein and rich trace elements, and the nutritional value is higher than that of rice and wheat.

[0003] The co-dominant molecular marker method developed based on proso millet regulatory gene Hd1 is mainly used for accurate identification of crop genotypes, and serves the practice of molecular breeding. The function is mainly to use the co-dominant marker to directly distinguish the homozygous genotype (HH / hh) and the heterozygous genotype (Hh), so as to lock the plant carrying the target gene Hd1 without relying on phenotype observation, and significantly improve the selection efficiency by 45%; the molecular mechanism of Hd1 gene in crop regulation of heading date response to photoperiod; However, the existing co-dominant molecular marker method has the following technical problems in use: (1) The genetic diversity of proso millet is extremely low, and the polymorphic sites are seriously insufficient: natural variation is scarce, the genetic diversity of proso millet is only 20% of that of rice, and the Hd1 gene may have undergone strong artificial selection in the domestication process, resulting in a significant reduction in natural variation; the polymorphic sites are concentrated in the non-functional region, even if there are polymorphisms, they may be concentrated in the region that has no significant effect on the function of the gene, resulting in weak correlation between the marker and the agronomic traits; (2) The genomic data is not perfect, and the marker development depends on homologous alignment: the genomic data of proso millet is far from perfect compared with crops such as rice and corn, and only a pan-genome map is available, and high-precision haplotype analysis is lacking, so if the primers of rice Hd1 are directly used, the sequence differentiation of proso millet Hd1 gene may not be effectively amplified.

[0004] Therefore, the present application provides a co-dominant molecular marker method developed based on proso millet regulatory gene Hd1 to solve the above problems. SUMMARY

[0005] The present application aims to provide a co-dominant molecular marker method developed based on proso millet regulatory gene Hd1 to solve the problems in the background.

[0006] To achieve the above purpose, the present application provides the following technical scheme: a co-dominant molecular marker method developed based on proso millet regulatory gene Hd1, and the specific step process of the co-dominant molecular marker method is as follows: Step one, Hd1 gene sequence acquisition and cloning based on homologous alignment: Obtain the Hd1 gene sequence of rice-related species from the NCBI database, align with the reference genome of proso millet through BLAST, and locate the proso millet Hd1 homologous region; Design degenerate primers for the conserved domain of the Hd1 gene, control the length of the primers at 18-25 bp, and the GC content at 40%-60%, avoid continuous single-base repeats, use proso millet genomic DNA as a template, and use high-fidelity polymerase for PCR amplification, clone the amplification product into a T vector and sequence verification; Cross-species marker migration verification: test the CAPS marker of rice Hd1 in proso millet, optimize the amplification effect by adjusting the annealing temperature; Step two, polymorphism site mining and co-dominant marker development: Sanger sequencing or second-generation sequencing of the Hd1 gene of 20-30 proso millet varieties with large genetic background differences, combined with proso millet pan-genome data, identify single nucleotide polymorphisms (SNPs), insertions / deletions (InDels), and gene structure variations; Select insertion / deletion sites with a length of ≥5 bp, design primers to make the length difference of the amplification product ≥10 bp; for SNPs without natural enzyme cutting sites, design primers to introduce mismatched bases to create enzyme cutting sites; Transcriptome sequencing of different tissues of proso millet to screen differential expression regions of the Hd1 gene, design EST-SSR or InDel markers; Step three, preferentially develop markers in the coding region and promoter region of the Hd1 gene: use Hd1 gene markers of millet, rice and other related species for cross-species verification; integrate PAVs and TE-derived variations in pan-genome data to develop markers based on structural variations; Step four, marker detection: capillary electrophoresis of fluorescent marker PCR products, design specific primers for SNP sites, distinguish genotypes by melting curve analysis, and perform association analysis of the developed markers with proso millet heading date, photoperiod sensitivity and other traits.

[0007] Preferably, the conserved domain of the Hd1 gene in step one includes a B-box zinc finger domain and a CCT domain.

[0008] Preferably, the high-fidelity polymerase in step one is Phusion.

[0009] Preferably, in step three, the specific implementation of preferentially developing markers in the coding region and promoter region of the Hd1 gene is: SNP in the promoter region may affect gene expression, screen variations in differentially expressed regions through transcriptome data, design marker RYW-Hd1-P1, and distinguish genotypes by HRM analysis.

[0010] Preferably, the specific implementation of the step three of utilizing the Hd1 gene marker of millet, rice and other related species for cross-species verification is that the InDel marker Si93368 (91bp / 90b) of rice Hd1 is tested in millet, the amplification effect is optimized by adjusting the primer sequence (such as F: 5'-GCTAGCTAGCTAGC-3', R: 5'-GATCGATCGATCG-3'), and finally a 105 bp / 95 bp polymorphic fragment is detected in millet.

[0011] Preferably, in the step three, the specific implementation of integrating PAVs and TE-derived variations in pan-genome data to develop markers based on structural variations is that the DNA transposon insertion event near the Hd1 gene can be designed with primers Hd1-TE-F / R, the amplification fragment is 250 bp in the insertion type and 200 bp in the deletion type, and the genotypes are distinguished by fluorescence-labeled capillary electrophoresis.

[0012] Compared with the prior art, the beneficial effects of the present application are: 1) By sequencing the Hd1 gene of 20-30 millet varieties, combining with pan-genome data, identifying low-frequency PAVs and TE-derived variations, supplementing the deficiencies of traditional SNPs / InDels, and preferentially developing markers in coding regions and promoter regions, the variations in these regions directly affect the gene function, and the polymorphism detection efficiency is improved; 2) Cross-species verification is performed by utilizing the Hd1 marker of rice, millet and other related species, the millet marker is quickly developed by adjusting the primers and amplification conditions, the differential expression region of Hd1 is identified by transcriptome sequencing, and the EST-SSR or InDel marker is designed. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 The present method is a step flow chart. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0015] Embodiment one: Please refer to Figure 1 The present application provides a technical solution: A co-dominant molecular marker method developed based on the millet regulatory gene Hd1, and the specific steps of the co-dominant molecular marker method are as follows: Step one, Hd1 gene sequence acquisition and cloning based on homologous alignment: Obtain the Hd1 gene sequence of rice-related species from the NCBI database, align with the reference genome of proso millet through BLAST, and locate the homologous region of proso millet Hd1. For example, the CCT domain of rice Hd1 gene is highly conserved in proso millet and can be used as an anchor region Design degenerate primers targeting the conserved domain of the Hd1 gene. The length of the primer is controlled at 18-25 bp, the GC content is 40%-60%, and continuous single-base repeats are avoided. Use proso millet genomic DNA as a template and perform PCR amplification with high-fidelity polymerase. The reaction conditions are as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55-60°C annealing for 30 s, 72°C extension for 1 min, 35 cycles; 72°C final extension for 10 min. Clone the amplification product into a T vector and perform sequencing verification. Cross-species marker migration verification: Test the CAPS marker of rice Hd1 in proso millet by adjusting the annealing temperature to optimize the amplification effect. For example, the SNP (C→T) in the 2nd exon of the rice Hd1 gene causes the disappearance of the MspI enzyme cutting site, and primers Hd1-CAPS-F / R can be designed to verify the enzyme cutting polymorphism in proso millet. Step two, polymorphic site mining and co-dominant marker development: Perform Sanger sequencing or second-generation sequencing on the Hd1 gene of 20-30 proso millet varieties with large genetic background differences, and identify single nucleotide polymorphisms (SNPs), insertions / deletions (InDels), and gene structure variations in combination with proso millet pan-genomic data. For example, PAVs (such as TE-derived insertions / deletions) in the Hd1 gene region of the pan-genome can be used as a source of polymorphic sites Select insertions / deletions sites with a length of ≥5 bp, and design primers to make the length difference of the amplification product ≥10 bp. For example, a 12 bp insertion / deletion is found in the 3rd intron of the Hd1 gene, and primers Hd1-InDel-F / R are designed. The amplification fragment is 150 bp for the insertion type and 138 bp for the deletion type, and genotypes can be distinguished by 8% non-denaturing polyacrylamide gel electrophoresis. For SNPs without natural enzyme cutting sites, design primers to introduce mismatched bases to create enzyme cutting sites. For example, at the SNP (C→T) in the 2nd exon of the Hd1 gene, design primers Hd1-dCAPS-F / R. The amplification product is cut by HindIII, and the wild type (C) produces 144 bp and 24 bp fragments, while the mutant type (T) only shows a 168 bp band.

[0016] Perform transcriptome sequencing on different tissues of proso millet, screen for differentially expressed regions of the Hd1 gene, and design EST-SSR or InDel markers. For example, a SSR site is found in the 3' untranslated region (UTR) of the Hd1 gene, and a co-dominant marker RYW-Hd1-1 is developed. Polymorphisms are detected by capillary electrophoresis. Step 3: Prioritize the development of markers in the coding and promoter regions of the Hd1 gene: Utilize Hd1 gene markers from closely related species such as millet and rice for cross-species validation; Integrate PAVs and TE-derived variants from pan-genome data to develop markers based on structural variations; Step 4, Label Detection: Capillary electrophoresis was performed on the fluorescently labeled PCR products. Specific primers were designed for SNP sites, and genotypes were distinguished by melting curve analysis. The developed labels were then correlated with traits such as millet heading date and photoperiod sensitivity.

[0017] In step one, the conserved domains of the Hd1 gene include the B-box zinc finger domain and the CCT domain. Their functions are as follows: To ensure the functionality and specificity of the amplified sequence and avoid interference from non-target sequences: The Hd1 gene is a core regulatory gene for plant photoperiod regulation and flowering (heading) stage. Its function depends on the integrity of its conserved domains: The B-box zinc finger domain is a characteristic structure of the zinc finger protein family and mainly participates in protein-protein interactions or specific binding to DNA, which is the basis for Hd1 to exert its transcriptional regulatory function; the CCT domain mainly participates in nuclear localization, protein dimerization, and interaction with downstream target genes, directly affecting the transmission of flowering signals by Hd1. Designing primers that limit the amplified sequences to these two domains can ensure that the PCR amplified fragment is the functional core region of the Hd1 gene, rather than a non-functional homologous sequence or pseudogene, thereby avoiding non-specific amplification from interfering with the accuracy of subsequent marker development; Improving the conservation and amplification efficiency of cross-species primers: The Hd1 gene is highly conserved in monocotyledonous plants (such as rice, millet, and foxtail millet), with low variation rates in the amino acid sequences of its B-box and CCT domains and the coding gene sequence. Degenerate primers designed based on these conserved regions can better match the homologous sequences of the foxtail millet Hd1 gene, reduce primer binding failures caused by sequence differences between species, and significantly improve the success rate of "cross-species marker migration" from closely related species such as rice to foxtail millet. Ensuring the association between polymorphic sites and traits and improving the practicality of markers: The functional variation of the Hd1 gene mainly originates from the sequence variation of its functional domains. Developing markers limited to the B-box and CCT domains can ensure that the discovered polymorphic sites are directly associated with the functional differences of Hd1, rather than neutral variations in irrelevant regions. This allows the codominant molecular markers developed subsequently to be more accurately associated with target traits such as millet heading date and photoperiod sensitivity, thus improving the practicality of markers in breeding. The high-fidelity polymerase used in step one is Phusion. Its function is as follows: Reducing the mismatch rate of PCR amplification: High-fidelity polymerases (such as Phusion) have 3'→5' exonuclease activity, which can recognize and remove mismatched bases during amplification, making the error rate of PCR products much lower than that of ordinary Taq enzymes (usually 10-50 times lower). Since subsequent steps require cloning, sequencing and polymorphism site mining based on the amplified Hd1 gene sequence, the sequence accuracy directly affects the reliability of marker development. If there are errors in the amplified sequence, it may lead to misjudgment of SNPs, insertions / deletions and other polymorphic sites, ultimately affecting the accuracy of marker-trait association analysis. Improving the efficiency and specificity of long-fragment amplification: The Hd1 gene contains conserved domains and potential long-fragment regulatory regions, while high-fidelity polymerases such as Phusion have stronger amplification capabilities for complex templates and can effectively reduce non-specific amplification products. Degenerate primers may exhibit some non-specific binding, while the high specificity of high-fidelity enzymes can reduce heterogeneous band interference, ensuring that the amplification products are mainly the target Hd1 gene fragment, facilitating subsequent cloning and sequencing verification; Adapting to the amplification requirements of complex templates: Millet genomic DNA may contain secondary structures, and ordinary Taq polymerases are prone to stalling or detachment when amplifying such regions, leading to amplification failure or incomplete fragments. High-fidelity polymerases such as Phusion, however, possess strong thermal stability and chain elongation capabilities, enabling efficient amplification of complex templates at relatively high annealing temperatures (typically 55-72℃), ensuring the complete amplification of the full-length Hd1 gene. This provides a complete sequence basis for subsequent steps such as cross-species marker migration validation and structural variation analysis. In step three, the specific implementation of prioritizing the development of markers in the coding and promoter regions of the Hd1 gene is as follows: SNPs in the promoter region may affect gene expression. Variants in differentially expressed regions are screened using transcriptome data, and the marker RYW-Hd1-P1 is designed. This is then combined with HRM analysis to distinguish genotypes. Its function is as follows: Targeting functional variations to enhance the association between markers and traits: The promoter is a key regulatory region for gene transcription initiation. SNPs (single nucleotide polymorphisms) in its sequence can directly alter the expression level of the Hd1 gene (e.g., expression timing and intensity) by affecting processes such as transcription factor binding and RNA polymerase recruitment. As a key gene regulating heading date and photoperiod sensitivity, differences in the expression level of the Hd1 gene directly lead to variations in these agronomic traits. By screening differentially expressed regions using transcriptome data, promoter SNPs that have been proven to be associated with differential Hd1 gene expression (i.e., these variants are potential causes of gene expression differences) can be preferentially identified. The marker RYW-Hd1-P1, designed based on such variants, is not a randomly selected neutral site, but a "functional marker" directly associated with gene function. Therefore, it has a higher correlation with target traits such as heading date and photoperiod sensitivity, which can significantly improve the accuracy and reliability of subsequent association analyses. HRM technology is adapted to the detection needs of codominant markers, improving the accuracy of genotype differentiation: The core requirement of codominant markers is the ability to accurately distinguish between homozygous dominant, homozygous recessive, and heterozygous genotypes (e.g., the SNP targeted by RYW-Hd1-P1 may have three genotypes: AA, aa, and Aa). HRM technology, by analyzing the differences in melting curves of PCR products (based on the GC content, length, and base composition of DNA fragments), can achieve high-resolution differentiation of subtle sequence differences such as SNPs.

[0018] The specific implementation method for cross-species verification using Hd1 gene markers from closely related species such as millet and rice in step three is as follows: The InDel marker Si93368 (91bp / 90b) of rice Hd1 was tested in millet. The amplification effect was optimized by adjusting the primer sequences F: 5'-GCTAGCTAGCTAGC-3', R: 5'-GATCGATCGATCG-3', and finally a 105bp / 95bp polymorphic fragment was detected in millet. The function is as follows: Shortening the marker development cycle and reducing development costs: The Hd1 gene in closely related species such as rice and millet has been studied extensively, and the InDel markers developed for these species have generally been verified to be associated with traits such as heading date and photoperiod sensitivity. Directly testing the applicability of these markers in millet through cross-species validation eliminates the need for de novo full-sequence scanning and polymorphism screening of the millet Hd1 gene, significantly shortening marker development time and reducing experimental costs. To ensure the association between the marker and Hd1 gene function and improve marker reliability: The rice InDel marker Si93368 originates from the Hd1 gene region, and its polymorphism is itself related to functional variations of Hd1. In millet, after primer optimization, a 105bp / 95bp polymorphic fragment was detected, indicating that this region also exhibits sequence differences in the millet Hd1 gene, and this difference is highly likely related to the functional conservation of Hd1. Compared to randomly developed markers, this cross-species validated marker has a clearer association with Hd1 gene function, and subsequent association analyses with traits such as millet heading date and photoperiod sensitivity are also more reliable. Clearly identifiable co-dominant polymorphisms were obtained to meet detection requirements: the optimized 105bp / 95bp fragments detected in millet showed a length difference of 10bp, indicating a clear InDel polymorphism. This difference is sufficient to clearly distinguish different genotypes using conventional detection methods such as capillary electrophoresis, perfectly meeting the core requirement of co-dominant markers to "simultaneously identify homozygotes and heterozygotes."

[0019] In step three, the specific implementation of integrating PAVs and TE-derived variants from pan-genome data to develop markers based on structural variations is as follows: primers Hd1-TE-F / R can be designed for DNA transposon insertion events near the Hd1 gene to amplify fragments of 250 bp for insertion and 200 bp for deletion, and genotypes can be distinguished by fluorescently labeled capillary electrophoresis.

[0020] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0021] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A codominant molecular marker method based on the millet regulatory gene Hd1, characterized in that, The specific steps of this co-dominant molecular marker method are as follows: Step 1: Obtaining and cloning the Hd1 gene sequence based on homology alignment: Obtain the Hd1 gene sequence of closely related rice species from the NCBI database, and locate the homologous region of Hd1 in millet by BLAST alignment with the reference genome of millet. Degenerate primers were designed targeting the conserved domains of the Hd1 gene, with primer lengths controlled at 18-25 bp and GC content at 40%-60%, avoiding continuous single-base repetitions. Using millet genomic DNA as a template, PCR amplification was performed using high-fidelity polymerase. The amplified products were cloned into a T vector and sequenced for verification. Cross-species marker migration validation: The CAPS marker of rice Hd1 was tested in millet, and the amplification effect was optimized by adjusting the annealing temperature; Step 2: Polymorphism site mining and codominant marker development: Sanger sequencing or next-generation sequencing was performed on the Hd1 gene of 20-30 millet varieties with significant differences in genetic background. Combined with millet pan-genome data, single nucleotide polymorphisms, insertions / deletions and gene structural variations were identified. Select insertion / deletion sites with a length ≥5bp and design primers to make the amplification product length difference ≥10bp; For SNPs that lack natural cleavage sites, primers are designed to introduce mismatched bases to create cleavage sites. Transcriptome sequencing was performed on different tissues of millet to screen differentially expressed regions of the Hd1 gene and design EST-SSR or InDel markers. Step 3: Prioritize the development of markers in the coding and promoter regions of the Hd1 gene: Utilize Hd1 gene markers from closely related species such as millet and rice for cross-species validation; Integrate PAVs and TE-derived variants from pan-genome data to develop markers based on structural variations; Step 4, Label Detection: Capillary electrophoresis was performed on the fluorescently labeled PCR products. Specific primers were designed for SNP sites, and genotypes were distinguished by melting curve analysis. The developed labels were then correlated with traits such as millet heading date and photoperiod sensitivity.

2. The codominant molecular marker method based on the millet regulatory gene Hd1 as described in claim 1, characterized in that: In step one, the conserved domains of the Hd1 gene include the B-box zinc finger domain and the CCT domain.

3. The codominant molecular marker method based on the millet regulatory gene Hd1 as described in claim 1, characterized in that: The high-fidelity polymerase used in step one is Phusion.

4. The codominant molecular marker method based on the millet regulatory gene Hd1 as described in claim 1, characterized in that: In step three, the specific implementation of prioritizing the development of markers in the coding region and promoter region of the Hd1 gene is as follows: SNPs in the promoter region may affect gene expression. By screening for variants in differentially expressed regions through transcriptome data, the marker RYW-Hd1-P1 is designed, and genotypes are distinguished by combining HRM analysis.

5. The codominant molecular marker method based on the millet regulatory gene Hd1 as described in claim 1, characterized in that: The specific implementation method of cross-species verification using Hd1 gene markers from closely related species such as millet and rice in step three is as follows: the InDel marker Si93368 of rice Hd1 was tested in millet, and the amplification effect was optimized by adjusting the primer sequence. Finally, a 105bp / 95bp polymorphic fragment was detected in millet.

6. The codominant molecular marker method based on the millet regulatory gene Hd1 as described in claim 1, characterized in that: In step three, the specific implementation of integrating PAVs and TE-derived variants from pan-genome data to develop markers based on structural variations is as follows: primers Hd1-TE-F / R can be designed for DNA transposon insertion events near the Hd1 gene, amplifying fragments of 250bp for insertion and 200bp for deletion, and distinguishing genotypes by fluorescently labeled capillary electrophoresis.

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