SNP molecular markers associated with soybean canopy coverage and flowering period and applications

Abstract

The present application relates to the field of biotechnology, and particularly relates to a SNP molecular marker associated with soybean canopy coverage and flowering period and application. The present application provides a SNP molecular marker associated with soybean canopy coverage and flowering period, wherein the SNP molecular marker contains a nucleotide sequence with polymorphism of T and C at position 342 of the sequence shown in SEQ ID NO. 1. The SNP molecular marker found by the present application has very important guiding significance for molecular identification and assisted breeding of soybean.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a SNP molecular marker associated with soybean canopy coverage and flowering period and its application. Background Technology

[0002] Soybean is a globally important dual-purpose crop for both food and oil, and a major source of high-quality protein for humans and forage. Temporal genetic analysis of canopy cover can lay the foundation for determining the ideal soybean plant architecture. The ideal high-yielding soybean requires rapid leaf area expansion in the early stages, a short peak period, and a slow decline in the later stages (Gai Junyi et al., 1990), i.e., "early canopy closure and late senescence." Furthermore, as a short-day crop, soybean requires strict photoperiodic conditions, resulting in a narrow range of adaptability and strong regionality, which limits the introduction, breeding, and large-scale cultivation of soybean varieties. Temporal genetic analysis of canopy cover and flowering time throughout the entire growth cycle is expected to provide a theoretical basis for breeding ideal plant architecture and widely adaptable high-yielding soybeans.

[0003] Due to the inefficiency of manual phenotyping, there are few reports on the genetic analysis of temporal canopy cover. In recent years, with the maturity of unmanned aerial vehicle (UAV) field phenotyping platforms, progress has been made in the study of temporal soybean canopy cover. Xavier et al. (2017) used this technology to discover 19 QTLs for canopy cover. In contrast, the gene loci related to soybean flowering and growth stages have been studied more extensively in soybean, with 12 genes having been functionally validated: E1 and E2 (Bernard, 1971), E3 (Buzzell, 1971), E4 (Buzzell and Volden, 1980), E5 (Dissanayaka et al., 2016), E6 (Bonato and Vello, 1999), E7 (Cober and Voldeng, 2001), E8 (Cober et al., 2010), E9 (Kong et al., 2014), J (Ray et al., 1995), PRR3b (Li et al., 2020), and PRR3a (Lu et al., 2020).

[0004] With the development and use of molecular markers linking or associated with QTLs of important yield traits, marker-assisted selection has become one of the important methods in rapid breeding (Cregan et al., 1999). The greatest advantage of marker-assisted selection is that it can identify plants with the target phenotype by confirming whether they carry the target genotype without having to assess phenotypic traits.

[0005] If a single SNP locus can be identified that is associated with both soybean canopy coverage and flowering time, it could provide strong technical support for soybean molecular identification and assisted breeding. Summary of the Invention

[0006] This invention provides SNP molecular markers and applications associated with soybean canopy coverage and flowering time, enabling rapid detection of soybean canopy coverage and flowering time, and providing strong technical support for soybean in molecular identification and assisted breeding.

[0007] This invention provides an SNP molecular marker associated with soybean canopy coverage and flowering time, wherein the SNP molecular marker contains a nucleotide sequence with polymorphisms of T and C at position 342 of the sequence shown in SEQ ID NO.1.

[0008] According to the present invention, a SNP molecular marker associated with soybean canopy coverage and flowering time is provided. The genotype of the site with the polymorphism is TT, which corresponds to a phenotype with relatively low canopy coverage and early flowering time; the genotype of the site with the polymorphism is CC, which corresponds to a phenotype with relatively high canopy coverage and late flowering time.

[0009] This invention provides primers for amplifying the above-mentioned SNP molecular markers.

[0010] Preferably, the primers used to amplify the above-mentioned SNP molecular markers are KASP primer combinations.

[0011] More preferably, the KASP primer combination used to amplify the above-mentioned SNP molecular markers includes the following primers: allele forward primer 1 as shown in SEQ ID NO.2: tcccgattcaccacagttgtaA; allele forward primer 2 as shown in SEQ ID NO.3: tcccgattcaccacagttgtaG; and universal reverse primer as shown in SEQ ID NO.4: gagggcagcaatggtaggagg.

[0012] Preferably, allele forward primer 1 as shown in SEQ ID NO.2 and allele forward primer 2 as shown in SEQ ID NO.3 contain different fluorescent groups.

[0013] Further preferred embodiments include allele forward primer 1 shown in SEQ ID NO.2, which contains the fluorescent group FAM, and allele forward primer 2 shown in SEQ ID NO.3, which contains the fluorescent group HEX.

[0014] The present invention provides a kit comprising the primers described above.

[0015] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in identifying soybean canopy coverage phenotypes.

[0016] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in identifying soybean flowering phenotypes.

[0017] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in the identification, improvement, or targeted breeding of soybean germplasm resources. For example, by crossing and backcrossing southern soybean materials carrying superior alleles with other materials (northern or international varieties), soybean materials carrying superior alleles can be created.

[0018] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in soybean molecular marker-assisted breeding and whole-genome selection breeding.

[0019] This invention provides the application of the above-mentioned SNP molecular markers, primers, and kits in screening and / or cultivating soybeans with high canopy coverage and late flowering.

[0020] According to the above-described application of the present invention, the soybean can be any soybean variety. The molecular markers, primers, and reagent kits described in this invention are universal and can be applied to any soybean variety grown in any environment.

[0021] This invention provides a method for identifying soybean canopy coverage and / or flowering phenotype, the method comprising the following steps:

[0022] 1) Detect the genotypes of the polymorphic sites of the above-mentioned SNP molecular markers in the sample to be tested;

[0023] 2) Determine the canopy coverage and flowering period of the sample to be tested based on the genotype obtained in step 1);

[0024] The specific determination method is as follows: if the obtained genotype is TT, then the soybean to be tested is a candidate soybean with relatively low canopy coverage and early flowering period; if the obtained genotype is CC, then the soybean to be tested is a candidate soybean with relatively high canopy coverage and late flowering period.

[0025] The beneficial effects of this invention are:

[0026] (1) This invention, through genome-wide association analysis of cultivated soybean varieties in my country, found that the SNP locus Chr08-11602352 is significantly associated with canopy coverage and flowering time throughout the entire growth period. The SNP locus discovered in this invention has very important guiding significance for the molecular identification and assisted breeding of soybeans.

[0027] (2) The method for detecting soybean canopy coverage and flowering period described in this invention is simple, fast and accurate.

[0028] (3) Further statistical analysis has shown that the primers provided by this invention can specifically detect the genotype at the Chr08-11602352 locus in soybeans, and can also serve as a new genetic resource for marker-assisted selection in breeding at different growth stages, which has very important positive significance. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0030] Example 1: Mining of SNP sites

[0031] Download the Glyma.08G150600 gene sequence from Phytozome (http: / / www.phytozome.net). The SNP site Chr08-11602352 is located at position 342 of the Glyma.08G150600 gene nucleotide sequence (SEQ ID NO.1). Its basic information is shown in Table 1.

[0032] Table 1. Basic information on single nucleotide polymorphism sites of Chr08-11602352

[0033]

[0034] This invention, through genome-wide association analysis of soybean cultivars in my country, identified a single SNP locus that is significantly associated with soybean canopy coverage and flowering time. The SNP locus is Chr08:11602352 in the genome version Glycine maxWm82.a2.v1 (Soybean, http: / / www.phytozome.net). The alleles at this locus are T and C, with two main genotypes: TT and CC.

[0035] The SNP site and its flanking sequences at both ends are shown in SEQ ID NO.1.

[0036] SEQ ID NO.1:

[0037] >Glyma.08g150600position=Gm08:11602011..11602587(+strand)

[0038]

[0039] Note: [T / C] represents SNP "Chr08:11602352". The SNP polymorphism site in SEQ ID NO.1 of the sequence listing is T, and its actual polymorphism is T / C.

[0040] Example 2: Application of SNP sites in identifying or assisting in the identification of soybean canopy coverage and flowering time.

[0041] 1. The Chr08-11602352 locus genotype was significantly associated with canopy coverage and soybean flowering time.

[0042] (1) Identifying the canopy coverage and flowering period of soybean varieties

[0043] To verify the reliability of SNP loci, flowering time and canopy coverage were investigated for 528 soybean local varieties in southern China whose genetic relationships were clustered. The investigation locations and traits were: Hefei Base of Anhui Academy of Agricultural Sciences (116.5645°E 33.26584°N) (HF) (flowering time in 2018 and 2019) and Nanchang Base of Jiangxi Academy of Agricultural Sciences (NC) (115.8935°E 28.6896°N) (flowering time and canopy coverage in 2020). Row length was 1.5 meters (two rows), plant spacing was 0.1 meters, and row spacing was 0.55 meters. Local standard management practices were adopted to ensure normal growth and maturity of the experimental materials. Field phenotypic identification was conducted according to the "Soybean Germplasm Resource Description Specifications and Data Standards" to determine the flowering period (Qiu et al., 2006, flowering period is when 50% of the plants begin to flower). Soybean canopy coverage was determined at 61, 63, 65, 68, and 70 days after sowing using images acquired by a DJI Phantom 4 multispectral drone. Canopy coverage was extracted using the formula (EGI, (2G-RB) / G) (Meyer and Neto, 2008), where EGI was set to 0.05. Immature materials and those with excessively low emergence rates were treated as deletions.

[0044] (2) The genotype of the above 528 soybean samples was detected using the genotype identification method provided in Example 1. The results are shown in Table 2.

[0045] Table 2. Genotypes of soybean Chr08-11602352 locus and their correlation with flowering time and canopy coverage in 528 soybean accessions.

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Note: Two traits: FT and CC represent flowering time and canopy coverage, respectively; DAS represents days after sowing. HF and NC represent Hefei and Nanchang, respectively. NA indicates missing information.

[0067] The soybean varieties mentioned in Table 2 are all recorded in the following references: Wang Guoxun. Catalogue of Chinese Soybean Variety Resources. Beijing: China Agriculture Press, 1982; Chang Ruzhen and Sun Jianying. Catalogue of Chinese Soybean Variety Resources: Continued Volume 1. Beijing: Agriculture Press, 1991; Chang Ruzhen, Sun Jianying, Qiu Lijuan and Chen Yiwu. Catalogue of Chinese Soybean Variety Resources: Continued Volume 2. Beijing: China Agriculture Press, 1996.

[0068] The genotypes of the Chr08-11602352 locus in 528 materials were detected using whole-genome resequencing, including 416 TT genotypes and 112 CC genotypes.

[0069] In the Hefei environment, in 2018, the TT genotype had 316 flowering phenotypes with a flowering time of (17–63) days and an average flowering time of (36.76±8.1) days; the CC genotype had 56 flowering phenotypes with a flowering time of (28–64) days and an average flowering time of (42.43±9.3) days. In 2019, the TT genotype had 329 flowering phenotypes with a flowering time of (12–64) days and an average flowering time of (36.38±7.2) days; the CC genotype had 64 flowering phenotypes with a flowering time of (27–61) days and an average flowering time of (42.38±8.9) days. Wilcoxon Rank Sum Test (WLS) showed that the flowering time of the TT genotype material in 2018 was significantly earlier than that of the CC genotype material (P<2.8E-5); the flowering time of the TT genotype material in 2019 was significantly earlier than that of the CC genotype material (P<1.2E-6).

[0070] In the Nanchang environment, 407 flowering phenotype materials with the TT genotype were observed in 2020, with flowering times ranging from 24 to 65 days and an average flowering time of 38.7 ± 7.2 days. 106 flowering phenotype materials with the CC genotype were observed, with flowering times ranging from 27 to 58 days and an average flowering time of 42.6 ± 6.7 days. A Wilcoxon Rank Sum Test (one-tailed test) showed that the flowering time of the TT genotype materials was significantly earlier than that of the CC genotype materials (P < 2.6e-7).

[0071] In the Nanchang environment, 416 accessions with the TT genotype and 112 accessions with the CC genotype exhibited canopy coverage phenotypes in 2020. At 61 days post-sowing, the canopy coverage for the TT genotype ranged from 0.28 to 0.98, with an average of 0.74 ± 0.09 days. The canopy coverage for the CC genotype ranged from 0.53 to 0.92, with an average of 0.78 ± 0.07 days. At 63 days post-sowing, the canopy coverage for the TT genotype ranged from 0.28 to 0.94, with an average of 0.74 ± 0.09 days. The canopy coverage of the CC genotype was (0.44–0.91) days, with an average canopy coverage of (0.78 ± 0.07) days. At 65 days post-sowing, the canopy coverage of the TT genotype was (0.43–0.94) days, with an average canopy coverage of (0.77 ± 0.10) days. The canopy coverage of the CC genotype was (0.49–0.94) days, with an average canopy coverage of (0.81 ± 0.08) days. At 68 days post-sowing, the canopy coverage of the TT genotype was (0.35–0.95) days, with an average canopy coverage of (0.73 ± 0.10) days. The canopy coverage of the CC genotype ranged from 0.44 to 0.93, with a mean canopy coverage of 0.77 ± 0.08 days. At 70 days post-sowing, the canopy coverage of the TT genotype ranged from 0.40 to 0.96, with a mean canopy coverage of 0.77 ± 0.11 days. The canopy coverage of the CC genotype ranged from 0.52 to 0.96, with a mean canopy coverage of 0.82 ± 0.08 days. Wilcoxon rank-sum test (one-tailed test) showed that the canopy coverage of the TT genotype was significantly lower than that of the CC genotype at 61 days post-sowing (1.4E-7), 63 days post-sowing (9.3E-7), 65 days post-sowing (1.1E-6), 68 days post-sowing (3.2E-07), and 70 days post-sowing (1.6E-09).

[0072] Therefore, it can be determined that using the SNP sites provided by this invention to detect the soybean canopy coverage and flowering period of the soybean under test is feasible and accurate.

[0073] The specific method for detecting soybean canopy coverage and flowering time of soybeans using the SNP loci provided by this invention is as follows: The whole genome resequencing method provided in Example 1 is used to identify the genomic DNA of the soybeans to be tested and obtain the sample genotype: if the obtained genotype is TT, then the soybean to be tested is a candidate with relatively low canopy coverage and early flowering; if the obtained genotype is CC, then the soybean to be tested is a candidate with relatively high canopy coverage and late flowering.

[0074] The genotype at the Chr08-11602352 locus in soybean can serve as a new genetic resource for marker-assisted selection in breeding soybeans with different planting densities and growth stages, and has very important guiding significance.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. sequence list <110> Institute of Crop Science, Chinese Academy of Agricultural Sciences <120> SNP molecular markers associated with soybean canopy coverage and flowering time and their applications <130> KHP221116118.3 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 577 <212> DNA <213> Artificial Sequence <400> 1 atgagtggta gggattctgg gaaggtgaag tggttcaatg atcagaaggg gtttggattc 60 ataaaccctg atgaagccaa tgaggatctc ttcgttcacc aatctcagat caaatttgac 120 cgttaagttc gccattaaat ttgaatctga tggacacgtt aggactattg atgtcactag 180 ccccgacaac gccaacattc agggaactag acacagtggt gatggtggcc gaagctatgg 240 cgggggacga ggagggggtg gaggtggata tagcgacgat gacggctacg gtggtggtga 300 tggctatggt ggaggtggtg gctacgatgg tgacaggctt gytacaactg tggtgaatcg 360 ggacatctgg ctagggacta cagccaagga ggaagtagag acaggtacaa cagaggcaat 420 gggtggtggt ggcaagtatg gaggcagcaa tggtaggagg tacgatggtg gtggaggagg 480 cggtggtgga ggaggaggtg gtggcggcgg aggaagctgc tacagctgtg gagagtctgg 540 gcattttgcc agagattgcc catcaagtgc tcgttga 577 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <400> 2 tcccgattca ccacagttgt aa 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 tcccgattca ccacagttgt ag 22 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gaggcagcaa tggtaggagg 20

Claims

1. A SNP molecular marker associated with soybean canopy coverage and flowering period, characterized in that, The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.

1.

2. Use of the SNP molecular marker, primer or kit for amplifying the SNP molecular marker of claim 1 in identifying soybean canopy coverage phenotype. The kit comprises primers for amplifying the SNP molecular marker of claim 1.

3. Use of the SNP molecular marker, primer or kit for amplifying the SNP molecular marker of claim 1 in identifying soybean flowering period phenotype. The kit comprises primers for amplifying the SNP molecular marker of claim 1.

4. Use of the SNP molecular marker, primer or kit for amplifying the SNP molecular marker of claim 1 in identifying, improving or targeted breeding of soybean canopy coverage or flowering period related germplasm. The kit comprises primers for amplifying the SNP molecular marker of claim 1.

5. Use of the SNP molecular marker, primer or kit for amplifying the SNP molecular marker of claim 1 in soybean canopy coverage or flowering period related molecular marker assisted breeding, whole genome selection breeding. The kit comprises primers for amplifying the SNP molecular marker of claim 1.

6. Use of the SNP molecular marker, primer or kit for amplifying the SNP molecular marker of claim 1 in screening and / or cultivating soybean with high canopy coverage and late flowering period. The kit comprises primers for amplifying the SNP molecular marker of claim 1.

7. A method of identifying soybean canopy coverage and / or flowering period phenotypes, characterized by, The method comprises the following steps: 1) detecting the genotype of the polymorphic site of the SNP molecular marker of claim 1 in the sample to be tested; 2) determining the canopy coverage and flowering period of the sample to be tested according to the genotype obtained in step 1); The specific determination method is: if the obtained genotype is TT, the sample to be tested is a candidate soybean with relatively low canopy coverage and early flowering period; if the obtained genotype is CC, the sample to be tested is a candidate soybean with relatively high canopy coverage and late flowering period.

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