A set of ILP molecular marker primers for tea trees in Yunnan camellia and their applications

Abstract

This invention discloses a set of molecular marker primers for intron length polymorphism (ILP) in tea trees, applicable to Yunnan camellia, and their applications, relating to the field of molecular biology. Based on the completed whole-genome sequencing of 'Shuchazao', 'Longjing 43', and 'Ancient Tea Tree DASZ', this set of 25 pairs of highly polymorphic and universal ILP molecular marker primers addresses the extreme scarcity of genetic information resources in Yunnan camellia. The specific nucleotide sequences are shown in SEQ ID NO. 1–50. The ILP molecular marker primers provided by this invention can be used for genetic diversity analysis, population structure analysis, variety identification, and marker-assisted breeding of Yunnan camellia.

Description

Technical Field

[0001] This invention relates to population genetics of Yunnan camellia, specifically to a set of ILP molecular marker primers for tea trees that are universally applicable to Yunnan camellia and their applications. Background Technology

[0002] Camellia reticulata, the Yunnan camellia, is one of the most famous and economically valuable species in the Camellia genus, renowned for its flowers and camellia oil. Native to southwestern China, it possesses distinct regional characteristics, adapting well to the mountainous terrain and unique plateau monsoon climate of the southwest. It plays a vital role in ornamental purposes, the camellia oil industry, and specialty tourism. However, compared to other Camellia species, the genomic information of the Yunnan camellia is scarce, leading to slow progress in population genetics research. The decoding of the tea tree genome and the release of high-quality chromosome-level genomes have provided valuable information resources for conducting population genetics studies of the Yunnan camellia. This invention aims to develop a set of intron length polymorphism (ILP) molecular marker primers based on the tea tree genome information. These primers exhibit high polymorphism and universality in the Yunnan camellia, and can be used to effectively assess the genetic diversity of the Yunnan camellia, providing a scientific basis for Yunnan camellia breeding and other research. Summary of the Invention

[0003] This invention provides a set of ILP molecular marker primers for tea trees that are universally applicable to Yunnan camellia, for use in population genetics studies of Yunnan camellia.

[0004] To achieve the above objectives, the present invention provides a set of ILP molecular marker primers applicable to Yunnan camellia. This set of primers contains 25 pairs of primers, the nucleotide sequences of which are shown in SEQ ID NO.1 to 50, as detailed in Table 1 below.

[0005] Table 1. Sequence information of 25 pairs of ILP molecular marker primers

[0006]

[0007]

[0008]

[0009] Furthermore, the aforementioned Yunnan camellia includes

[0010] Xuejiao (C. reticulata 'Xuejiao'), Zipao (C. reticulata 'Zipao'), Jing'an Tea (C. reticulata 'Jing'ancha'), Zaotaohong (C. reticulata 'Zaotaohong'), Zaomudan (C. reticulata 'Zaomudan'), Fengshan Tea (C. reticulata 'Fengshancha'), Shizitou (C. reticulata 'Shizitou'), Dali Tea (C. reticulata 'Dalicha'), Tongzimi (C. reticulata 'Tongzimi') an'), Pine Cone Scale (C. reticulata 'Songzilin'), Brocade Robe Red (C. reticulata 'Jinpaohong'), Willow Leaf Silver Red (C. reticulata 'Liuyeyinhong'), Hemp Leaf Peach Red (C. reticulata 'Mayetaohong'), Yunnan Western Crane's Crest Red (C. reticulata 'Hedinghong'), Fragrant Concubine (C. reticulata 'Xiangfei'), Agate (C. reticulata 'Manao'), Peony Queen (C. reticulata 'Mudankui'), Golden Powder Beauty (C. reticulata 'ret') C. reticulata 'Jinfenjiao'), Peony Tea (C. reticulata 'Mudancha'), Five-Colored Red Peony (C. reticulata 'Wusechidan'), Lotus Fairy (C. reticulata 'Hehuaxianzi'), Baiyizaotaohong (C. reticulata 'Baiyizaotaohong'), Baozhucha (C. reticulata 'Baozhucha'), Small Osmanthus Leaf (C. reticulata 'Xiaoguiye'), Dahongpao (C. reticulata 'Dahongpao'), Thick-Leaf Disc Wing (C. reticulata ta'Houyediechi'), Great Agate (C. reticulata 'Damanao'), Chrysanthemum Petal (C. reticulata 'Juban'), Hemp Leaf Silver Red (C. reticulata 'Mayeyinhong'), Buddha Seat Lotus (C. reticulata 'Fozuolian'), Large Silver Red (C. reticulata 'DayinHong'), Small Silver Red (C. reticulata 'XiaoyinHong'), Large Peach Red (C. reticulata 'Dataohong'), Brahma Butterfly (C. reticulata 'Fandie'), Deer City Spring (C.reticulata'Luchengchun'), Datoutiao', Lifang', Jiejiegao', Weixihong', Liangtaohong', Yumeigui', Yinfenmudan', Dong Varieties of *C. reticulata* 'Dongling', *C. reticulata* 'Lingyesongzike', *C. reticulata* 'Biyu', *C. reticulata* 'Dahongliujiao', *C. reticulata* 'Duxindiechi', *C. reticulata* 'Manyi', *C. reticulata* 'Ruyi', and *C. reticulata* 'Taohongpao'.

[0011] The tea tree ILP molecular marker primers provided by this invention can be used for any of the following purposes: genetic diversity analysis of Yunnan camellia, body structure analysis of Yunnan camellia, molecular marker-assisted breeding of Yunnan camellia, or identification of Yunnan camellia varieties.

[0012] The present invention also provides a kit for identifying Camellia yunnanensis, which contains the above-mentioned 25 pairs of primers. The kit can also be used for any of the following: genetic diversity analysis of Camellia yunnanensis, body structure analysis of Camellia yunnanensis, molecular marker-assisted breeding of Camellia yunnanensis, or identification of Camellia yunnanensis varieties.

[0013] The ILP molecular marker primers of the present invention have the following advantages:

[0014] The ILP molecular marker primers of this invention are the first to be developed at the whole genome level of tea plants. The primers have high polymorphism and high versatility, providing reliable information resources for genetic diversity analysis, molecular marker-assisted breeding, and variety identification of Yunnan camellia. Attached Figure Description

[0015] Figure 1 Electrophoresis results of partial screening of tea plant ILP molecular marker primers amplified in Camellia yunnanensis.

[0016] Figure 2The results show the amplification of primers Tea_ILP1982, Tea_ILP1986, and Tea_ILP2581 in Yunnan Camellia germplasm resources among 25 pairs of polymorphic primers.

[0017] Figure 3 Phylogenetic tree results obtained from 50 Yunnan camellia varieties after identification using 25 pairs of primers.

[0018] Figure 4 The results of population structure analysis of 50 Yunnan camellia varieties obtained after identification using 25 pairs of primers.

[0019] Figure 5 The results of principal component analysis were obtained after identifying 50 Yunnan camellia varieties using 25 pairs of primers. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Note: Methods not specifically described in this application are conventional techniques in the field, and reagents and consumables not specifically described are conventional reagents and consumables in the field.

[0022] Experimental Example 1: Extraction of Genomic DNA from Camellia yunnanensis

[0023] 1. Select tender leaves of Yunnan camellia;

[0024] To verify the amplification efficiency and polymorphism of tea tree ILP markers in Yunnan camellia, six Yunnan camellia (C. reticulata Lindl.) varieties were randomly selected for DNA extraction. The specific varieties and sample sources are shown in Table 2 below.

[0025] Table 2. Basic Information of 6 Yunnan Camellia Varieties

[0026]

[0027] 2. DNA was extracted from Yunnan mountain tea leaves using the CTAB method:

[0028] (1) Take 0.2g of fresh tender buds and leaves and put them into a mortar. Add 400μL of 2% CTAB extract. After the material is fully ground, add 200μL of 2% CTAB extract. Then, pour the solution obtained after grinding into a 1.5mL test tube.

[0029] (2) Heat the water bath to a constant temperature of 65°C, put the ground extract into the water bath and heat it. During this period, take out the test tube and shake it once every 10 minutes, and repeat the cycle 5 times.

[0030] (3) Then remove the test tube and cool it to room temperature. Add an equal volume of 24:1 (chloroform:isoamyl alcohol) to the extract and invert the tube for 4-5 minutes. After mixing thoroughly, place the test tube in a centrifuge and centrifuge at 10,000 rpm for 10 minutes at room temperature (24-25°C).

[0031] (4) After centrifugation, take 400 μL of supernatant and put it into a new centrifuge tube. Add the same volume of 24:1 (chloroform:isoamyl alcohol) and repeat the above steps to mix by inverting and centrifugation.

[0032] (5) After the second centrifugation is completed, add 2 to 2.5 times the volume of pre-cooled anhydrous ethanol to 300 μL of the supernatant, shake gently until flocculent matter appears, and then place in a refrigerator (-20℃) and let stand for 30 min.

[0033] (6) Take out the test tube and put it into a centrifuge. Centrifuge at 10,000 rpm for 5 minutes at room temperature (24-25℃).

[0034] (7) Discard the supernatant and collect the precipitate. Rinse with 75% alcohol and let it dry.

[0035] (8) Repeat step (7) above 2 to 3 times. Finally, after the alcohol has dried, add 100 μL of RNase enzyme and store it in a refrigerator at -20°C for later use.

[0036] Experimental Example 2: Development of Molecular Marker Primers

[0037] Development of ILP molecular marker primers for tea plants

[0038] 1. Search for introns in tea trees:

[0039] This was achieved using an IPv2.0 program independently developed by the Zu Feng team at the Institute of Economic Crops, Yunnan Academy of Agricultural Sciences (Software Copyright Registration No.: 2021SR1113216). The basic process involves importing 'Shucha Zao' (pcsb.ahau.edu.cn:8080 / CSS / (scaffold-level genome)), 'Longjing 43' (https: / / bigd.big.ac.cn / search / ?dbId=gwh&q=GWHACFB0000000), and 'Ancient Tea Tree DASZ' (http...) into the server. The annotation file for the tea plant genome (link: https: / / figshare.com / articles / journal_contribution / Assembly_and_annotation_of_DASZ_genome / 12560462 / 1) was retrieved, and the maximum length of introns was set to 300 bp. Then, the IPv2.0pl script was run to retrieve the location, length, and sequence information of introns and their genes. Markers that showed a single band in e-PCR and were different on all three reference genomes were selected for synthesis and validation.

[0040] 2. Design of primers for ILP molecular marker amplification in tea plants:

[0041] After successfully uploading the tea plant genome sequence information file, the intron information file obtained in the previous step is used in the IP_p3in.pl script to generate the sequences required for primer design. Finally, the results file generated by the IP_p3out script is used to design tea plant ILP molecular marker primers using Primer 3.0 software. To further narrow down the primer selection range and effectively utilize the ILP molecular marker primers, the designed tea plant ILP molecular marker primers are validated by e-PCR amplification on the whole genome sequence. Finally, the target tea plant ILP molecular marker primers are screened out. The specific method is as follows:

[0042] (1) Based on the genome annotation information of 'Shucha Zao' mentioned above, a total of 229,441 introns were detected in 56,073 genes. 127,360 intron fragments were less than 300 bp, and 3,132 introns were not read. A total of 105,127 pairs of tea plant ILP molecular marker primers were designed, of which 53,016 pairs showed a single band in e-PCR. These 53,016 pairs of tea plant ILP molecular marker primers were compared with 31,975 pairs of primer sequences in the RNA database that showed a single band in e-PCR, resulting in a total of 23,948 pairs of tea plant ILP molecular marker primers. Subsequently, 479 Tea_ILP markers were randomly selected from these 23,948 pairs for e-PCR. Finally, 39 markers showing ≥2 bands in e-PCR were selected.

[0043] (2) Based on the gene annotation information of 'Longjing 43', there are a total of 145,229 introns in 32,666 genes, of which 50,516 intron fragments are less than 300 bp. A total of 48,232 pairs of tea tree ILP molecular marker primers were designed. Using the designed tea tree ILP molecular marker primers, e-PCR analysis was performed on the genomes of 'Longjing 43', 'Yunkang 10' (www.plantkingdomgdb.com / tea_tree / ), and 'Shuchazao' (pcsb.ahau.edu.cn:8080 / CSS / (scaffold-level genome)). A total of 25,469 pairs of tea tree ILP molecular marker primers showing 1, 2, and 3 bands in e-PCR were obtained. Then, 1,393 pairs of Tea_ILP markers showing 1 band in e-PCR and different across the three reference genomes were selected. Finally, 91 pairs of Tea_ILP markers were selected from these 1,393 pairs for synthesis verification.

[0044] (3) Based on the genome annotation information of 'Ancient Tea Tree DASZ', there are a total of 144,668 introns in 26,328 genes. Among them, 56,074 intron fragments are less than 300bp, and 2,547 introns for which no primers were generated. A total of 53,527 pairs of tea tree ILP molecular marker primers were designed. Using the designed markers, e-PCR analysis was performed on the genomes of six chromosome-level genes: 'DASZ', 'Longjing 43', 'Shuchazao' (https: / / github.com / JiedanChen / TeaGenomeData (chromosome-level genome)), 'Biyun' (www.plantkingdomgdb.com / CSS-BY), 'Huangyan' (https: / / ngdc.cncb.ac.cn / search / ?dbId=&q=GWHBAUV00000000), and 'Tieguanyin' (https: / / ngdc.cncb.ac.cn / search / ?dbId=gwh&q=GWHASIV00000000). Primers for tea plant ILP molecular markers, common to all reference genomes, were screened, resulting in a total of 5772 primer pairs. From these, 1342 Tea_ILP marker pairs with differences in base size were then selected. Finally, 100 pairs of Tea_ILP tags will be selected from these 1342 pairs for subsequent experimental verification.

[0045] 3. Amplification of ILP markers in tea trees in Yunnan camellia:

[0046] The 230 pairs of ILP molecular marker primers developed above were synthesized and amplified in Camellia yunnanensis (Table 2 above), yielding 25 pairs of polymorphic ILP molecular marker primers, as shown in Table 1 above. The PCR amplification results of 16 of these marker primer pairs are shown in Table 1 above. Figure 1 As shown. The specific PCR amplification procedure is as follows:

[0047] The reaction mixture consisted of 10 μL of 50 ng / μL DNA and 2 μL of 10×PCR Buffer (Mg2+). 2+ 1 μL, 0.8 μL of 2.5 μM dNTPs, 0.1 μL of 5 U / μL Taq enzyme, 0.2 μL of 10 μM primer, 5.9 μL of ddH2O, and 1-2 drops of paraffin oil.

[0048] The reaction program was as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 30 s, annealing at 65℃ for 45 s, extension at 72℃ for 1 min, for a total of 13 cycles, with a touch-down temperature reduction of 0.7℃ per cycle; then, a large cycle was performed: denaturation at 94℃ for 30 s, annealing at 56℃ for 45 s, extension at 72℃ for 1 min, for a total of 23 cycles; extension at 72℃ for 5 min, and storage at 4℃.

[0049] Experiment Example 3: Application and Verification of ILP Molecular Marker Primers in Tea Plants

[0050] Because tea tree ILP markers have high universality and polymorphism in Yunnan camellia, they can quickly and efficiently detect the genotype of Yunnan camellia varieties, providing support for research on population genetic diversity and parent selection in Yunnan camellia. Given the scarcity of Yunnan camellia genomic information, which has slowed progress in population genetics research, this invention aims to verify the reliability and application value of the 25 pairs of tea tree ILP molecular marker primers developed in this invention for the identification of Yunnan camellia. Fifty local Yunnan camellia varieties from different sources were selected. The samples originated from Kunming City, Yunnan Province, and all 50 varieties are unique to Yunnan, as detailed in Table 3 below.

[0051] Table 3. Names and origins of 50 Yunnan camellia varieties

[0052]

[0053]

[0054] Detecting the genetic diversity of Yunnan Camellia varietal populations using tea tree ILP molecular marker primers can guide the selection of breeding parents for Yunnan Camellia. Among the 25 polymorphic primer pairs screened above, the amplification results of primer pairs Tea_ILP1982, Tea_ILP1986, and Tea_ILP2581 in the aforementioned 50 Yunnan Camellia germplasm resources are as follows: Figure 2 As shown, where Figure 2 In the diagram, A represents the amplification band of the Tea_ILP1982 labeled primer. Figure 2 In the image, B represents the amplification band of the Tea_ILP1986 labeled primer. Figure 2 C in the text represents the amplification band of the Tea_ILP2581 labeled primer.

[0055] Following the experimental methods of Examples 1 and 2, the ILP genotypes of the 50 Yunnan camellia varieties listed in Table 3 were obtained, and then genetic diversity was assessed. Each band of the ILP molecular marker primer amplification product was considered a locus. Bands with clear and high-resolution gel electrophoresis imaging were statistically analyzed using the 0,1 method: bands were marked as "1", and weak bands (no band or difficult to distinguish) were marked as "0", and a 0,1 matrix was constructed. The genetic similarity coefficient and Nei's genetic distance among the varieties were calculated using NTSYSpc-2.1 software. Then, the phylogenetic tree was plotted in MEGA 7.0 using the NJ method. The results are shown below. Figure 3 As shown, the 50 Yunnan camellia varieties mentioned above can be grouped into three major categories. Category I includes five Yunnan camellia varieties: Xuejiao, Fengshancha, Xiangfei, Damanao, and Dahong Liujiao. Category II includes 42 camellia varieties such as Zipao, Zao Mudan, and Manao. These 42 varieties can be further subdivided into two subcategories: Category II-1 includes 31 Yunnan camellia varieties such as Maye Taohong and Liuye Yinhong, while Category II-2 includes 11 Yunnan camellia varieties such as Zao Taohong and Shizitou. Category III has the fewest Yunnan camellia varieties, with only three: Jing'ancha, Lifang, and Liang Taohong.

[0056] The original 0 and 1 type data were analyzed using the population structure analysis software Structure v2.3.4. The K value was set to 1–10, and the optimal K value was determined using the online website Structure Harvester. The MCMC (markov chain montecarlo) was set to 10,000. A population structure diagram was generated. (See [link to documentation]). Figure 4 Finally, principal component analysis was performed on the Yunnan camellia population using the "vegan" package in R, and a two-dimensional plot of the principal component analysis was drawn using the "ggplot2" package. The results are as follows: Figure 5 As shown in the figure. Among them, the phylogenetic tree is used to describe the branching tree of the differentiation order of variety populations and represent the evolutionary relationship between populations; population structure and principal component analysis are used to analyze the genetic structure of the population. Figure 3 , Figure 4 Camellias of Yunnan with the same petal type can be clustered together, especially Camellias of Yunnan with double petals, which have a high degree of genetic similarity and are more closely related when clustered. Figure 5The principal component analysis demonstrates the reliability of the results, showing that the 50 Yunnan Camellia varieties are highly similar and genetically closely related. The phylogenetic tree, population structure, and principal component analysis revealing the kinship among the Yunnan Camellia variety populations will provide a scientific reference for parent selection in the next stage of breeding new Yunnan Camellia varieties. In summary, the tea plant ILP molecular marker primers provided in this application are reliable and effective in the application of Yunnan Camellia, and can be used for genetic diversity analysis, population structure analysis, variety identification, and molecular marker-assisted breeding of Yunnan Camellia.

[0057] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A set of ILP molecular marker primers for tea plants commonly used in Yunnan camellia, characterized in that, This primer set contains 25 primer pairs, with nucleotide sequences as shown in SEQ ID NO. 1~2, 3~4, 5~6, 7~8, 9~10, 11~12, 13~14, 15~16, 17~18, 19~20, 21~22, 23~24, 25~26, 27~28, 29~30, 31~32, 33~34, 35~36, 37~38, 39~40, 41~42, 43~44, 45~46, 47~48, and 49~50.

2. The molecular marker primer according to claim 1, characterized in that, The Yunnan camellia mentioned above includes the following: Xuejiao (C. reticulata 'Xuejiao'), Zipao (C. reticulata 'Zipao'), Jing'an Tea (C. reticulata 'Jing'ancha'), Zaotaohong (C. reticulata 'Zaotaohong'), Zaomudan (C. reticulata 'Zaomudan'), Fengshan Tea (C. reticulata 'Fengshancha'), Shizitou (C. reticulata 'Shizitou'), Dali Tea (C. reticulata 'Dalicha'), Tongzimian (C. reticulata 'Tongzimian'), Songzilin (C. reticulata 'Songzilin'), Jinpaohong (C. reticulata 'Jinpaohong'), Liuyeyinhong (C. reticulata 'Liuyeyinhong'), Mayetaohong (C. reticulata 'Mayetaohong'), Dianxi Hedinghong (C. reticulata 'Hedinghong'), Xiangfei (C. reticulata 'Xiangfei'), Agate (C. reticulata 'Manao'), Mudankui (C. reticulata 'Mudankui'), Jinfenjiao (C. reticulata 'Jinfenjiao'), Mudancha (C. reticulata 'Mudancha'), Wuse Chidan (C. reticulata 'Wusechidan'), Hehuaxianzi (C. reticulata 'Hehuaxianzi'), Baiyizaotaohong (C. reticulata 'Baiyizaotaohong'), Baozhucha (C. reticulata 'Baozhucha'), Xiaoguiye (C. reticulata 'Xiaoguiye'), Dahongpao (C. reticulata 'Dahongpao'), Houyediechi (C. reticulata 'Houyediechi'), Damanao (C. reticulata 'Houyediechi') 'Damanao'), Chrysanthemum Petal (C. reticulata 'Juban'), Hemp Leaf Silver Red (C. reticulata 'Mayeyinhong'), Buddha Seat Lotus (C. reticulata 'Fozuolian'), Large Silver Red (C. reticulata 'DayinHong'), Small Silver Red (C. reticulata 'XiaoyinHong'), Large Peach Red (C.C. reticulata 'Dataohong', C. reticulata 'Fandie', C. reticulata 'Luchengchun', C. reticulata 'Datoutiao', C. reticulata 'Lifang', C. reticulata 'Jiejiegao', C. reticulata 'Weixihong', C. reticulata 'Liangtaohong', C. reticulata 'Yumeigui', C. reticulata 'Yinfenmudan', C. reticulata 'Dongling', C. reticulata 'Lingyesongzike', C. reticulata 'Biyu', C. reticulata 'Biyu', C. reticulata 'Dahongliujiao'), 'Duxindiechi' (C. reticulata 'Duxindiechi'), 'Manyi' (C. reticulata 'Manyi'), 'Ruyi' (C. reticulata 'Ruyi'), 'Taohongpao' (C. reticulata 'Taohongpao').

3. The application of the molecular marker primer as described in claim 1, characterized in that, The application is selected from any of the following: 1) Genetic diversity analysis of Camellia sinensis in Yunnan; 2) Analysis of the population structure of Camellia yunnanensis in Yunnan; 3) Molecular marker-assisted breeding of Camellia yunnanensis; or 4) Identification of Yunnan camellia varieties.

4. A reagent kit for identifying Yunnan camellia, characterized in that, This kit contains the molecular marker primers as described in claim 1.

5. The application of the reagent kit as described in claim 4, characterized in that, The application is selected from any of the following: 1) Genetic diversity analysis of Camellia sinensis in Yunnan; 2) Analysis of the population structure of Camellia yunnanensis in Yunnan; 3) Molecular marker-assisted breeding of Camellia yunnanensis; or 4) Identification of Yunnan camellia varieties.

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