Co-segregation gene molecular marker for identifying tagetes patula cotyledon character, detection primer and kit and application

Abstract

The application discloses a co-segregation gene molecular marker for identifying a tagetes bract character, a detection primer and a kit and application. The co-segregation gene molecular marker for identifying the tagetes bract character has a nucleotide sequence shown in SEQ ID NO. 1 or both of the two nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2 in a tagetes single plant without a bract, and has a nucleotide sequence shown in SEQ ID NO. 2 in a tagetes single plant with a bract. The application further provides a PCR detection primer for detecting the co-segregation gene molecular marker and application of the co-segregation gene molecular marker in tagetes bract character breeding. The detection method established by using the co-segregation gene molecular marker identified in the application can directly use PCR products to quickly identify the presence or absence of the tagetes bract, the detection efficiency reaches 100% in an F2 separation population, early screening can be carried out at a seedling stage, and unfavorable characters can be removed, so that a breeding period is effectively shortened and cost is reduced.

Description

Technical Field

[0001] This invention relates to molecular markers for cosegregating genes in marigolds to identify developmental traits, and particularly to molecular markers, primers, and kits for identifying cosegregating genes in marigold bracts and their applications, belonging to the field of molecular markers for cosegregating genes in marigold bracts and their applications. Background Technology

[0002] Bracts are modified leaf-like structures within the inflorescence that do not promote plant growth. In a broader sense, any leaf-like structure related to the inflorescence is called a bract. The majority of bracts clustered around the periphery of the inflorescence are called the involucre, while the smaller bracts attached to the peduncle are called bracteoles. In many plants, the bracts resemble stipules and gradually wither and fall off after the flower buds and blossoms unfold. Only in a few plants are the bracts preserved, playing different roles in different plants.

[0003] Asteraceae plants have extremely complex capitula, composed of dozens or even hundreds of florets. The involucre of Asteraceae plants is located on the outer edge of the capitula, protecting the growth and development of all the florets, as well as the maturation of the seeds. The bracteoles, which originally surrounded the florets, have gradually degenerated or even disappeared with the evolution of Asteraceae plants, thus providing more space for the florets to grow and develop.

[0004] Marigold (Tagetes erecta) is an annual herbaceous flowering plant belonging to the Asteraceae family, possessing significant ornamental and economic value. Currently, cultivated marigold cultivars all have only outer involucral bracts in their capitulum, lacking bracteoles. Our research group previously discovered a mutant, MT, which produces a bracteole on the outer side of each floret forming the capitulum. Genetic studies have shown that a recessive gene controls the occurrence of bracteoles. In MT, the presence of bracteoles encroaches on the growth and development space of the florets, leading to a reduction in the number of florets and floret morphology deformities, severely impacting the ornamental value of the marigold. Because this is a recessive trait, preventing its infiltration has become a critical challenge in marigold breeding.

[0005] Gene molecular markers are effective gene markers developed based on sequence differences in a gene and closely linked to a specific trait. Developing gene molecular markers closely linked to the marigold bract trait for marker-assisted breeding can enable seedling detection, overcome the shortcomings of traditional breeding methods, and improve breeding efficiency. Summary of the Invention

[0006] One objective of this invention is to provide a molecular marker for cosegregating genes to identify the bract traits of marigolds;

[0007] A second objective of this invention is to provide detection primers for amplifying the molecular markers of the co-segregated genes;

[0008] The third objective of this invention is to apply the aforementioned co-segregating gene molecular markers or detection primers to identify the bract traits of marigolds.

[0009] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0010] This invention first provides a cosegregating gene molecular marker for identifying the bract-like trait of marigolds, named MADS32; the nucleotide sequence of the cosegregating gene molecular marker MADS32 in marigold plants without bracts is shown in SEQ ID NO.1, or has both SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequence of the cosegregating gene molecular marker MADS32 in marigold plants with bracts is shown in SEQ ID NO.2.

[0011] The cosegregating gene molecular marker MADS32 provided by this invention has multiple base mutations and short fragment insertions or deletions in the marigold sequence with bracts, and a 141bp insertion was found in MT, and its cosegregation with the bract trait was observed. This shows that the cosegregating gene molecular marker MADS32 provided by this invention can be used to identify the presence or absence of the marigold bract trait.

[0012] The present invention further provides PCR detection primers for detecting the co-segregated gene molecular marker MADS32.

[0013] As a preferred embodiment, the PCR detection primers for detecting the co-segregated gene molecular marker MADS32 consist of the upstream detection primer shown in SEQ ID NO.3 and the downstream detection primer shown in SEQ ID NO.4.

[0014] The PCR detection primers provided by this invention can be used as a primer set for amplifying the co-segregating gene molecular marker MADS32, and then used to identify the presence or absence of marigold bract traits. Accordingly, the application of the primer set in identifying marigold bract traits falls within the protection scope of this invention.

[0015] This invention further provides a PCR detection kit for identifying the characteristics of marigold bracts, comprising: PCR Master Mix (including Taq enzyme, dNTPs, Mg) 2+ The PCR detection primers consist of deionized water and detection primers; wherein the PCR detection primers are designed using the co-segregated gene molecular marker MADS32 as the target; as a preferred embodiment, the PCR detection primers consist of an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4.

[0016] The present invention further provides the application of the co-segregating gene molecular marker MADS32 in identifying the presence or absence of marigold bracts, comprising the following steps: (1) extracting DNA from the marigold sample to be tested as an amplification template; (2) performing PCR amplification using upstream and downstream primers designed with the co-segregating gene molecular marker MADS32 as the target gene and detecting the amplification product using agarose gel; (3) if the PCR amplification product is a single band of 472bp or two bands of 472bp and 612bp are amplified simultaneously, then the marigold sample to be tested is a marigold without bracts; if the PCR amplification product is a single electrophoretic band of 612bp, then the marigold sample to be tested is a marigold with bracts.

[0017] As a preferred embodiment of the present invention, the PCR amplification reaction system is as follows: PCRMaster Mix 12.5 μL (including Taq enzyme, dNTPs and Mg) 2+ ), 1 μL DNA template, 1 μL upstream primer, 1 μL downstream primer, and 9.5 μL deionized water.

[0018] As a preferred embodiment of the present invention, the PCR amplification reaction program is as follows: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 10 min; and 20℃ for 5 min.

[0019] To obtain a co-segregating gene molecular marker for the bract trait in marigolds, this invention constructs an F2 segregating population of marigolds with the bract trait and develops the co-segregating gene molecular marker MADS32 based on the differential sequence of the MADS32 gene. The results show that detection primers designed using this co-segregating gene molecular marker MADS32 as a target can effectively distinguish the presence or absence of bracts in marigolds. Marigolds with bracts amplify a 612bp band; marigolds without bracts amplify a single 472bp band or both 472bp and 612bp bands simultaneously. Those amplifying both 472bp and 612bp bands are heterozygous marigold plants without bracts.

[0020] The co-segregating gene molecular marker MADS32 identified in this invention achieved a detection efficiency of 100% in the F2 segregating population of marigold bract traits.

[0021] The co-segregating gene molecular marker MADS32 identified by this invention was validated in the F2 population of marigolds with segregating bract traits. The PCR amplification products of 608 single plants without bracts were either a single band of 472 bp or two bands of 472 bp and 612 bp. The PCR amplification products of 172 single plants with bracts were a single band of 612 bp. The detection results showed that the plant phenotype was completely consistent with the co-segregating gene molecular marker MADS32, with no exchange occurring and a detection efficiency of 100%.

[0022] The detection method established using the co-segregating gene molecular marker MADS32 identified in this invention can directly and rapidly screen for the presence or absence of marigold bract traits using PCR products, allowing for early screening at the seedling stage, greatly shortening the breeding cycle and effectively reducing costs.

[0023] This invention relates to the definition of key terms and abbreviations.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0025] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more of the selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell. Probes 8:91-98 (1994)).

[0026] Gene molecular markers: Effective gene markers developed based on sequence differences of a gene and closely linked to a certain trait.

[0027] Gene structure: A gene generally contains four regions: the coding region, which includes exons and introns; the leader region, located upstream of the coding region, which is equivalent to the 5-pinched terminal non-coding region of RNA; the tail region, located downstream of the 3-pinched coding region of RNA, which is equivalent to the terminal non-coding region; and the regulatory region, which includes promoters and enhancers, etc. Attached Figure Description

[0028] Figure 1 The images show marigold capitula with and without bracteoles; a is the capitula of S5 without bracteoles, and b is the capitula of MT with bracteoles (indicated by arrows).

[0029] Figure 2 The image shows the Manhattan plot based on the ED method; the horizontal axis represents chromosome length (Mb); the vertical axis represents the ED value; the points in the figure represent the location of each SNP / InDel and the result value calculated by the ED method, and the red line in the figure represents the threshold line.

[0030] Figure 3 Differential promoter sequence analysis of the MADS32 gene of marigold S5 and MT.

[0031] Figure 4 Figure showing the validation results of the co-segregating gene molecular marker MADS32 in marigold parents and F2 population. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0033] Example 1: Identification of gene molecular markers cosegregating with the trait of marigold bracts

[0034] 1. Construction of a segregating population of marigold bract traits

[0035] In this embodiment, the marigold inbred line S5 without bracts was used as the female parent, and the marigold mutant MT with bracts was used as the male parent. Figure 1 ), hybridization yields F1, and self-pollination of individual F1 plants yields the F2 segregating population.

[0036] 2. Analysis of the inheritance patterns of marigold bract traits

[0037] Statistical analysis of the bract trait in the F2 segregating population revealed 608 plants without bracts and 172 plants with bracts. According to the chi-square test, the ratio of plants without bracts to plants with bracts was approximately 3:1, which conforms to Mendel's laws of inheritance. This indicates that a single gene controls the bract trait in marigolds, with the absence of bracts being a dominant trait and the presence of bracts being a recessive trait.

[0038] 3. Pool construction and BSR-seq sequencing

[0039] Thirty plants with bracts and 30 plants without bracts were selected from the F2 segregating population. Three 1mm flower buds were collected from each plant and mixed to construct two F2 pools, labeled "pool-F2-MT" and "pool-F2-S5". Samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for RNA-seq sequencing using the Illumina NovaSeq 6000 platform, with the marigold genome sequence as the reference genome. The quality-controlled sequencing reads were aligned to the reference genome using BWA software. The alignment results (BAM file) were processed using GATK's Best Practices workflow, and SNP loci were detected using GATK's Haplotyper method.

[0040] 4. Screening of SNP sites closely linked to bract traits

[0041] The Euclidean Distance (ED) algorithm was used to identify significantly different SNP markers between pools, and these were used to evaluate regions associated with the trait. A quantile threshold of 99.5% was used for candidate region screening. The qualified SNP loci were mostly clustered in the 34.26 Mb-47.87 Mb region on chromosome 5 of marigold. Figure 2 Based on the SNP sites within the region, restriction enzyme sites were located using SnapeGene software, and primers were designed using Primer Premier 5.0 software to develop three effective CAPS markers. Simultaneously, based on the specific site differences of the SNPs in the sequence, primers were designed using Primer Premier 5.0 software, and three marker sites were developed using DNA sequencing technology. Validation was performed on 780 individual plants to achieve fine mapping, locking the target genes within the range of 37.53Mb-38.02Mb, which contains 43 genes (Table 1).

[0042] Table 1. ID numbers and predicted gene functions of 43 genes in the 37.53Mb-38.02Mb region of chromosome 5 of Marigold.

[0043]

[0044]

[0045] 5. Cloning and sequence difference analysis of the marigold MADS32 gene promoter

[0046] Functional annotation, sequence alignment, and qRT-PCR expression analysis were performed on 43 genes within the interval. It was found that the expression level of an SVP-like gene, MADS32, was downregulated 18-fold in MT compared to S5. Using DNA from marigold S5 and MT as templates, the promoter sequence of the MADS32 gene was cloned. The results showed that the promoter sequence of the MADS32 gene differed between marigold S5 and MT. The nucleotide sequence in marigold S5 without bracts was SEQ ID NO.1, while the nucleotide sequence in marigold MT with bracts was SEQ ID NO.2. Comparison of sequence information revealed multiple base mutations and short insertions or deletions at SEQ ID NO.1 and SEQ ID NO.2, and a 141bp insertion was found in MT. Figure 3 Gene marker primers were designed on both sides of the differentially expressed sequences using PrimerPremier 5.0 software. The sequences were distinguished by the length of the amplified fragments. The nucleotide sequences of the gene marker primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0047] 5. Validation of the co-segregating gene molecular marker MADS32 in the F2 population

[0048] The co-segregating gene marker MADS32 was detected in 20 individual plants from the F2 segregating population, indicating that MADS32 co-segregated with the bracteole trait. PCR amplification products from marigold plants with bracteoles showed a 612 bp band, while PCR amplification products from marigold plants without bracteoles showed a 472 bp band, or both 472 bp and 612 bp bands. Figure 4 ).

[0049] 6. Co-isolated gene molecular marker MADS32 and PCR amplification system and procedure

[0050] The total PCR reaction volume was 25 μL, containing 1 μL of template DNA at a concentration of 100 ng / μL. PCR Master Mix 12.5 μL (containing Taq enzyme, dNTPs, Mg) 2+ ), 1 μL each of 10 μmol / L upstream and downstream primers, and 9.5 μL of ddH2O.

[0051] The PCR reaction program was 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 10 min; and incubation at 20℃ for 5 min. The amplified products were detected by 1% agarose gel electrophoresis.

[0052] Example 1: Validation experiment using the co-segregating gene molecular marker MADS32 to identify the bract traits of 780 marigold F2 populations.

[0053] (1) Take 4-5 fresh marigold leaves, extract marigold genomic DNA to be tested using the 2×CTAB method, test the DNA quality using 1% agarose gel, test the DNA concentration using a UV spectrophotometer, and then dilute the extracted DNA to 100 ng / uL and aliquot it for later use.

[0054] (2) Using the marigold genome DNA to be tested as a template and the identified co-segregating gene molecular marker MADS32 as a target, upstream and downstream primers were designed. The base sequences of the upstream and downstream primers are as follows:

[0055] MADS32-F (upstream primer): CATCAATCGACTCGTATTCTCACTC

[0056] (SEQ ID NO.3)

[0057] MADS32-R (downstream primer): GTTGAAATTAGTGATCTAATAGTGAA (SEQ ID NO.4)

[0058] (3). The PCR reaction system consisted of 1 μL of 100 ng / μL DNA template. PCR Master Mix 12.5 μL (containing Taq enzyme, dNTPs, Mg) 2+ ), upstream primer 1μL, downstream primer 1μL, ddH2O 9.5μL.

[0059] (4). The PCR reaction program is as follows: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 10 min; 20℃ for 5 min.

[0060] (5) The amplification products were detected by 1% agarose gel electrophoresis. If the PCR product could amplify a 472bp band or two bands of 472bp and 612bp at the same time, it was marigold without bracts. Among them, the plant that amplified two bands at the same time was a marigold heterozygote. If a 612bp amplification band was amplified after agarose gel detection, it was marigold with bracts.

[0061] The co-segregating gene molecular marker MADS32 was used to validate 780 F2 segregating populations. The PCR products of 608 plants without bracts were either a single 472bp band or two bands of 472bp and 612bp. The PCR products of 172 single plants with bracts were a single 612bp band. The results showed that the marigold plant phenotype was completely consistent with the co-segregating gene molecular marker MADS32, with no exchange occurring and a detection efficiency of 100%.

Claims

1. The co-segregating gene molecular marker MADS32 for identifying the bract traits of marigolds, characterized in that, The nucleotide sequence of the co-segregating gene molecular marker MADS32 in marigold plants without bracts is shown in SEQ ID No. 1 or has both SEQ ID No. 1 and SEQ ID No. 2; the nucleotide sequence of the co-segregating gene molecular marker MADS32 in marigold plants with bracts is shown in SEQ ID No.

2.

2. The application of the co-segregating gene molecular marker MADS32 as described in claim 1 in the selection or detection of the presence or absence of marigold bract traits.

3. The application of the PCR detection primers for the co-segregating gene molecular marker MADS32 as described in claim 1 in the selection or detection of the presence or absence of marigold bract traits.

4. The application according to claim 3, characterized in that, include: (1) Extract DNA from marigold samples to be tested as amplification template; (2) A PCR amplification reaction system was established using upstream and downstream primers designed with the co-segregating gene molecular marker MADS32 as the target gene, and PCR amplification was performed; (3) If the PCR amplification product is a single band of 472 bp, or two bands of 472 bp and 612 bp are amplified simultaneously, then the marigold sample to be tested is a marigold without bracts; if the PCR amplification product is a single electrophoretic band of 612 bp, then the marigold sample to be tested is a marigold with bracts; the nucleotide sequence of the upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.

4.

5. The application according to claim 4, characterized in that, The PCR amplification reaction system was: 2×Hieff ® PCRMaster Mix 12.5 μL, DNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, deionized water 9.5 μL; The PCR amplification reaction program was as follows: 94℃ for 4 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 10 min; and 20℃ for 5 min.

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