SNP molecular markers at the major QTL locus for cold tolerance at bud stage in peanut and its application

Abstract

The present invention discloses a SNP molecular marker of a major QTL site for cold tolerance at the bud stage of peanut and its application, belonging to the field of molecular marker technology. The present invention locates a major QTL site for cold tolerance at the bud stage in peanut. The site is located between base positions 155637831 and 155854093 on chromosome 19 of the peanut genome, with a physical distance of about 216 kb, and has a regulatory effect on the cold tolerance of peanut at the bud stage. On this basis, the present invention develops a SNP molecular marker that is tightly linked to the major QTL site for cold tolerance at the bud stage of peanut, and designs a corresponding primer set based on the SNP molecular marker. By using the primer set designed by the present invention, the SNP molecular marker that is tightly linked to the major QTL site for cold tolerance at the bud stage of peanut can be cloned, thereby providing a reliable genetic method for identifying and breeding cold-resistant peanut varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular markers, and in particular relates to SNP molecular markers of a major QTL site of cold tolerance in the bud stage of peanuts and applications thereof. Background Art

[0002] Low temperature stress is a significant adverse factor limiting the growth, development, and yield of peanuts. Chilling damage has varying degrees of adverse impact on agricultural production, significantly limiting the scope of crop cultivation while also causing yield reductions, quality degradation, and even complete crop failure. Global crop losses from chilling damage amount to hundreds of billions of dollars annually. Peanut (Arachis hypogaea L.) is an important oilseed and cash crop worldwide. Peanut cold tolerance is a quantitative trait controlled by multiple genes, with a highly complex genetic regulatory mechanism. With the development and integration of molecular marker technology and quantitative genetics, complex quantitative traits can now be decomposed into individual quantitative trait loci (QTLs), allowing the multiple genes controlling these traits to be studied similarly to qualitative traits. Progress has been made in mapping QTLs for cold tolerance in rice, both domestically and internationally. For example, Yan Changjie et al., using DH populations of both indica and japonica rice, discovered a major QTL for cold tolerance at the bud stage located on chromosome 7; Chen Wei et al., using RIL populations, identified four QTLs for cold tolerance at the bud stage in rice; Qiao Yongli et al., using F2 segregating populations, identified three QTLs related to cold tolerance at the bud stage; and Zhang Luxia et al., using RIL populations constructed from a hybrid of indica and japonica subspecies, identified three QTLs for cold tolerance at the bud stage. With the continuous development and improvement of high-throughput, low-cost sequencing technologies, peanut genomics research has made rapid progress, facilitating the discovery and mapping of QTLs for several peanut traits. However, there are currently few reports on QTLs and molecular markers for cold tolerance in peanuts. Summary of the Invention

[0003] This study mapped a major QTL locus, qRGRB09, controlling cold tolerance at the bud stage in peanut. This locus is located between bases 155637831 and 155854093 on chromosome 19 of the peanut genome, with a physical distance of approximately 216 kb. It explains 10.85% to 24.07% of phenotypic variation and has a regulatory effect on cold tolerance at the bud stage. Based on the foregoing findings, the present invention proposes the following technical solutions:

[0004] The present invention provides SNP molecular markers tightly linked to the above-mentioned main effect QTL site qRGRB09 for cold tolerance at the bud stage of peanut. The SNP molecular markers and the main effect QTL site qRGRB09 for cold tolerance at the bud stage of peanut are co-located on chromosome 19 of the peanut genome, including G22096 and G22097, both of which are co-dominant molecular markers; their nucleic acid sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0005] The present invention also provides a primer set for amplifying the above-mentioned SNP molecular marker; wherein, the primer set for amplifying SEQ ID NO: 7 is shown as SEQ ID NOs: 11 to 13; the primer set for amplifying SEQ ID NO: 8 is shown as SEQ ID NOs: 14 to 16.

[0006] The present invention provides a detection kit comprising any one of the above primer sets, and further comprising other PCR amplification reagents, such as ddH2O, magnesium ions, DNA polymerase, etc.

[0007] The present invention provides the use of the aforementioned SNP molecular markers and / or primer sets and / or detection kits for identifying cold tolerance in peanuts. The present invention also provides the use of the aforementioned SNP molecular markers and / or primer sets and / or detection kits for breeding cold-tolerant peanut varieties or lines. The present invention also provides the use of the aforementioned SNP molecular markers and / or primer sets and / or detection kits for peanut molecular breeding, cultivating transgenic peanuts, or improving peanut germplasm resources.

[0008] The present invention provides a method for identifying cold resistance of peanuts, comprising the following steps:

[0009] The genome of the peanut material to be tested is amplified using any of the above primer sets, and the amplified fragment is detected; if the amplified fragment is consistent with the SNP molecular marker corresponding to the primer set, the peanut material to be tested is identified as a cold-tolerant peanut; if no amplified fragment appears or the amplified fragment is inconsistent with the SNP molecular marker corresponding to the primer set, the peanut material to be tested is identified as a non-cold-tolerant peanut.

[0010] In the above identification method, amplification can be performed using a conventional PCR procedure, and detection of the amplified fragment can be performed using conventional methods such as gel electrophoresis or gene sequencing.

[0011] The present invention provides the application of the major effect QTL locus qRGRB09 in regulating the cold tolerance of peanut.

[0012] The beneficial effects of the present invention are:

[0013] The present invention has, for the first time, located a major QTL locus, qRGRB09, controlling cold tolerance at the bud stage in peanut. This locus is located between base positions 155637831 and 155854093 on chromosome 19 of the peanut genome, with a physical distance of approximately 216 kb. It explains 10.85% to 24.07% of phenotypic variation and has a regulatory effect on cold tolerance at the bud stage in peanut. Based on this, the present invention has developed a single-nucleotide polymorphism (SNP) molecular marker tightly linked to the major QTL locus for cold tolerance at the bud stage in peanut. A corresponding primer set was designed based on the SNP molecular marker. The primer set designed by the present invention enables the cloning of SNP molecular markers tightly linked to the major QTL locus for cold tolerance at the bud stage in peanut, thus providing a reliable genetic method for identifying and breeding cold-resistant peanut varieties. Furthermore, the SNP molecular marker can be used to accurately predict cold tolerance at the bud stage in peanut breeding materials and enable marker-assisted selection in early generations, unaffected by environmental factors, significantly improving breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Statistics for genetic map information;

[0015] Figure 2 This is the genetic map result diagram;

[0016] Figure 3 This is the preliminary mapping of the major QTL for peanut cold tolerance;

[0017] Figure 4 This is the fine mapping map of the major QTL for peanut cold tolerance;

[0018] Figure 5 This is the amplification result of the primer set for SNP molecular marker G22097. DETAILED DESCRIPTION

[0019] The present invention uses the peanut varieties Huayu 44 and DF12, which have significant differences in cold resistance during the bud stage, as research materials; among them, Huayu 44 is a cold-resistant variety cultivated by the Shandong Peanut Research Institute, and DF12 is a cold-sensitive variety introduced by the Hainan Tropical Ocean University; the above two varieties are both stored in the seed bank of the Economic Crops Research Institute of Shanxi Agricultural University.

[0020] The steps for locating the major QTL locus and screening its linked SNP molecular markers are as follows:

[0021] 1. Construction and trait determination of peanut cold-tolerant recombinant inbred line (RIL) population

[0022] (1) RIL group construction

[0023] Huayu 44 and DF12 were crossed to obtain F0 seeds. False hybrids were removed, and recombinant inbred line (RIL) populations were constructed using the single-seed descent method to obtain high-generation RIL populations. These RIL populations and their parents were planted in five locations: Ledong in 2019, Fenyang in 2020, Ledong and Nanbin, and Fenyang in 2021.

[0024] (2) Identification of cold-resistance phenotype

[0025] The germination rate was used to characterize the cold resistance of the budding period. 20 seeds of the parent and RIL population with uniform size, intact seed coat and plump seed kernel were selected, and the seeds were soaked for 2 minutes with carbendazim (100-fold dilution of 80% WP wettable powder), rinsed 3 times with sterilized pure water, placed in a sterilized culture dish (two layers of filter paper were placed at the bottom), and then soaked for 8 hours at room temperature with sterilized pure water. Then, the seeds were placed in a 12°C artificial climate box (BIC-300, relative humidity 70%). After dark cultivation for 72 hours, the climate box was adjusted to 2°C low temperature stress for cultivation for 72 hours, and then placed in a 25°C climate box for constant temperature germination. The seeds were soaked for 8 hours at room temperature and then germinated at 25°C for 72 hours as the control. The results were repeated 3 times, and the average value was used as the statistical unit. The phenotypic data of the cold resistance of the RIL population and the parent during the budding period in each planting environment were obtained.

[0026] 2. Genomic library construction

[0027] (1) DNA extraction

[0028] The optimized CTAB method was used to extract genomic DNA from the leaves of Huayu 44, DF12 and RIL population materials (200 families were randomly selected). The DNA quality was detected by 1% agarose gel electrophoresis, and its concentration was determined by UV spectrophotometer. The DNA concentration was uniformly adjusted to 100 ng / L and stored at -20℃ for future use.

[0029] (2) Construction of genomic library

[0030] Qualified DNA samples were randomly fragmented into 350-bp fragments using a Covaris shredder. The DNA fragments were then subjected to end-repair, PloyA tailing, sequencing adapter addition, purification, and PCR amplification to complete library preparation. The constructed library was sequenced using an Illumina Nova 6000 in PE150 mode.

[0031] (3) Whole genome resequencing

[0032] After the genomic library was constructed, preliminary quantification was performed using Qubit 2.0, diluting the genomic library to 1 ng / μL. The insert size of the genomic library was then tested using an Agilent 2100. Once the insert size met expectations, the effective concentration of the genomic library was accurately quantified using Q-PCR (the effective concentration of the genomic library was greater than 2 nM) to ensure the quality of the genomic library. If the library passed the test, high-throughput sequencing (performed by Hangzhou Lianchuan Biotechnology Co., Ltd.) was performed based on the effective concentration of the genomic library and data output requirements. Genotypic data for each family and parent in the RIL population were obtained.

[0033] 3. Mapping of the main QTL loci for cold tolerance at the budding stage of peanut

[0034] The offline data were quality-controlled, and low-quality sequences and adapter sequences were removed to generate CleanData. The CleanData data were aligned to a reference genome (the Arachis hypogaea Tifrunner.gnm2 genome, available for download at: https: / / www.peanutbase.org / data / public / Arachis_hypogaea / Tifrunner.gnm2.J5K5 / ). SNPs (single nucleotide polymorphisms) and indels (insertions and deletions) were detected using GATK software, and the detected variant sites were quality-filtered. After obtaining polymorphic SNP markers, binning and bin marker filtering were performed, followed by genotyping and coding, and a genetic map was constructed using Joinmap. The BC1 population genotype data were input into IciMapping software to construct a genetic map.

[0035] Genetic map information: contains a total of 2494 Bin marker loci, distributed in 20 linkage groups, covering a genetic map distance of 1216.317 cM, such as Figure 1 As shown. According to the linkage group map size, the map is drawn, as shown Figure 2 shown.

[0036] The genotype, genetic map, and phenotypic data of cold tolerance at the bud stage of the RIL population were input into the R / qtl software, and the composite interval mapping (CIM) method was used for QTL mapping. A stable major effect QTL was located on the B09 linkage group, as shown in Figure 3This major QTL was detected in all five environments (Ledong in 2019, Fenyang in 2020, Ledong and Nanbin, and Fenyang in 2021), explaining 20.57%, 10.85%, 15.99%, 12.43%, and 24.07% of the phenotypic variation, respectively. This major QTL locus was named qRGRB09.

[0037] 4. Identify SNP molecular markers that are closely linked to the major QTL loci

[0038] Based on the major QTL locus for cold tolerance at the bud stage of peanut identified above, 10 SNP molecular markers (numbered from G22090 to G22099) were developed within and flanking the major QTL locus. Their nucleic acid sequences are shown below:

[0039] G22090:

[0040]

[0041] G22091:

[0042]

[0043] G22092:

[0044]

[0045]

[0046] G22093:

[0047]

[0048] G22094:

[0049]

[0050] G22095:

[0051]

[0052] G22096:

[0053]

[0054] G22097:

[0055]

[0056] G22098:

[0057]

[0058]

[0059] G22099:

[0060]

[0061] The sequence set forth in SEQ ID NO:1 has a T / C polymorphism at base 101, the sequences set forth in SEQ ID NOs:2 to 5 all have a G / A polymorphism at base 101, the sequence set forth in SEQ ID NO:6 has a C / G polymorphism at base 101, the sequence set forth in SEQ ID NO:7 has a G / A polymorphism at base 101, the sequence set forth in SEQ ID NO:8 has a C / T polymorphism at base 101, the sequence set forth in SEQ ID NO:9 has a C / A polymorphism at base 101, and the sequence set forth in SEQ ID NO:10 has a C / T polymorphism at base 101. In the sequence listing, polymorphic sites are indicated by N.

[0062] Using 200 family materials from the RIL population that were not involved in resequencing as the test objects, KASP high-throughput genotyping was used to conduct QTL localization in the local interval, combined with the cold-tolerant phenotype of the tested materials. Ultimately, the main effect QTL locus was narrowed down to between SNP molecular markers G22096 and G22097. This main effect QTL locus is located between base positions 155637831 and 155854093 on chromosome 19 of the peanut genome, with a physical distance of approximately 216 kb. Its fine mapping data are shown in Table 1 and Figure 4 As shown:

[0063] Table 1

[0064]

[0065] The present invention has developed a related primer set based on the above-mentioned SNP molecular marker G22096 and SNP molecular marker G22097, as shown below:

[0066] (1) Primer set for amplifying G22096

[0067] G22096-F1: 5'-GAAGGTCGGAGTCAACGGATTGGCGACGGTGAGATGCATTA-3' (SEQ ID NO: 11)

[0068] G22096-F2: 5'-GAAGGTGACCAAGTTCATGCTGCGACGGTGAGATGCATTG-3' (SEQ ID NO: 12)

[0069] G22096-R:5'-ACTCCCCTTTTCTATCGCACC-3'(SEQ ID NO:13)

[0070] (2) Primer set for amplifying G22097

[0071] G22097-F1:

[0072] 5'-GAAGGTCGGAGTCAACGGATTTTTACATATAGGTTATTATCTCATAAGACATTA-3'(SEQ IDNO:14)

[0073] G22097-F2:

[0074] 5'-GAAGGTGACCAAGTTCATGCTTTACATATAGGTTATTATCTCATAAGACATTG-3'(SEQ IDNO:15)

[0075] G22097-R:5'-GTCTATAACAATACCTTGGAAGAACAT-3'(SEQ ID NO:16)

[0076] The primer set can clone the corresponding SNP molecular marker. Therefore, in practical applications, the primer set can be used to identify whether a peanut variety contains the corresponding SNP molecular marker, thereby determining whether the peanut variety contains the major QTL locus and whether it is cold-resistant.

[0077] Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. Below, the present invention will be further described in detail with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0078] Example 1

[0079] The primer set of SNP molecular marker G22097 was used to amplify the following peanut varieties:

[0080] Qinshui peanuts, Licheng local peanuts, Jishan small vines, Houma large grains, Wuxiang colored grains, Yushe peanuts, Yuncheng large vine peanuts, Hongdong peanuts 4, Hongzhao large grains -1, Daning Yibazhua, Linfen Yiwofeng, Xieyu large grains, Xiaoyi peanuts, Jixian large grain vines, Yuncheng peanuts, Pinglu large grains, Qixian peanuts, Fenxi small grains, Xiangfen oil peanuts, Changzi peanuts, Quwo Yiwofeng, Yongji Cadilong, Linxian multi-grain peanuts and Yuci peanuts.

[0081] 1. Reaction product detection

[0082] The PCR products were detected by 8% native polyacrylamide gel electrophoresis (PAGE).

[0083] Clean the glass plate, gasket and ear plate with clean water, especially make sure there is no residual glue on the glass plate and ear plate, and then fix them in place with clips in turn;

[0084] Use 5 mL (about the amount of one glass plate) of non-denatured polyacrylamide solution for bottom sealing. Add 1% volume of 20% ammonium persulfate (50 μL) and 1‰ volume of TEMED (5 μL) to the solution and mix well to accelerate the solidification of the non-denatured polyacrylamide solution.

[0085] The proportions of 8% or 10% non-denaturing polyacrylamide solution (40 mL per gel) are shown in Table 2:

[0086] Table 2

[0087] Components Volume (8%) 40% polyacrylamide (Acr:Bis=39:1) 8mL 10xTBE 10mL <![CDATA[H2O]]> 28mL total 40mL

[0088] Before pouring the glue, check whether the bottom glue is solidified. Take 40mL of 8% non-denatured polyacrylamide solution, add 1% volume of 20% ammonium persulfate (400μL) and 1‰ volume of TEMED (40μL), mix well, and pour into the sealed glass plate. Do not pour too fast during the glue pouring process to avoid the formation of bubbles. After the glue is filled, insert the comb to a depth of about 1 / 3-1 / 2 of the comb teeth;

[0089] After the gel solidifies, rinse with tap water. Remove the comb and continue rinsing to avoid residual gel. Install the glass plate into the electrophoresis tank and secure it with a clamp. Add 1xTBE electrophoresis buffer to the electrophoresis tank:

[0090] 10xTBE ​​recipe:

[0091] Table 3

[0092] Reagents Dosage Tris 540g Boric acid 275g <![CDATA[EDTA-2Na]]> 37.22g <![CDATA[ddH2O]]> 5L

[0093] In the small groove of 8% non-denaturing polyacrylamide gel plate, use a pipette to draw 2μL of PCR amplification product and spot it. After adding each sample, replace a pipette tip to prevent contamination. When adding samples, do not damage the gel surface around the sample well.

[0094] After turning on the power, adjust the voltage output and adjust the voltage according to the experimental needs. Generally, 120V constant voltage electrophoresis is performed for about 4 hours.

[0095] 2. Rapid silver staining

[0096] Remove the polyacrylamide gel from the glass plate and place it in 500 mL of 0.1% silver nitrate solution. Shake gently on a shaker for 10 to 15 minutes (the amplitude should not be too large).

[0097] Pour away the silver nitrate solution and rinse once with deionized water;

[0098] Add developer (10 g NaOH, 0.2-0.4 g Na2CO3, 800 μL formaldehyde to 500 mL deionized water and shake to mix), and place on a shaker and shake gently for 10-15 minutes;

[0099] When clear DNA bands were observed, the developing solution was discarded, the cells were rinsed with tap water, photographed, and the band type statistics were performed (silver staining and development of the gel were both performed in a fume hood).

[0100] The electrophoresis results are as follows Figure 5 As shown:

[0101] Qinshui peanut, Licheng local peanut, Jishan small vine, Houma large grain, Wuxiang colored grain, Yushe peanut, Yuncheng large vine peanut, Hongdong peanut 4, Hongzhao large grain-1, Daning Yibazhua, Qixian peanut, Linfen Yiwofeng, Xieyu large grain, Xiaoyi peanut, Jixian large grain seedling, Yuncheng peanut and Pinglu large grain had no obvious bands. In contrast, Fenxi small grain, Xiangfen oil peanut, Changzi peanut, Quwo Yiwofeng, Yongji Padilong, Linxian multi-grain and Yuci peanut had relatively clear bands between 100bp and 250bp, indicating that these seven varieties are cold-resistant varieties, which is basically consistent with the results of the previous laboratory cold-resistant phenotypic identification [see reference: Bai Dongmei, et al. Identification of cold-resistant budding and SSR genetic diversity of Shanxi peanut local varieties. Acta Agronomica Sinica, 2018(10):1459-1467.], indicating that the G22097 primer set can effectively identify cold-resistant budding peanuts.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A SNP molecular marker tightly linked to the main QTL locus for cold tolerance at the bud stage of peanut, characterized by: The nucleic acid sequence of the SNP molecular marker is selected from any one or both of SEQ ID NO: 7 and SEQ ID NO: 8; Among them, the main effect QTL site is located between base positions 155637831 and 155854093 on chromosome 19 of the peanut genome, with a physical distance of 216kb, which can explain 10.85% to 24.07% of the phenotypic variation.

2. The primer set for amplifying the SNP molecular marker according to claim 1, characterized in that: The primer set for amplifying SEQ ID NO: 7 is shown in SEQ ID NOs: 11 to 13; the primer set for amplifying SEQ ID NO: 8 is shown in SEQ ID NOs: 14 to 16.

3. A detection kit, characterized in that Contains the primer set according to claim 2.

4. Use of the primer set according to claim 2 and / or the detection kit according to claim 3 in identifying cold resistance of peanut.

5. Use of the primer set according to claim 2 and / or the detection kit according to claim 3 in breeding cold-resistant peanut varieties or lines.

6. A method for identifying cold resistance of peanuts, characterized in that: Here are the steps: Amplifying the genome of the peanut material to be tested using any primer set according to claim 2, and detecting the amplified fragment; if the amplified fragment is consistent with the SNP molecular marker corresponding to the primer set, the peanut material to be tested is identified as a cold-resistant peanut; If no amplified fragment appears or the amplified fragment is inconsistent with the SNP molecular marker corresponding to the primer set, the peanut material to be tested is identified as a cold-intolerant peanut.

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