Molecular markers, primer pairs, and uses thereof, and methods for identifying gender in cycadaceae plants

Abstract

The application discloses a molecular marker, a primer pair, application thereof and a gender identification method for plants in the Cycadaceae family, relates to the technical field of plant gender identification, and specifically provides a molecular marker, a primer pair and a gender identification method for plants in the Cycadaceae family. The molecular marker can be linked with Y chromosomes of plants in the Cycadaceae family, and the molecular marker contains a nucleotide sequence as shown in SEQ ID NO. 1. The molecular marker can be closely linked with Y chromosomes of plants in the Cycadaceae family, so that the purpose of rapidly detecting the gender of plants in the Cycadaceae family is achieved, and the efficiency of garden research and management is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of plant sex identification technology, and particularly relates to a method for identifying the sex of Cycadaceae plants using molecular markers, primer pairs, and other methods. Background Technology

[0002] Cycadaceae Pers., belonging to the phylum Gymnosperms, class Cycadales, and order Cycadales, are commonly known as "iron trees." They are mainly distributed in my country, South Asia, Southeast Asia, Pacific islands, Australia, and Africa, with over 100 species in one genus worldwide.

[0003] Cycads are dioecious plants, characterized by slow growth and difficulty in distinguishing male from female plants. Previously, sex could only be determined when the plant flowered, but mature trees typically flower only after ten years or more and under suitable conditions. Due to the lack of simple and effective methods for sex identification in cycads, there are currently no international reports on the sex ratios and distribution patterns of different wild cycad populations. Furthermore, there is a lack of evidence for sex matching in reintroduction practices. Therefore, sex identification of cycads has always been a technical challenge in the scientific conservation and horticultural cultivation of cycads. Summary of the Invention

[0004] The first objective of this invention is to provide a molecular marker that can be linked to the Y chromosome of cycads for rapid sex identification of cycads.

[0005] The technical solution adopted in the embodiments of the present invention is as follows:

[0006] A molecular marker that is linked to the Y chromosome of cycads;

[0007] The molecular marker contains a sequence that encodes a nucleotide sequence as shown in SEQ ID NO.1.

[0008] Compared with existing technologies, the molecular markers provided in this invention can be closely linked to the Y chromosome of Cycadaceae plants, thereby achieving the purpose of rapid detection of sex in Cycadaceae plants.

[0009] The second objective of this invention is to provide a primer pair, the specific technical solution of which is as follows:

[0010] A primer pair, including primer 1 and primer 2, is used to amplify DNA from the genome of a male Cycadaceae plant as a template to obtain the molecular marker described above.

[0011] Primer 1 contains a sequence encoding a nucleotide sequence as shown in SEQ ID NO. 2;

[0012] Primer 2 contains a sequence that encodes a nucleotide sequence as shown in SEQ ID NO. 3.

[0013] The primer pair provided by the second objective of this invention can amplify the DNA of the male genome of cycads as a template and obtain the aforementioned molecular markers. This allows for rapid identification of the sex of cycads, effectively improving the identification of the sex of cycads and facilitating the rapid development of horticultural research and management.

[0014] A third objective of this invention is to provide the application of the aforementioned molecular markers in identifying the sex of Cycadaceae plants.

[0015] The fourth objective of this invention is to provide a method for identifying the sex of Cycadaceae plants, the specific technical solution of which is as follows:

[0016] This includes the step of detecting whether the sex chromosomes of the cycad species being tested are linked to the molecular markers described above.

[0017] Alternatively, the steps include performing PCR amplification on the Cycadaceae plant to be tested using the primers described above, and determining whether the PCR amplification product contains the molecular markers described above.

[0018] The beneficial effects of the embodiments of the present invention are as follows:

[0019] Compared with existing technologies, the method for sex identification of Cycadaceae plants provided in this invention can quickly identify the sex of Cycadaceae plants of any age without waiting for them to flower or transplanting them to a suitable flowering environment. This greatly shortens the observation time for sex identification of Cycadaceae plants, which is beneficial to improving the efficiency of garden research and management. In addition, it has important practical significance for the sex selection and proportion allocation of Cycad ornamental garden plants, as well as for understanding and protecting endangered Cycad germplasm resources. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0021] Figure 1 This is a schematic diagram showing the results of PCR amplification of DNA from the genomes of five Cycadaceae plants using primer pairs, as described in an embodiment of the present invention. Detailed Implementation

[0022] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0023] This application provides a primer pair that can perform polymerase chain reaction (PCR) using the genomic DNA of male Cycadaceae plants as a template to obtain a molecular marker containing a coding nucleotide sequence as shown in SEQ ID NO. 1. This molecular marker can be tightly linked to the Y chromosome of Cycadaceae plants, thereby enabling rapid sex identification of Cycadaceae plants. The molecular marker provided in this embodiment can link genomic DNA sequences to the sex chromosomes of Cycadaceae plants, thus facilitating the establishment of molecular marker-based sex identification for Cycadaceae plants. Furthermore, since the molecular marker is located on the Y chromosome of Cycadaceae plants, the sex of Cycadaceae plants can be obtained simply, rapidly, and with high throughput, which is beneficial for sex identification in conservation biology and horticultural cultivation practices of Cycadaceae plants.

[0024] Specifically, the primer pair includes primer 1 and primer 2, wherein primer 1 contains a sequence encoding a nucleotide sequence as shown in SEQ ID NO. 2; and primer 2 contains a sequence encoding a nucleotide sequence as shown in SEQ ID NO. 3.

[0025] In some embodiments, the encoding nucleotide sequence of the molecular marker is the sequence shown in SEQ ID NO.1. Of course, in some embodiments, the encoding nucleotide sequence of the molecular marker can be obtained by DNA chemical synthesis. In some embodiments, the molecular marker is a DNA fragment in the cycad genome that encodes the nucleotide sequence shown in SEQ ID NO.1, meaning that the nucleotide sequence other than the 5' end and / or 3' end of SEQ ID NO.1 is also a sequence in the cycad genome. In some embodiments, the molecular marker is the upstream and downstream sequences of the 5' end and / or 3' end of SEQ ID NO.1 in the cycad genome.

[0026] It should be noted that as long as the genomic DNA of the male Cycas revoluta strain to be amplified or detected contains this molecular marker, the sequence shown in SEQ ID NO.1 can be detected or amplified. The length of the upstream and downstream sequences at the 5' and / or 3' ends of SEQ ID NO.1 is appropriate and not particularly limited. For example, the length of the molecular marker is less than 10,000 bp, less than 5,000 bp, less than 2,000 bp, less than 1,500 bp, less than 1,200 bp, less than 1,000 bp, or less than 800 bp, etc., are all acceptable.

[0027] In some embodiments, the encoding nucleotide sequence of primer 1 is the sequence shown in SEQ ID NO. 2; and the encoding nucleotide sequence of primer 2 is the sequence shown in SEQ ID NO. 3. In other embodiments, primers 1 and 2 may each have 1 to 10 bases added to their 5' or 3' ends. The type of added bases can be determined based on the base types in the regions on the cycad genomic DNA that match SEQ ID NO. 2 and SEQ ID NO. 3, according to base pairing principles. The resulting primer pairs are substantially identical to the amplification products of SEQ ID NO. 2 and SEQ ID NO. 3 (the DNA sequences between the upstream and downstream primers are identical). Therefore, primer pairs that have 1 to 10 bases added to the 5' or 3' ends of SEQ ID NO. 2 and SEQ ID NO. 3 and can amplify substantially identical DNA fragments are all included in the primer pairs of the present invention.

[0028] The molecular markers provided in this application can be used for sex identification of Cycadaceae plants.

[0029] Specifically, the method for identifying the sex of Cycadaceae plants includes the step of detecting whether the sex chromosome of the Cycadaceae plant to be tested is linked to the molecular marker; or, the step of performing PCR amplification on the Cycadaceae plant to be tested using the primers described above, and determining whether the PCR amplification product contains the molecular marker.

[0030] In some embodiments, the Cycadaceae plants to be tested include any one of the following: Cycas debaoensis, Cycas panzhihuaensis, Cycas sexseminifera, Cycas segmentifida, and Cycas szechuanensis.

[0031] To better illustrate the technical solution of the present invention, further explanations and descriptions are provided below through specific embodiments.

[0032] Example

[0033] The method for sexing plants in the Cycadaceae family includes the following steps:

[0034] 1. Steps for extracting genomic DNA from leaves of Cycadaceae plants of different sexes

[0035] Genomic DNA was extracted from leaves of 10 male and female plants from five Cycadaceae species (C. debaoensis, C. panzhihuaensis, C. sexseminifera, C. segmentifida, and C. szechuanensis) using the cetyltrimethylammonium bromide (CTAB) method. The specific extraction method for each plant is as follows:

[0036] (1) Weigh 1.0g of fresh leaves, cut them into pieces and put them into a mortar. Grind them with liquid nitrogen and then add 3mL of 1.5×CTAB. Grind them into a homogenate and transfer it into a 15mL centrifuge tube. Then add 1mL of 1.5×CTAB to the mortar to rinse and transfer it into the centrifuge tube. Mix well and then heat in a 65℃ water bath for 30min, shaking slowly from time to time.

[0037] The formulation for 1.5×CTAB is as follows (1L):

[0038] CTAB 15g; 1mol / L Tris hydrochloric acid (pH=8.0) 75mL; 0.5mol / L EDTA 30mL; NaCl 61.4g;

[0039] After placing the above components in a volumetric flask, add deionized water to bring the volume to 1L. Before use, add mercaptoethanol to a final concentration of 0.2% (2mL).

[0040] (2) After cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1), mix gently until the lower layer turns dark green.

[0041] (3) Centrifuge at 10,000 rpm for 10 min, transfer the upper aqueous phase to a 15 mL centrifuge tube, add 2 volumes of pre-cooled anhydrous ethanol, mix and let stand for 5 min. Then place at -20℃ for 30 min to allow DNA precipitation.

[0042] (4) Centrifuge at 10,000 rpm for 10 min, discard the supernatant, add 1 mL of 75% ethanol, wash the precipitate once, invert the centrifuge tube to dry the DNA, and add 200 μL of TE buffer to dissolve the DNA.

[0043] (5) Genomic DNA was detected using 0.8% agarose gel.

[0044] (6) Store the genomic DNA of each individual at -20°C for later use.

[0045] 2. Preparation steps of molecular markers

[0046] Using the extracted leaves of cycads of different sexes as templates, PCR amplification was performed using primer pairs (including SEQ ID NO.2 and SEQ ID NO.3).

[0047] The PCR reaction system is as follows:

[0048] 20.2 μL of sterile water;

[0049] 10×buffer (containing Mg) 2+ 2.5 μL;

[0050] dNTPs (25mM) 0.15μL;

[0051] Taq enzyme (5 U / μL) 0.15 μL;

[0052] 0.5 μL of forward primer;

[0053] 0.5 μL of reverse primer;

[0054] Template 1.0 μL;

[0055] Total volume 25 μL;

[0056] The PCR reaction procedure is as follows:

[0057] Pre-denaturation at 94℃ for 5 minutes; denaturation at 94℃ for 30 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 40 seconds, run for 35 cycles; final extension at 72℃ for 3 minutes. PCR amplification products can be stored at 4℃.

[0058] Molecular markers were obtained through the above amplification process, and the amplified products were purified. Sequencing was then performed after purification, and the results are shown in SEQ ID NO.1.

[0059] The PCR products were subjected to agarose gel electrophoresis, yielding partial results of amplification of 10 male and female individuals from 5 cycad species using molecular marker primer pairs (including primer 1, SEQ ID NO:2, and primer 2 SEQ ID NO:3). Figure 1 As shown.

[0060] from Figure 1It can be seen that lanes 1 and 2 are PCR amplification products of *Cycas debaoensis*; lanes 3 and 4 are PCR amplification products of *Cycas panzhihuaensis*; lanes 5 and 6 are PCR amplification products of *Cycas shishanensis*; lanes 7 and 8 are PCR amplification products of *Cycas repens*; and lanes 9 and 10 are PCR amplification products of *Cycas xianhuensis*. Lanes 1, 3, 5, 7, and 9 show the PCR amplification products of individual male plants from the five Cycadaceae families, while lanes 2, 4, 6, 8, and 10 show the PCR amplification products of individual female plants from the five Cycadaceae families. Lane M is the DNA ladder, with molecular weights including 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp. Figure 1 It can be seen that the amplification products of male Cycadaceae plants are around 750bp and 500bp, while the amplification products of female Cycadaceae plants are only around 500bp.

[0061] In summary, the identification method of this invention can quickly detect the sex of any Cycadaceae plant, eliminating the need to determine sex based on whether the plant is flowering, thus effectively improving the efficiency of garden research and management.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention. sequence list <110> Shenzhen Fairy Lake Botanical Garden (Shenzhen Landscape Architecture Research Center) Shenzhen BGI Life Science Research Institute <120> Molecular markers, primer pairs and their applications, and methods for sex identification in Cycadaceae plants. <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 739 <212> DNA <213> Artificial Sequence <400> 1 aagtgacatt ctccaagcgt agaggagggc tcctgaagaa ggcgcacgaa ctctccgtgt 60 tatgtgacgc agaggtcgct ctgaataat tttcaagcac cggaaagctc ttcgagtatg 120 caagctcgag tagcaggtta gtaacggttt ggatcagcct ataatgtgag actgtttcta 180 ctttctggcc ctcatatcgt gttttcttt ttcttctccc tccgaggaca cgcacatata 240 ttttacatgg cttctactca ttaaactgaa attcaagcct atctgttatg tcgaaaagca 300 taaagagaat tctagagaga tatcagaggg tctccggagt acgactttgg gattatgagc 360 atcaagtaag acaaatcttg tgcatttcgc cttttattgt cgatgattta ccgtagtcat 420 tgtttaggtg ggtaccgtaa tgcacaaact aatttgggt cgtgggggtt gctaattggc 480 caaacagcag ttgttttgtg agatgacaag attgaagaat gagaatgaaa aacttcaaaa 540 tgctttgagg tattataact gtattatttt tcaaaaattt cgatgattct attgttggtg 600 tatttgataa aaataaaaag agcaatctac catcgaatat attgtaggca tatgacgggg 660 gaggacctca attcgctgtc cacgaacgag ctgcatcatc tagagcaaaa tcttgaaatt 720 tcagcaactc gtgttcgaa 739 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cacactctat acagatcagc 20 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 gcatatcctc ttggagtgtt c 21

Claims

1. The application of a primer pair for detecting molecular markers in the preparation of a sex identification reagent for Cycadaceae plants, characterized in that, The molecular marker is linked to the Y chromosome of Cycadaceae plants; The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and the Cycadaceae plant is any one of Cycas debaoensis, Cycas panzhihuaensis, Cycas shishanensis, Cycas cypriniformis, and Cycas xianhuensis. The primer pair includes primer 1 and primer 2, the nucleotide sequence of primer 1 is shown in SEQ ID NO.2, and the nucleotide sequence of primer 2 is shown in SEQ ID NO.

3.

2. A method for identifying the sex of Cycadaceae plants, characterized in that, include: The primer pair described in claim 1 was used to perform PCR amplification on the Cycadaceae plant to be tested. The amplification product size of the male Cycadaceae plant to be tested was around 750bp and 500bp, and the amplification product size of the female Cycadaceae plant to be tested was around 500bp. The Cycadaceae plants to be tested are any one of Cycas debaoensis, Cycas panzhihuaensis, Cycas shishanensis, Cycas cypriniformis, and Cycas xianhuensis.

3. The method for identifying the sex of Cycadaceae plants as described in claim 2, characterized in that, The Cycadaceae plants to be tested include Cycadaceae plants of any age.

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