Molecular marker primers associated with drought stress in tea trees and their applications

Abstract

This invention discloses molecular marker primers associated with drought stress in tea plants and their applications, belonging to the field of molecular marker technology. The molecular marker primer sets of this invention consist of SSR molecular marker primer pairs and ILP molecular marker primer pairs; the SSR molecular marker primer pairs are at least one pair of the five primer pairs shown in SEQ ID NO.1-SEQ ID NO.10; the ILP molecular marker primer pairs are at least one pair of the five primer pairs shown in SEQ ID NO.11-SEQ ID NO.20. This invention associates SSR and ILP molecular marker primers of tea plants with drought stress in tea plants, which is beneficial for molecular-assisted breeding, enhancing the stress resistance of tea plants, and preserving genetic resources.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to molecular marker primers related to drought stress in tea plants and their applications. Background Technology

[0002] The tea plant (Camellia sinensis) is an important global natural resource and a healthy, non-alcoholic beverage plant (Wang et al., 2013). Numerous studies have shown that tea leaves are rich in various active ingredients (polyphenols, amino acids, caffeine, etc.) and possess strong biopharmacological activities, such as antioxidant, anti-tumor, anti-radiation, weight loss, and anti-cardiovascular disease effects (Wei et al., 2007; Qiao et al., 2011). Currently, tea is increasingly popular as a healthy beverage.

[0003] Tea trees thrive in shade and tolerate moisture, but are extremely sensitive to water availability. China's tea-growing regions have complex terrains, including plains, hills, mountains, and plateaus. Rainfall is unevenly distributed, with most rainfall occurring in summer, while autumn and winter bring less rain and frequent droughts. With the effects of global warming, spring rainfall is also decreasing, leading to longer and more severe droughts in tea gardens, significantly impacting spring tea production and drastically reducing the economic benefits of tea cultivation. Therefore, research on the drought resistance of tea trees has received increasing attention from scholars in recent years.

[0004] Drought stress is a significant limiting factor affecting the growth and development of tea trees, as well as the yield and quality of tea. Under drought stress, the root system, photosynthesis, respiration, relative water content, membrane integrity, pigment content, osmotic potential, carbohydrate status, antioxidant metabolism, nutrient content, and plant hormones of tea trees are all affected, as are the transcriptional levels of regulatory and functional genes.

[0005] Currently, the main approach to drought-resistant tea breeding is still to introduce and screen drought-resistant gene resources and create new drought-resistant varieties using traditional breeding research methods. Therefore, drought resistance evaluation is crucial for screening and breeding new drought-resistant germplasm. However, on the one hand, because tea is a perennial woody plant, directly evaluating its drought resistance is too time-consuming and costly; on the other hand, there is a lack of molecular markers closely linked to the yield and quality traits of tea under drought stress. Further in-depth research is needed to utilize molecular markers to assist in the breeding of drought-resistant tea varieties. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a set of molecular marker primers related to drought stress in tea trees and their applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a molecular marker primer pair consisting of an SSR molecular marker primer pair and an ILP molecular marker primer pair;

[0009] The SSR molecular marker primer pair is at least one of the five primer pairs shown in SEQ ID NO.1-SEQ ID NO.10;

[0010] The ILP molecular marker primer pair is at least one of the five primer pairs shown in SEQ ID NO.11-SEQ ID NO.20.

[0011] Preferably, the molecular marker primer pair consists of the SSR molecular marker primer pair shown in SEQ ID NO.1-SEQ ID NO.2 and the ILP molecular marker primer pair shown in SEQ ID NO.11-SEQ ID NO.12.

[0012] A second aspect of the present invention provides the application of the above-mentioned molecular marker primer combination in the evaluation of drought resistance in tea plants.

[0013] A third aspect of the present invention provides the application of the above-mentioned molecular marker primer combination in the cultivation of tea varieties with improved drought resistance.

[0014] In a fourth aspect, the present invention provides a kit for identifying the drought resistance of tea trees, wherein the kit uses the above-mentioned molecular marker primer combination as the active ingredient.

[0015] The beneficial effects of this invention are:

[0016] This invention associates SSR and ILP molecular marker primers of tea plants with drought stress, which is beneficial for molecular-assisted breeding, enhancing the stress resistance of tea plants, and preserving genetic resources. The drought stress-related molecular marker combinations of this invention can be applied to the identification of drought-resistant tea plants in some arid and low-rainfall areas in northern China. Because tea plants are susceptible to drought stress under natural environmental conditions, which adversely affects their growth and development, the molecular markers developed using this invention can accelerate the breeding of drought-resistant tea varieties, improve the drought resistance of tea plants, and lay a solid scientific foundation for the genetic breeding of tea plants. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, 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 application pertains.

[0018] As mentioned earlier, since tea trees are perennial woody plants, directly evaluating their drought resistance takes too long and is too costly. On the other hand, there is a lack of molecular markers that are closely linked to the yield and quality traits of tea trees under drought stress. Further in-depth research is needed to use molecular markers to assist in the breeding of drought-resistant tea varieties.

[0019] Based on this, this invention combines SSR and ILP molecular marker primers of tea trees with the abiotic stress resistance of tea trees to develop and design a set of molecular marker primers related to drought stress in tea trees, which can be used for evaluating the drought resistance of tea trees and breeding drought-resistant tea tree varieties. This invention is thus proposed.

[0020] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0021] Example 1: Design of molecular marker primers associated with drought stress in tea trees

[0022] 1. SSR locus and intron locus scanning

[0023] Download the genome sequence and annotation information of the tea variety "Camellia sinensis" from the public data platform (http: / / tpia.teaplant.org / download.html). The version number of the "Camellia sinensis" genome sequence is: CSS_ChrLev_20200506.gff3.gz TPIA.

[0024] The SSR and intron sites in the genome sequence are scanned to obtain the corresponding site information. It is generally believed that the minimum length of an SSR site is 12 bp, that is, a single nucleotide repeat unit has at least 12 repeats, a dinucleotide repeat unit has at least 6 repeats, and a trinucleotide repeat unit has at least 4 repeats.

[0025] The corresponding site information we obtained through scanning is as follows:

[0026]

[0027] 2. Primer design

[0028] Based on the site information obtained in the previous step, the Perl program was used to extract DNA sequences of 60 bp length on both sides of the site as primer precursors.

[0029] The information we obtained regarding the primer precursor is as follows:

[0030]

[0031]

[0032]

[0033] Primers are designed using eprimer3 software. The designed primers exist in pairs. The length of the first primer is about 20 bp, usually 15 bp-25 bp, and the length of the second primer is also about 20 bp, usually 15 bp-25 bp.

[0034] The information for the primers we designed is as follows:

[0035]

[0036]

[0037]

[0038] 3. Initial validation of ePCR

[0039] Based on the downloaded tea plant genome sequence, electronic simulated PCR was performed on the designed primers using ePCR software on the Linux platform. If a pair of primers amplified a band in the downloaded electronic simulated PCR of the tea plant variety, then the pair of primers is a molecular marker.

[0040] USSR1 1 136 3684805 3684940 CSS0045397.1 USSR2 1 124 5919147 5919270 CSS0041720.1 USSR3 1 125 11081875 11081999 CSS0037063.1 USSR4 1 132 157378909 157379040 CSS0010970.1 USSR5 1 124 209570622 209570745 CSS0041358.1 USSR6 1 115 8018265 8018379 CSS0007179.1 USSR7 1 129 21623781 21623909 CSS0043642.1 USSR8 1 128 31913791 31913918 CSS0048341.1 USSR9 1 124 42157970 42158093 CSS0044171.1 USSR10 1 132 45549303 45549434 CSS0004337.1 USSR11 1 152 162646322 162646473 CSS0033345.1 UILP1 1 617 2833650 2834266 CSS0028077.1 UILP2 1 212 73528454 73528665 CSS0001894.1 UILP3 1 5098 67660044 67665141 CSS0010965.1 UILP4 1 1285 4548561 4549845 CSS0037513.1 UILP5 1 192 14412422 14412613 CSS0002779.1 UILP6 1 219 12609589 12609807 CSS0004378.1 UILP7 1 217 146051340 146051556 CSS0019515.1 UILP8 1 2128 100538760 100540887 CSS0009675.2 UILP9 1 304 199292499 199292802 CSS0007696.1 UILP10 1 650 209365789 209366438 CSS0000679.1 UILP11 1 5933 221855975 221861907 CSS0048682.1

[0041] After ePCR validation, we obtained a total of 11 pairs of SSR molecular marker primers and 11 pairs of ILP molecular marker primers.

[0042] 4. Screening for differentially expressed genes involved in drought stress in tea trees

[0043] Data on drought stress in tea plants were downloaded from the NCBI public data platform (project number PRJNA545401). Using software such as hisat2, stringtie, and Python on the Linux platform, the expression levels of genes involved in drought stress in tea plants were obtained. Differentially expressed genes were screened using R packages, resulting in 6501 genes upregulated and 7287 genes downregulated under drought stress from a total of 37687 genes. Based on the differentially expressed genes, gene names were obtained, and then the sequences of all gene names were obtained using TBtools software based on the gene names and the genome file.

[0044] 5. ePCR re-validation

[0045] Based on the sequences of differentially expressed genes involved in drought stress in tea trees obtained in step 4, the SSR and ILP molecular marker primers obtained in step 3 are used for ePCR verification in the sequences of differentially expressed genes involved in drought stress in tea trees. If a pair of primers amplifies a band in electronic simulated PCR, then the pair of primers is the molecular marker primer related to drought stress.

[0046] Finally, a total of 5 pairs of SSR molecular marker primers and 5 pairs of ILP molecular marker primers related to drought stress in tea trees were screened. Among them:

[0047] The primer sequences for the USSR4 molecular marker are shown in SEQ ID NO.1 and SEQ ID NO.2; the primer sequences for the USSR5 molecular marker are shown in SEQ ID NO.3 and SEQ ID NO.4; the primer sequences for the USSR6 molecular marker are shown in SEQ ID NO.5 and SEQ ID NO.6; the primer sequences for the USSR10 molecular marker are shown in SEQ ID NO.7 and SEQ ID NO.8; and the primer sequences for the USSR11 molecular marker are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0048] The primer sequences for the U ILP2 molecular marker are shown in SEQ ID NO.11 and SEQ ID NO.12; the primer sequences for the U ILP3 molecular marker are shown in SEQ ID NO.13 and SEQ ID NO.14; the primer sequences for the U ILP5 molecular marker are shown in SEQ ID NO.15 and SEQ ID NO.16; the primer sequences for the U ILP7 molecular marker are shown in SEQ ID NO.17 and SEQ ID NO.18; and the primer sequences for the U ILP8 molecular marker are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0049] The specific information for these 10 pairs of molecular marker primers is as follows:

[0050]

[0051] The gene names corresponding to these 10 pairs of molecular marker primers are as follows:

[0052]

[0053] Experimental example:

[0054] The five pairs of SSR molecular marker primers and five pairs of ILP molecular marker primers related to drought stress in tea trees obtained in Example 1 can be used to identify the drought resistance of tea varieties. The specific identification method is as follows:

[0055] Tender leaves from the seedling stage of tea plant varieties were collected, and DNA was extracted. Using the extracted DNA as a template, one pair of SSR molecular marker primers and one pair of ILP1 molecular marker primers were randomly selected from five pairs to form molecular marker primer combinations, and amplification was performed separately.

[0056] If both the SSR molecular marker primers and the ILP1 molecular marker primers produce amplification bands, the drought resistance of the corresponding tea variety is identified as "strong"; if only one pair of primers produces amplification bands, the drought resistance of the corresponding tea variety is identified as "moderate"; if no amplification bands appear, the drought resistance of the corresponding tea germplasm resource is identified as "weak".

[0057] Referring to the method in the literature "Study on drought resistance identification of tea varieties (lines) in Qianmei" (Guizhou Agricultural Sciences, 2003, 31(1): 12-13), the product of water retention capacity and drought resistance coefficient of biological yield was used as an indicator to measure the drought resistance of tea varieties. Among them:

[0058] Water retention capacity = 1 - (fresh weight - 24h water loss weight) / (fresh weight - dry weight); The test method is as follows: at 8:00 am, 8 mature leaves of the experimental tea tree variety are cut intact, surface moisture and dust are wiped off and weighed, and the leaves are naturally dehydrated in a dark incubator at 24℃ and 63% relative humidity. After 24 hours, the leaves are weighed again, and then the leaves are fixed at 105℃ for 30 minutes and dried at 105℃ to constant weight.

[0059] The drought tolerance coefficient of biological yield = 1 - (non-drought 100-bud weight measurement value - drought-treated 100-bud weight measurement value) / non-drought 100-bud weight measurement value.

[0060] The tea germplasm collected from Rizhao Shenggushan Tea Plantation was also divided into three levels according to this index based on drought resistance: a product number ≥ 0.5 indicates strong drought resistance; 0.3 ≤ product number < 0.5 indicates moderate drought resistance; and a product number < 0.3 indicates weak drought resistance.

[0061] Five pairs of SSR molecular marker primers and five pairs of ILP molecular marker primers related to drought stress in tea trees, obtained from Example 1, were paired and combined to identify the drought resistance of the aforementioned tea germplasm. The results were compared with the drought resistance results measured by the product of water retention capacity and biological yield, and the consistency between the two measurement methods was calculated. The results are as follows:

[0062]

[0063]

[0064] The above results show that the identification results of drought resistance of tea trees using the SSR molecular marker primers and ILP molecular marker primers combination of the present invention are basically consistent with the drought resistance identification results in the prior art, indicating that the molecular marker primer combination of the present invention has practical application value; moreover, different SSR molecular marker primers and ILP molecular marker primer combinations have certain differences in the consistency rate with the identification results of existing methods, with the USSR4+UILP2 combination showing the best effect.

[0065] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A molecular marker primer combination, characterized in that, The molecular marker primer combination is any one of the following (1)-(9): (1) The molecular marker primer pair USSR4+UILP2 is composed of a USSR4 molecular marker primer pair and a UILP2 molecular marker primer pair; the primer sequences of the USSR4 molecular marker primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer sequences of the UILP2 molecular marker primer pair are shown in SEQ ID NO.11 and SEQ ID NO.12; (2) The molecular marker primer pair USSR5+UILP3 is composed of a USSR5 molecular marker primer pair and a UILP3 molecular marker primer pair; the primer sequences of the USSR5 molecular marker primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, and the primer sequences of the UILP3 molecular marker primer pair are shown in SEQ ID NO.13 and SEQ ID NO.14; (3) The molecular marker primer pair USSR6+UILP5 is composed of a USSR6 molecular marker primer pair and a UILP5 molecular marker primer pair; the primer sequences of the USSR6 molecular marker primer pair are shown in SEQ ID NO.5 and SEQ ID NO.6, and the primer sequences of the UILP5 molecular marker primer pair are shown in SEQ ID NO.15 and SEQ ID NO.16; (4) Molecular marker primer pair USSR10+UILP7, consisting of USSR10 molecular marker primer pair and UILP7 molecular marker primer pair; the primer sequences of the USSR10 molecular marker primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8, and the primer sequences of the UILP7 molecular marker primer pair are shown in SEQ ID NO.17 and SEQ ID NO.18; (5) Molecular marker primer pair USSR11+UILP8, consisting of USSR11 molecular marker primer pair and UILP8 molecular marker primer pair; the primer sequences of the USSR11 molecular marker primer pair are shown in SEQ ID NO.9 and SEQ ID NO.10, and the primer sequences of the UILP8 molecular marker primer pair are shown in SEQ ID NO.19 and SEQ ID NO.20; (6) Molecular marker primer pair USSR4+UILP3, consisting of USSR4 molecular marker primer pair and UILP3 molecular marker primer pair; the primer sequences of the USSR4 molecular marker primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer sequences of the UILP3 molecular marker primer pair are shown in SEQ ID NO.13 and SEQ ID NO.14; (7) Molecular marker primer pair USSR4+UILP5, consisting of USSR4 molecular marker primer pair and UILP5 molecular marker primer pair; the primer sequences of the USSR4 molecular marker primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer sequences of the UILP5 molecular marker primer pair are shown in SEQ ID NO.15 and SEQ ID NO.16; (8) Molecular marker primer pair USSR4+UILP7, consisting of USSR4 molecular marker primer pair and UILP7 molecular marker primer pair; the primer sequences of the USSR4 molecular marker primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer sequences of the UILP7 molecular marker primer pair are shown in SEQ ID NO.17 and SEQ ID NO.18; (9) Molecular marker primer pair USSR4+UILP8, consisting of USSR4 molecular marker primer pair and UILP8 molecular marker primer pair; the primer sequences of the USSR4 molecular marker primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer sequences of the UILP8 molecular marker primer pair are shown in SEQ ID NO.19 and SEQ ID NO.

20.

2. The application of the molecular marker primer combination according to claim 1 in the evaluation of drought resistance in tea trees, characterized in that, If both SSR and ILP molecular marker primers produce amplification bands, the drought resistance of the corresponding tea variety is identified as "strong"; if only one pair of primers produces amplification bands, the drought resistance of the corresponding tea variety is identified as "moderate". If no amplification band appears, the drought resistance of the corresponding tea germplasm resource is identified as "weak".

3. The application of the molecular marker primer combination according to claim 1 in cultivating tea varieties with improved drought resistance, characterized in that, If both SSR and ILP molecular marker primers produce amplification bands, the drought resistance of the corresponding tea variety is identified as "strong"; if only one pair of primers produces amplification bands, the drought resistance of the corresponding tea variety is identified as "moderate". If no amplification band appears, the drought resistance of the corresponding tea germplasm resource is identified as "weak".

4. A reagent kit for identifying the drought resistance of tea trees, characterized in that, The kit uses the molecular marker primer combination described in claim 1 as the active ingredient.

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