Indel site related to soybean stem diameter, molecular marker and application of Indel site

Abstract

The invention discloses an Indel site related to soybean stem diameter, a molecular marker and application of the Indel site and the molecular marker, and belongs to the field of molecular marker assisted breeding. Carrying out PCR (Polymerase Chain Reaction) amplification and detection by using an amplification primer of the molecular marker 17-10.64 by taking the molecular marker 17-10.64 as a template; when the product fragment is 199bp, judging that the soybean to be identified is a soybean strain with thin stalks; and when the product fragment is 255bp, judging that the to-be-identified soybean is a soybean strain with thicker stalks. The molecular marker 17-10.64 contains an Indel site related to the soybean stem diameter, the Indel site is located at the No.17 chromosome 1, 064, 063 bp of the soybean, and the polymorphism is an amplified fragment difference of 56bp. According to the method, the soybean breeding time is shortened, the soybean breeding efficiency is improved, and powerful support is provided for efficiently developing and utilizing wild soybean germplasm resources and rapidly breeding lodging-resistant high-quality soybean varieties with thick and strong stalks.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding, and in particular relates to an Indel site related to soybean stem thickness, a molecular marker and an application thereof. Background Art

[0002] Soybean (Glycine max (L.) Merr.) is an important dual-purpose crop for food and oil, and is an important source of high-quality plant protein for humans. In recent years, the self-sufficiency rate of soybeans has dropped below 20%, and the oil yield and yield per unit area of ​​soybeans are lower than those of imported soybeans. Dependence on imported soybeans has seriously affected food security. Studies have shown that in the process of domestication from wild species to cultivated species, the genetic diversity of cultivated soybeans has decreased by 30%. The narrow genetic basis of the cultivated soybean population has become an important factor restricting the increase in soybean yield and quality. Therefore, broadening the genetic basis of cultivated soybeans through genetic improvement is of great significance to alleviating soybean production problems.

[0003] The annual wild soybean (Glycine soja Sieb.et Zucc.) is a wild species closely related to cultivated soybean. The two can hybridize normally and most of the offspring are fertile. Wild soybean has excellent traits such as multiple flowers and pods, high protein, high isoflavones, disease and insect resistance, and stress resistance, providing a valuable source of genes for high-yield and high-quality soybean breeding. However, since wild soybean also has unfavorable traits such as slender stems and creeping nature, its application in breeding is restricted. The thick stem trait of soybeans is usually manifested in the soybean seedling stage. This trait can easily lead to plant lodging, reduce yield and quality, and double the workload of soybean field management.

[0004] The traditional breeding method is used to breed cultivated soybeans that have the excellent qualities of wild soybeans and grow upright. The breeding process is time-consuming and laborious, and cannot meet the high demand for expanding soybean germplasm resources in current breeding work. With the development and application of molecular markers, molecular marker-assisted selection can help breeders select excellent target lines at the gene level while filtering out inferior lines with thin stems, avoiding a large amount of inefficient field phenotypic identification and management work. However, different molecular markers also have their own problems in application. For example, the polymorphism of RFLP (Restriction Fragment Length Polymorphism) markers in the soybean genome is low, which limits the construction of high-density genetic maps of soybeans (Keim P, Diers B W, Olson T C, et al. RFLP mapping in soybean: association between markers located variation in quantitative traits. [J]. Genetics, 1990, 126 (3): 735–742.). At the same time, this method requires the use of polyacrylamide gel electrophoresis, and the experimental operation process is complex, unstable, and highly dependent on technology; SSR (Simple Sequence Repeat) markers can be realized through simple and easy high-concentration agarose gel electrophoresis, and this marker is widely distributed in the soybean genome, has co-dominance, and its polymorphism is much higher than that of RFLP markers, but this marker method has the limitation of uneven genome distribution (Wang Zhen. Construction of soybean SSR genetic map and QTL analysis of important agronomic traits [D]. Guangxi University, 2004.); SNP (Single Nucleotide Polymorphism) markers have the highest polymorphism, the most uniform distribution, and the best accuracy, but it requires whole-genome resequencing of the population, and the research funding investment is relatively large (Yuan Jinhong et al. Research progress of SNP analysis based on whole-genome resequencing in crop gene positioning [D]. Journal of Plant Physiology, 2015.). Therefore, a simple, efficient, cost-effective, and easy-to-operate method for identifying soybean stem thickness traits is needed. Summary of the invention

[0005] In order to solve the problems in existing molecular marker-assisted breeding methods, such as complicated RFLP test steps, long test cycle, strong technology dependence, uneven distribution of SSR in the genome and certain preference, and SNP requiring whole genome sequencing and high research funding investment, the present invention provides an Indel site associated with soybean stem thickness, a molecular marker and applications thereof.

[0006] The present invention uses an Indel molecular marker located on soybean chromosome 17 and closely related to the soybean stem thickness trait to establish a method for efficiently identifying and utilizing wild soybean introgression line breeding. By detecting the size of the amplified fragment of the Indel molecular marker 17-10.64, soybean materials carrying stem thickness allele variation can be selected. The present invention can effectively solve the problem of being troubled by the thinness and lodging of soybean stalks in the utilization of wild soybean germplasm resources, and provides strong technical support for the efficient development and utilization of wild soybean germplasm resources and the rapid selection of high-quality soybean varieties with thick stalks and resistance to lodging in the hybrid offspring of wild soybeans and cultivated soybeans. At the same time, the present invention shortens the soybean breeding time and improves the soybean breeding efficiency.

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0008] In a first aspect, the present invention provides an Indel site associated with soybean stem thickness, wherein the Indel site is located at 1,064,063 bp on soybean chromosome 17, and the polymorphism is a 56 bp difference in the amplified fragment.

[0009] As a preferred embodiment, the phenotypic contribution rate of the Indel site to the soybean stem thickness trait is 9.37%.

[0010] In a second aspect, the present invention provides an application of an Indel site associated with soybean stem thickness in identifying the soybean stem thickness trait.

[0011] In a third aspect, the present invention provides an Indel molecular marker 17-10.64 containing an Indel site associated with soybean stem thickness.

[0012] In a fourth aspect, the present invention provides the use of Indel molecular marker 17-10.64 in identifying soybean stem thickness trait.

[0013] In a fifth aspect, the present invention provides amplification primers for Indel molecular marker 17-10.64, and the sequence information of the amplification primers is as follows:

[0014] F: 5'-GGAACAATCGTGGAAGGATTCT-3';

[0015] R: 5'-GACATACTCAATTGGATAACTATTTTTTATCT-3'.

[0016] In a sixth aspect, the present invention provides the use of amplification primers of Indel molecular marker 17-10.64 in identifying the soybean stem thickness trait.

[0017] In a seventh aspect, the present invention provides a method for identifying the thick stem trait of soybean using amplification primers of Indel molecular marker 17-10.64, which specifically comprises the following steps:

[0018] (1) Extract the genomic DNA of soybean leaves to be identified using the TIANGEN plant genomic DNA extraction kit operating instructions;

[0019] (2) using the soybean leaf genomic DNA to be identified as a template, and performing PCR amplification using the amplification primers of the Indel molecular marker 17-10.64 to obtain a PCR amplification product;

[0020] (3) Detect the size of the PCR amplification product fragment using 1.2% agarose gel electrophoresis;

[0021] When the fragment size of the PCR amplification product is 199 bp, the soybean to be identified is judged to be a soybean variety with thinner stems. When the fragment size of the PCR amplification product is 255 bp, the soybean to be identified is judged to be a soybean variety with thicker stems.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides a simple, easy, economical and efficient soybean stem thickness trait identification method by screening out Indel molecular markers associated with soybean stem thickness, and the prediction and selection of soybean stem thickness can be achieved by only performing PCR amplification on the sequence at the position of the Indel molecular marker and detecting the size of the PCR product fragment by 1.2% agarose gel electrophoresis, thereby achieving economical, efficient, simple and easy soybean breeding auxiliary selection, and providing a more feasible and practical solution for the efficient selection of soybean stem thickness trait and the efficient utilization of wild soybean germplasm resources.

[0024] The Indel molecular marker screened by the present invention can be widely used to screen out individuals with thick stems from soybean materials with polymorphism at the molecular marker. Among them, the interval of 906,324bp-2,604,965bp of soybean chromosome 17 is an ideal marker interval for regulating soybean stem thickness, and the Indel molecular marker 17-10.64 located at 1,064,063bp has a genetic contribution rate of 9.37% to stem thickness.

[0025] The present invention reduces the selection cost in the soybean breeding process, the test operation is simple and easy, and the stem thickness of the soybean line can be identified by distinguishing the size of the fragment with the marker site, which greatly improves the utilization efficiency of wild soybean germplasm resources, broadens the soybean genetic basis, and accelerates the improvement and improvement of cultivated soybean quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The Indel molecular marker 17-10.64 in the present invention is in the two parents and part of the F 3 Amplification performance in individual strains of the segregating population.

[0027] Figure 2 To analyze the significance of predicting soybean stem thickness phenotype. DETAILED DESCRIPTION

[0028] 1. The present invention provides an Indel site associated with soybean stem thickness, the Indel site is located at 1,064,063 bp on soybean chromosome 17, and its polymorphism is a 56 bp difference in the amplified fragment.

[0029] Among them, the phenotypic contribution rate of the Indel site to the soybean stem thickness trait was 9.37%.

[0030] 2. The present invention provides an application of an Indel site associated with soybean stem thickness in identifying the soybean stem thickness trait.

[0031] 3. The present invention provides an Indel molecular marker 17-10.64 containing an Indel site associated with soybean stem thickness.

[0032] 4. The present invention provides the application of Indel molecular marker 17-10.64 in identifying soybean stem thickness trait.

[0033] 5. The present invention provides amplification primers for Indel molecular marker 17-10.64, and the sequence information of the amplification primers is as follows:

[0034] F: 5'-GGAACAATCGTGGAAGGATTCT-3';

[0035] R: 5'-GACATACTCAATTGGATAACTATTTTTTATCT-3'.

[0036] 6. The present invention provides the use of amplification primers of Indel molecular marker 17-10.64 in identifying the soybean stem thickness trait.

[0037] VII. The present invention provides a method for identifying the thick stem trait of soybean using amplification primers of Indel molecular marker 17-10.64, which specifically comprises the following steps:

[0038] (1) Extract the genomic DNA of soybean leaves to be identified using the TIANGEN plant genomic DNA extraction kit operating instructions;

[0039] (2) using the soybean leaf genomic DNA to be identified as a template, and performing PCR amplification using the amplification primers of the Indel molecular marker 17-10.64 to obtain a PCR amplification product;

[0040] (3) Detect the size of the PCR amplification product fragment using 1.2% agarose gel electrophoresis;

[0041] When the fragment size of the PCR amplification product is 199 bp, the soybean to be identified is judged to be a soybean variety with thinner stems. When the fragment size of the PCR amplification product is 255 bp, the soybean to be identified is judged to be a soybean variety with thicker stems.

[0042] In summary, the present invention provides an Indel site associated with soybean stem thickness, an Indel molecular marker 17-10.64 containing an Indel site associated with soybean stem thickness, and an amplification primer of the Indel molecular marker 17-10.64, which are mainly used in a method for identifying the soybean stem thickness trait, mainly including the selection of the soybean stem thickness trait and the early prediction of the soybean stem thickness trait.

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] 1 Experimental materials:

[0045] Parental materials: cultivated soybean Williams 82 as the female parent, wild soybean GS 98 as the male parent;

[0046] Soybean to be tested: F hybrid between female parent “Williams 82” and male parent “GS 98” 3 Generation of isolated populations.

[0047] 2 Experimental steps:

[0048] 2.1 Parental genome resequencing and sequence difference analysis between parents;

[0049] First, DNA libraries were constructed using maternal material "Williams 82" and paternal material "GS 98", and sequencing operations were performed, wherein the sequencing data volume was set to ~10Gb / sample, the sequencing platform used DNBSEQ-T7, and the sequencing strategy was set to double-end 150bp (PE150). Then, BWA (v0.7.17-r1188) software was used to align the high-quality sequences of the two parents (maternal "Williams 82" and paternal "GS 98") to the soybean reference genome (Glycine max cv.Wm82.v4); BCFtools (v1.3.1) was used for variation detection operations to mine SNPs and small fragments of Indel between parents; finally, Manta was used to detect large fragments of Indel between parents, and sites with Indel sizes between 50bp-800bp were screened, so as to use the Indel site for Indel molecular marker design.

[0050] 2.1 Sequencing of mixed pools of progeny populations and BSA-seq analysis;

[0051] First, based on the test data of the target trait (main stem thickness) of the soybean population at the V4 stage, the hybrid F of the female parent "Williams 82" and the male parent "GS 98" was selected. 3 The extreme phenotypic individuals of the target trait in the generation isolation population were constructed to construct DNA pools. Each DNA pool contained 30 strains, and the library construction and sequencing operations were performed on the DNA of the DNA pool. The sequencing data volume of each DNA pool was ~30Gb / pool. The sequencing platform used was DNBSEQ-T7, and the sequencing strategy was set to double-end 150bp (PE150). Then, the sequencing data of each DNA pool were filtered using BWA (v0.7.17-r1188) software and aligned to the soybean reference genome (Glycine max cv.Wm82.v4). BCFtools (v1.3.1) was used to analyze the comparison results of the sequencing data of each DNA pool, and the sequencing depth of the parental fragments of all SNP sites was determined based on the SNP differences between the parents. The R package QTLseqr (v0.7.5.2) was used to analyze the ΔSNP index and G'value of each trait, thereby screening the areas with G' values ​​exceeding the threshold (adjusted p-value < 0.05) and ΔSNP index > 0 and confidence interval > 95%, which were initially defined as the QTL sites for the stem thickness trait, and finally 8-10 Indel molecular markers with a length greater than 50 bp were obtained.

[0052] 2.3 Screening of Indel molecular markers between parents;

[0053] First, the upstream and downstream sequence information of the Indel molecular marker obtained above was extracted, and the primers of the Indel molecular marker were designed using Primer5.0 software, with the primer annealing temperature of 59°C-61°C, the primer length range of 25bp-35bp, and the product length range of 150bp-800bp; then, PCR amplification was performed on the DNA of the two parents (the maternal "Williams 82" and the paternal "GS 98"), and then 1.2% agarose gel electrophoresis was performed to screen the Indel molecular marker that can effectively distinguish the two parents (the maternal "Williams 82" and the paternal "GS 98").

[0054] Among them, the selected Indel molecular marker 17-10.64 can effectively distinguish the two parents (the maternal parent "Williams 82" and the paternal parent "GS 98"). A large fragment of 255 bp was amplified in the maternal parent "Williams 82", and a small fragment of 199 bp was amplified in the paternal parent "GS 98". Figure 1 .

[0055] 2.4 Identification of the genotype of the offspring population;

[0056] The Indel molecular markers obtained by screening were used to identify the genotypes of all individuals in the progeny population. Combined with the phenotypic data, the QTL IciMapping (Version 4.2) software was used to construct and draw a genetic map. Finally, an Indel site 17-10.64 related to the soybean stem thickness trait was detected at 1,064,063bp on soybean chromosome 17 (see Table 1 for specific information), and the phenotypic contribution rate to the soybean stem thickness trait was 9.37%. Therefore, the Indel molecular markers closely linked to this Indel site 17-10.64 can be used as effective markers to assist in the selection of soybean stem thickness traits.

[0057] Table 1 Indel site information

[0058]

[0059] According to the Indel site information in Table 1, when the PCR amplification product fragment is a large fragment (255 bp), the soybean to be tested is judged to be a soybean variety with a thick stem trait; and when the PCR amplification product fragment is a small fragment (199 bp), the soybean to be tested is judged to be a soybean variety with a thin stem trait.

[0060] 3. Method for detecting soybean stem thickness using Indel molecular markers;

[0061] Soybean to be tested: F hybrid between female parent “Williams 82” and male parent “GS 98” 3There are 190 individual plants in the segregating population.

[0062] 3.1 Genomic DNA extraction;

[0063] Extract the genomic DNA of soybean leaves to be tested according to the operating instructions of TIANGEN Plant Genomic DNA Extraction Kit (DP305);

[0064] 3.2 Design of Indel molecular marker primers;

[0065] Indel molecular marker primer information is as follows:

[0066] F: 5'-GGAACAATCGTGGAAGGATTCT-3' (SEQ ID NO: 1);

[0067] R: 5'-GACATACTCAATTGGATAACTATTTTTTATCT-3' (SEQ ID NO: 2).

[0068] 3.3PCR amplification procedure;

[0069] Using the soybean genomic DNA as a template, the Indel molecular marker primers designed by the present invention are used to perform PCR amplification operations on all the soybeans to be tested, and PCR product fragments are obtained.

[0070] The total volume of the PCR reaction system is preferably 10 μL, including 1 μL 30 ng template, 5 μL nucleic acid dye (UElandy, Super GelBlueTM nucleic acid dye, S2019S), 2 μL ddH 2 O and 2 μL of 2 mM primer.

[0071] The PCR amplification program is preferably: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 40 s, 35 cycles; finally 72°C extension for 10 min; 4°C storage. The PCR amplification program is performed on a PCR amplification thermal cycler from ABI (Applied Biosystems, USA).

[0072] 3.3 Results

[0073] The statistical test results show that in the prediction of soybean stem thickness, the marker segment amplified a large fragment (255bp) of F 3 There were 60 plants in the first generation of segregation population, with stem diameter ranging from 1.15 mm to 2.29 mm, and an average of 1.55 ± 0.23 mm. A small fragment (199 bp) of F 3There were 86 plants in the segregating population, with stem diameters ranging from 1.03 mm to 1.81 mm, with an average value of 1.42 ± 0.16 mm. Figure 2 As shown, the one-way ANOVA multiple comparison showed that there was a significant difference between the two (P-value<0.01). Therefore, the Indel molecular marker 17-10.64 screened by the present invention can be used for auxiliary selection of soybean stem thickness trait.

[0074] The present invention discloses an Indel site, a molecular marker and an application thereof related to soybean stem thickness. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. An Indel site associated with soybean stem thickness, characterized in that: The Indel site is located at 1,064,063 bp on soybean chromosome 17, and the polymorphism is a difference of 56 bp in the amplified fragment.

2. The Indel site associated with soybean stem thickness according to claim 1, characterized in that: The phenotypic contribution rate of the Indel site to the soybean stem thickness trait is 9.37%.

3. Use of an Indel site associated with soybean stem thickness as claimed in claim 1 in identifying soybean stem thickness trait.

4. An Indel molecular marker 17-10.64 comprising the Indel site associated with soybean stem thickness as claimed in claim 1.

5. Use of the Indel molecular marker 17-10.64 as claimed in claim 4 in identifying the soybean stem thickness trait.

6. The amplification primers for Indel molecular marker 17-10.64 according to claim 4, characterized in that: The sequence information of the amplification primers is as follows: F: 5'-GGAACAATCGTGGAAGGATTCT-3'; R: 5'-GACATACTCAATTGGATAACTATTTTTTATCT-3'.

7. Use of the amplification primers of the Indel molecular marker 17-10.64 as claimed in claim 6 in identifying the soybean stem thickness trait.

8. A method for identifying soybean stem thickness trait using the amplification primers of the Indel molecular marker 17-10.64 according to claim 6, characterized in that: The following steps are involved: (1) Extract the genomic DNA of soybean leaves to be identified using the TIANGEN plant genomic DNA extraction kit operating instructions; (2) using the soybean leaf genomic DNA to be identified as a template, and performing PCR amplification using the amplification primers of the Indel molecular marker 17-10.64 to obtain a PCR amplification product; (3) Detect the size of the PCR amplification product fragment using 1.2% agarose gel electrophoresis; When the fragment size of the PCR amplification product is 199 bp, the soybean to be identified is judged to be a soybean variety with thinner stems. When the fragment size of the PCR amplification product is 255 bp, the soybean to be identified is judged to be a soybean variety with thicker stems.

Images