SNP molecular markers closely linked to the major QTL of spinach bolting and their applications

Abstract

This invention relates to the field of molecular genetics, specifically to a SNP molecular marker closely linked to a major QTL related to bolting in spinach and its application. The SNP molecular marker provided by this invention is KMBL29, with its polymorphic site located at position 47693313 on spinach chromosome 1, exhibiting an A / G polymorphism. A genotype of AA corresponds to bolting resistance in spinach; genotypes of AG or GG correspond to bolting susceptibility. This invention also provides specific primers based on KASP technology for genotyping this SNP molecular marker. This molecular marker and primers can be used to breed high-quality spinach varieties resistant to bolting, shortening the breeding cycle.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to SNP molecular markers closely linked to major QTLs in spinach bolting and their applications. Background Technology

[0002] Spinach (Spinacia oleracea L., 2n=2x=12) belongs to the subgenus Spinacia of the family Amaranthaceae. Native to Iran, it is an annual or biennial herbaceous plant primarily grown for its green leaves. Due to its wide adaptability, good storage life, long supply period, diverse cultivation methods, and relatively comprehensive nutrition, it is widely cultivated around the world.

[0003] Spinach is highly cold-resistant and adaptable, with a short growing season, allowing for multiple cropping within a year. However, its bolting behavior is sensitive to photoperiod, making spring- and summer-planted spinach prone to premature bolting. This reduces overall yield, lowers marketability, and hinders year-round production. Bolting is a quantitative trait, meaning its expression is easily influenced by environmental conditions. Chan-Navarrete et al. (2016) first reported a genetic linkage map in spinach containing six linkage groups (P01-P06) and 283 SNP markers. Among them, P01 contains two QTLs and P02 contains one QTL locus that is closely linked to bolting in spinach, which provides a possibility for studying bolting traits (Chan-Navarrete, R., Dolstra, O., van Kaauwen, M., van Bueren, ETL, & van der Linden, CG (2016). Genetic map construction and QTL analysis of nitrogen use efficiency in spinach (Spinacia oleracea L.). Euthytica, 208(3), 621-636.). In the sequencing analysis of 288 spinach germplasm materials from the United States using simplified genome sequencing technology, Chitwood et al. (2016) screened three SNP loci closely related to spinach bolting: AYZV02001321_398, AYZV02041012_1060, and AYZV02118171_95, located on chromosomes 2 and 4, respectively (Chitwood, J., Shi, A., Mou, B., Evans, M., Clark, J., Motes, D., ... & Hensley, D. (2016). Population structure and association analysis of bolting, plant height, and leaf erectness in spinach. Hort Science, 51(5), 481-486.).Xu et al. (2017) assembled the genome of the high-generation spinach inbred line Sp75 and obtained several bolting-related markers and quantitative trait loci (QTLs) in the 44.7-50.5 Mb region of chromosome 2. They screened out two major QTLs closely linked to spinach bolting, one of which contained the gene Spo00403, which was highly homologous to the Arabidopsis flowering control gene AGAMOUS-LIKE (Xu,C.,Jiao,C.,Sun,H.,Cai,X.,Wang,X.,Ge,C.,...&Wang,Q.(2017). Draft genome of spinach andtranscriptome diversity of 120Spinacia accessions.Nat Commun 8,15275.). Bhattarai et al. (2020) used genome-wide association analysis (GWAS) to identify SNPs associated with bolting and flowering on chromosomes 2, 3, and 5 (Bhattarai, G., Shi, A., Correll, JC, & Poudel, B. (2020, August). Identification of Genomic Regions Associated with Bolting and Flowering Time in Spinach. In 2020 ASHS Annual Conference. ASHS.).

[0004] Currently, there is still limited research on bolting traits in spinach, and it is necessary to develop more molecular markers related to bolting traits in spinach to facilitate the development of bolting-resistant spinach varieties. Summary of the Invention

[0005] The purpose of this invention is to provide an SNP molecular marker that is closely linked to the major QTL of spinach bolting, as well as the detection primers and applications of this molecular marker.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides an SNP molecular marker that is closely linked to the major QTL of spinach bolting, namely the molecular marker KMBL29, whose polymorphic site is located at position 47693313 on chromosome 1 of spinach, and the polymorphism is A / G.

[0008] The location information of this marker was determined based on the whole genome sequence of spinach published by Xu et al. (Xu,C.,Jiao,C.,Sun,H.,Cai,X.,Wang,X.,Ge,C.,...&Wang,Q.(2017). Draft genome of spinach and transcriptome diversity of 120Spinacia accessions.Nat Commun 8,15275.http: / / spinachbase.org / ).

[0009] The SNP molecular marker described in this invention contains a nucleotide sequence with a polymorphism of A / G at position 36 as shown in SEQ ID NO.1.

[0010] Specifically, the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the polymorphic site is located at position 36 of the sequence shown in SEQ ID NO.1, with a polymorphism of A / G.

[0011] SEQ ID NO.1:

[0012] 5'-ATTATAAATATGTTTAATCTTTATATACAAAGTTTGAGGTTATATGATCATTATCCTGAGCTGATTATATTGAATTACTC-3'.

[0013] The SNP molecular markers described in this invention can be obtained by amplification using primers with sequences as shown in SEQ ID NO.2 and SEQ ID NO.4, or by amplification using primers with sequences as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] The SNP molecular marker has a genotype of AA, which corresponds to spinach's resistance to bolting; the SNP molecular marker has a genotype of AG or GG, which corresponds to spinach's tendency to bolt.

[0015] In a second aspect, the present invention provides a specific primer set for detecting the above-mentioned SNP molecular markers, comprising a forward primer having a sequence as shown in SEQ ID NO. 2-3 and a reverse primer having a sequence as shown in SEQ ID NO. 4;

[0016] SEQ ID NO.2:

[0017] 5'-ATTATAAATATGTTTAATCTTTATATACAAAGTTTA-3'

[0018] SEQ ID NO.3:

[0019] 5'-ATTATAAATATGTTTAATCTTTATATACAAAGTTTG-3'

[0020] SEQ ID NO.4:

[0021] 5'-GAGTAATTCAATATAATCAGCTCCAGGATAATGAT-3'.

[0022] The primer set provided by this invention contains two forward primers and one reverse primer. The three primers are used together to detect the genotype of the SNP marker using KASP (Kompetitive Allele Specific PCR) technology.

[0023] To facilitate detection using KASP technology, a fluorescent tag sequence is also attached to the 5' end of the forward primer.

[0024] This invention does not impose any special restrictions on the fluorescent tag sequences to be connected, as long as the fluorescent tags corresponding to the two forward primers are different.

[0025] In one embodiment of the present invention, the 5' end of the primer shown in SEQ ID NO.2 is connected to the FAM fluorescent tag (universal tag A) sequence: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.7) to form SEQ ID NO.5, and the 5' end of the primer shown in SEQ ID NO.3 is connected to the HEX fluorescent tag (universal tag B) sequence: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO.8) to form SEQ ID NO.6.

[0026] The present invention also provides a kit containing the above-described specific primer set.

[0027] In addition to the specific primer set described above, the kit may also contain one or more reagents selected from the following: dNTPs, Mg 2+ DNA polymerase, ddH2O, and PCR reaction buffer; these reagents can be packaged individually or as a premixed solution.

[0028] Thirdly, the present invention provides any of the following applications of the above-mentioned SNP molecular markers or specific primer sets or the kits described herein:

[0029] (1) Application in identifying bolting characteristics of spinach;

[0030] (2) Application in molecular marker-assisted breeding of bolting-resistant spinach;

[0031] (3) Application in germplasm resource improvement of bolting trait in spinach.

[0032] In this invention, the application includes: using the DNA of the spinach plant to be tested as a template, performing PCR amplification with primers having sequences as shown in SEQ ID NO. 2-4, and determining the bolting characteristics of the spinach plant to be tested based on the PCR amplification products.

[0033] Preferably, the PCR amplification reaction program is as follows: (1) 94℃, 15 minutes; 1 cycle; (2) 94℃, 20 seconds, 61-55℃, 60 seconds, decreasing by 0.6℃ per cycle; 10 cycles; (3) 94℃, 20 seconds, 55℃, 60 seconds; 26 cycles; After the above reaction program is completed, fluorescence detection analysis is performed. If the typing result is not ideal, the following reaction program is performed again: (4) 94℃, 20 seconds, 57℃, 60 seconds; 3-9 cycles, and fluorescence detection analysis is performed again after the reaction program is completed.

[0034] Preferably, in the PCR amplification reaction system, the molar ratio of primers having the sequences shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 is 1:1:2.

[0035] The PCR reaction system is shown in Table 1 below:

[0036] Table 1

[0037] Components 96-well plate (μl) 384-well plate (μl) Template DNA 4.86 2.43 KASP Master mix 5 2.5 KASP Primer mix 0.14 0.07 Total volume 10 5

[0038] Specifically, the KASP genotyping method provided by this invention has high throughput and is simple to operate. It only requires adding a specific KASP Primer mix and a universal KASP Master mix to a PCR microplate containing a DNA sample for PCR amplification. The final results can be analyzed using a fluorescence detector.

[0039] The KASP Primer mix contains three specific primers: forward primers (SEQ ID NO.5 and SEQ ID NO.6) with universal tags A and B respectively, and a reverse primer (SEQ ID NO.4).

[0040] As a specific implementation method, the above-described application includes the following steps:

[0041] (1) Extract the DNA from the spinach to be tested;

[0042] (2) Dilute the DNA in step (1) to 20 ng / μL, and add specific KASP Primer mix and universal KASP Master mix to it for PCR amplification;

[0043] (3) The PCR products were analyzed by fluorescence detection at temperatures below 40°C.

[0044] The aforementioned specific KASP Primer mix contains SEQ ID NO. 4-6;

[0045] The aforementioned universal KASP Master mix contains the following components: universal TRET cassette fluorescent primers, ROX internal control dye, KlearTaq DNA polymerase, dNTPs, and MgCl2. Fluorescent reporter group A is FAM, and fluorescent reporter group B is HEX.

[0046] In this invention, if the fluorescence signal read is only the fluorescence signal corresponding to the primer with the sequence shown in SEQ ID NO.2, the spinach plant under test is determined to be resistant to bolting; if the fluorescence signal read is only the fluorescence signal corresponding to the primer with the sequence shown in SEQ ID NO.3, or if the fluorescence signal read is the fluorescence signal corresponding to the primer with the sequences shown in SEQ ID NO.2 and SEQ ID NO.3, the spinach plant under test is determined to be prone to bolting.

[0047] The beneficial effects of this invention are as follows: This invention provides an SNP molecular marker, KMBL29, tightly linked to the major QTL for spinach bolting. Using the three primers provided by this invention, bolting characteristics of spinach can be detected at the seedling stage, with high analytical throughput and high accuracy. The co-dominant SNP marker provided by this invention can greatly accelerate the breeding of high-quality, bolting-resistant spinach varieties. Attached Figure Description

[0048] Figure 1 This is the localization result of the major QTL related to bolting in Example 1 of the present invention; wherein, the right side is the name of the molecular marker, and the left side is the size of the genetic map distance, in cM.

[0049] Figure 2 This is the result of detecting the BC1 population using the molecular marker KMBL29 in Example 2 of the present invention.

[0050] Figure 3 This is a statistical graph showing the bolting time and phenotypic distribution of 95 BC1 plants in Example 3. The vertical axis is in days. Detailed Implementation

[0051] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all examples were conducted under conventional experimental conditions. All spinach varieties were provided by the Spinach Research Project of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Spinach inbred lines 12S3 and 12S4 were disclosed in Qian, W., Fan, G., Liu, D., Zhang, H., Wang, X., Wu, J., & Xu, Z. (2017). Construction of a high-density genetic map and the X / Y sex-determining gene mapping in spinach based on large-scale markers developed by specific-locus amplified fragment sequencing (SLAF-seq). BMC Genomics, 18(1), 1-10.

[0052] Example 1: Development of molecular markers tightly linked to major QTLs associated with spinach bolting resistance

[0053] 1. Construction of the genetic map of spinach

[0054] Using spinach inbred line 12S3 as the female and recurrent parent and 12S4 as the male parent, a BC1 population of 147 plants was obtained by hybridization. 12S3 was a bolting-resistant parent and 12S4 was a bolting-prone parent. SLAF-seq was performed on the parents to obtain 4048 SNPs (Qian, W., Fan, G., Liu, D., Zhang, H., Wang, X., Wu, J., & Xu, Z. (2017). Construction of a high-density genetic map and the X / Y sex-determining gene mapping in spinach basedon large-scale markers developed by specific-locus amplified fragment sequencing (SLAF-seq). BMC genomics, 18(1), 1-10.). 300 SNPs were evenly selected from these and designed as KASP primers. 180 of the KASP primers were successfully used for correct typing in the BC1 population. The genotyping results were imported into Joinmap 4.0, revealing 179 closely linked markers, thus constructing a linkage map containing 179 markers. This genetic map contains 6 linkage groups (LGs), with a total length of 583.84 cM and an average length of 3.26 cM.

[0055] 2. Investigation on bolting characteristics of spinach

[0056] All BC1 populations were planted at the experimental base of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The bolting date was defined as the date the flower stalk reached 5 cm in length, and the bolting time was the difference between the bolting date and the sowing date. The bolting time followed a normal distribution in the BC1 population.

[0057] 3. QTL localization and development of molecular markers

[0058] Based on the genetic map constructed from the BC1 population and the bolting time typographic data of the BC1 population, QTL detection was performed using interval mapping (IM) and multiple QTL model (MQM) in MapQTL 6.0 software. The LOD threshold was determined using a 1000-repeated permutation test. Significant QTLs were identified using the LOD threshold at the p<0.05 level. The major QTL for the final bolting time was located on LG3 (chromosome 1). Figure 1The KMBL29 marker is closely linked to this QTL (Table 2), and it was designed as a KASP primer. This primer set consists of forward primer 1 (SEQ ID NO. 2), forward primer 2 (SEQ ID NO. 3), and reverse primer (SEQ ID NO. 4). The two forward primers have allelic variations A / G at their ends, and the reverse primer sequence is selected to ensure the amplified fragment is 60-120 bp. The 5' ends of the forward primers are linked with fluorescent tag sequences. Forward primer 1 has a FAM fluorescent tag sequence 5'-GAAGGTGACCAAGTTCATGCT-3' linked to its 5' end, forming SEQ ID NO. 5, and forward primer 2 has a HEX fluorescent tag sequence 5'-GAAGGTCGGAGTCAACGGATT-3' linked to its 5' end, forming SEQ ID NO. 6.

[0059] The primer sequences mentioned above were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0060] Table 2. QTL mapping results of spinach bolting resistance.

[0061] Properties QTL Closely linked markers Location (cM) LOD value Explained variance (%) Additive effect Bolting time qBT1-1 KMBL29 47693313 10.2964 41.9909 -4.4059

[0062] Example 2: Validation of the molecular marker KMBL29 in a segregating population

[0063] This embodiment verifies the effectiveness of the molecular marker KMBL29 developed in Example 1. The specific method is as follows:

[0064] 1. Material phenotyping and genomic DNA extraction

[0065] Using spinach inbred line 12S3 as the female and recurrent parent, and 12S4 as the male parent, a BC1 population was obtained. In this invention, 95 plants were randomly selected from the BC1 population to investigate their bolting time. Simultaneously, whole-genome DNA was extracted using the CTAB method.

[0066] 2. Dilute DNA

[0067] Dilute the DNA extracted in step 1 to a concentration of 20 ng / μl.

[0068] 3. Preparation of KASP Primer mix

[0069] Take 12 μl (100 μM) of the forward primer (SEQ ID NO.5 and SEQ ID NO.6) and 30 μl (100 μM) of the reverse primer (SEQ ID NO.4), and make up to 100 μl with sterile ultrapure water.

[0070] 4. PCR amplification

[0071] The PCR amplification reaction system is shown in Table 3 below:

[0072] Table 3

[0073] Components 96-well plate (μl) 384-well plate (μl) Template DNA 4.86 2.43 KASP Master mix 5 2.5 KASP Primer mix 0.14 0.07 Total volume 10 5

[0074] At the same time, a blank control (NTC) without template DNA was set up in the reaction system, with one blank control set up for each plate.

[0075] KASP Master mix is ​​a product of LGC, a British company. Its catalog number is KBS-1016-002.

[0076] The PCR reaction conditions are shown in Table 4 below:

[0077] Table 4

[0078]

[0079] If the genotyping results are not ideal after fluorescence detection, the PCR reaction procedure shown in Table 5 below can be performed again.

[0080] Table 5

[0081]

[0082] Fluorescence detection and analysis were performed again after the second PCR reaction.

[0083] 5. Fluorescence scanning of PCR amplification products

[0084] PCR amplification products were scanned using an Applied Biosystems QuantStudio 6Flex scanner. Genotyping was achieved based on the different excitation and emission wavelengths of the two fluorescence sources (FAM and HEX). Samples aggregated on the Y-axis exhibited genotypes with two alleles, designated AG; samples aggregated in the middle showed genotypes linked to the HEX fluorescent tag sequence, designated AA; the black samples displayed in the lower left corner represented NTC. Figure 2 ).

[0085] Of the 95 selected BC1 plants, 53 had the AG genotype, corresponding to bolting times of 47-55 days, which, compared to the median bolting time of 56 days between the two parents, indicates an early bolting phenotype; the remaining 42 plants had the AA genotype, corresponding to bolting times of 58-65 days, which, compared to the median bolting time of 56 days between the two parents, indicates a late bolting phenotype. Figure 3 ).

[0086] Because this BC1 population is a backcross population, no non-recurrent parent genotype GGs were found.

[0087] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences <120> SNP molecular markers closely linked to the major QTL of spinach bolting and their applications <130> KHP211118170.2 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 80 <212> DNA <213> Artificial Sequence <400> 1 attataaata tgtttaatct ttatatacaa agtttgaggt tatatgatca ttatcctgag 60 ctgattatat tgaattactc 80 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <400> 2 attataaata tgtttaatct ttatatacaa agttta 36 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <400> 3 attataaata tgtttaatct ttatatacaa agtttg 36 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <400> 4 gagtaattca atataatcag ctcaggataa tgat 34 <210> 5 <211> 57 <212> DNA <213> Artificial Sequence <400> 5 gaaggtgacc aagttcatgc tattataaat atgtttaatc tttatataca aagttta 57 <210> 6 <211> 57 <212> DNA <213> Artificial Sequence <400> 6 gaaggtcgga gtcaacggat tattataaat atgtttaatc tttatataca aagtttg 57 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 gaaggtgacc aagttcatgc t 21 <210> 8 <211> twenty one <212> DNA <213> Artificial Sequence <400> 8 gaaggtcgga gtcaacggat t 21

Claims

1. An SNP molecular marker tightly linked to a major QTL for spinach bolting, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, and the polymorphic site is located at position 36 of the sequence shown in SEQ ID NO.1, with a polymorphism of A / G.

2. The SNP molecular marker according to claim 1, characterized in that, The SNP molecular marker has a genotype of AA, which corresponds to spinach's resistance to bolting; the SNP molecular marker has a genotype of AG or GG, which corresponds to spinach's tendency to bolt.

3. A specific primer set for detecting the SNP molecular marker as described in claim 1 or 2, characterized in that, It contains a forward primer having the sequence shown in SEQ ID NO.2-3 and a reverse primer having the sequence shown in SEQ ID NO.

4.

4. The specific primer set according to claim 3, characterized in that, The 5' end of the forward primer is also connected to a fluorescent tag sequence.

5. A kit containing the specific primer set as described in claim 3 or 4.

6. Any of the following applications of the specific primer set of claim 3 or 4 or the kit of claim 5: (1) Application in identifying bolting characteristics of spinach; (2) Application in molecular marker-assisted breeding of bolting-resistant spinach; (3) Application in germplasm resource improvement of bolting trait in spinach.

7. The application according to claim 6, characterized in that, The application includes: using the DNA of the spinach plant to be tested as a template, performing PCR amplification with primers having the sequence shown in SEQ ID NO.2-4, and determining the bolting characteristics of the spinach plant to be tested based on the PCR amplification product.

8. The application according to claim 7, characterized in that, The PCR amplification reaction procedure is as follows: (1) 94℃, 15 minutes; 1 cycle; (2) 94℃, 20 seconds, 61-55℃, 60 seconds, decreasing by 0.6℃ per cycle; 10 cycles; (3) 94℃, 20 seconds, 55℃, 60 seconds; 26 cycles; After the above reaction procedure is completed, fluorescence detection analysis is performed. If the typing result is not ideal, the following reaction procedure is performed again: (4) 94℃, 20 seconds, 57℃, 60 seconds; 3-9 cycles, and fluorescence detection analysis is performed again after the procedure is completed.

9. The application according to claim 8, characterized in that, In the PCR amplification reaction system, the molar ratio of primers having the sequences shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 is 1:1:

2.

10. The application according to any one of claims 7-9, characterized in that, If the fluorescence signal read is only the fluorescence signal corresponding to the primer with the sequence shown in SEQ ID NO.2, then the spinach plant under test is determined to be resistant to bolting; if the fluorescence signal read is only the fluorescence signal corresponding to the primer with the sequence shown in SEQ ID NO.3, or if the fluorescence signal read is the fluorescence signal corresponding to the primer with the sequences shown in SEQ ID NO.2 and SEQ ID NO.3, then the spinach plant under test is determined to be prone to bolting.

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