Indel site simultaneously related to pod number and single plant yield of soybean, molecular marker and application of Indel site and molecular marker

Abstract

The invention discloses an Indel site simultaneously related to the pod number and the single plant yield of soybean, a molecular marker and application of the Indel site and the molecular marker, and belongs to the field of molecular marker-assisted breeding. The Indel site is located at 19bp, 855bp and 388bp of a soybean No.6 chromosome, and the polymorphism is an amplified fragment difference of 89bp. The detection method comprises the following steps: extracting a soybean genome DNA to be identified and carrying out PCR amplification by taking the soybean genome DNA as a template; detecting the size of a PCR amplification product fragment by 1% agarose gel electrophoresis; when the fragment of the amplification product is a small fragment, the soybean to be identified is judged to be a soybean line with multiple pods and high yield, and when the fragment of the amplification product is a large fragment, the soybean to be identified is judged to be a soybean line with small pods and low yield. According to the method, the detection and breeding cost is reduced, the cultivation effect of the soybean high-per-unit-yield variety is improved, the progress of soybean yield improvement is accelerated, and the soybean breeding time is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular marker-assisted breeding, and specifically relates to an Indel site simultaneously related to soybean pod number and single-plant yield, a molecular marker and application thereof. Background Art

[0002] Soybean (Glycine max (L.) Merr.) is an important grain and oil crop worldwide, and is also an important source of plant protein for humans. In recent years, with the continuous improvement of people's demand for quality of life, the demand for soybean production has grown rapidly. Under the contradictory background of limited arable land and high demand for soybeans, effectively increasing soybean yield has become the key to solving this problem. The number of soybean pods and yield per plant are important factors affecting soybean yield. Both are typical quantitative trait inheritance, which are regulated by the joint action of several major genes and modifier genes. They are easily affected by growth environment conditions and have large differences in heritability under different genetic backgrounds and environments. Using traditional breeding methods to cultivate soybean varieties with high pod numbers and high yields is time-consuming and labor-intensive, with very low efficiency, and cannot meet the current development needs of soybean breeding.

[0003] With the development and application of various molecular markers, molecular marker-assisted selection helps breeders select target strains from the genetic level, avoiding time-consuming and laborious phenotypic identification. However, there are also many problems in the application of different molecular markers, such as: RFLP (restriction fragment length polymorphism) test process is complex and highly technology-dependent; SNP (single nucleotide polymorphism) requires sequencing and high investment, so there is an urgent need for a more concise, efficient, cost-saving and labor-saving method for identifying soybean pod number and single plant yield. The biggest advantage of Indel (insertion-deletion) markers is that they are low-cost and the test process is simple and easy to operate. By confirming the size of the fragment with the marker site, soybean varieties with multiple pods and high yield can be identified, thereby greatly improving breeding efficiency and accelerating the breeding process.

[0004] Traditional breeding is time-consuming and labor-intensive, and the artificial selection standards vary greatly, making it difficult to select dominant target varieties. Among molecular marker-assisted methods, FRLP is applied to polyacrylamide gel electrophoresis, and the experimental process is complex and time-consuming; the application of SNP markers is based on sequence sequencing, which requires a large amount of capital investment. In comparison, Indel markers can predict the number of pods and single plant yield and select dominant target traits simply by PCR amplification of the sequence at the location and detecting the size of the PCR product fragment by 1% agarose gel electrophoresis. This achieves economical, efficient, and simple breeding-assisted selection. Summary of the invention

[0005] In order to solve the problems of time-consuming and labor-intensive traditional breeding, large differences in artificial selection standards, difficulty in breeding superior varieties, and complex procedures, time-consuming and high costs in molecular marker-assisted methods, the present invention provides an Indel site, molecular marker and application thereof that are simultaneously related to soybean pod number and single plant yield.

[0006] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0007] The first object of the present invention is to provide an Indel site that is simultaneously associated with soybean pod number and single plant yield, wherein the Indel site is located at 19,855,388 bp on soybean chromosome 6, and the polymorphism is an 89 bp difference in the amplified fragment.

[0008] The second object of the present invention is to provide an application of an Indel site simultaneously associated with soybean pod number and single plant yield in soybean breeding for multiple pods and high yield traits and early prediction of soybean pod number and single plant yield traits.

[0009] The third object of the present invention is to provide a molecular marker Indel 6-19.85 containing the Indel site that is simultaneously related to soybean pod number and single plant yield.

[0010] The fourth object of the present invention is to provide the application of the molecular marker Indel 6-19.85 in the breeding of soybean with multiple pods and high yield and the early prediction of soybean pod number and single plant yield.

[0011] The fifth object of the present invention is to provide primers for amplifying the molecular marker Indel 6-19.85, comprising two primers:

[0012] F: 5'-CCCAATTCTCTGTTTGCACC-3';

[0013] R: 5'-TTCGGAAGCAGTTTAGTGGAG-3'.

[0014] The sixth object of the present invention is to provide the application of the amplification primers in the breeding of soybean with multiple pods and high yield per unit area and the early prediction of soybean pod number and yield per plant.

[0015] The seventh object of the present invention is to provide a method for detecting soybean pod number and single plant yield using the molecular marker Indel 6-19.85, which mainly comprises the following steps:

[0016] (1) extracting genomic DNA of soybean to be identified;

[0017] (2) Using the extracted genomic DNA as a template, PCR amplification is performed using amplification primers to obtain an amplification product; the sequence information of the amplification primers is as follows:

[0018] F: 5'-CCCAATTCTCTGTTTGCACC-3';

[0019] R: 5'-TTCGGAAGCAGTTTAGTGGAG-3';

[0020] (3) The size of the PCR amplification product fragment is detected by 1% agarose gel electrophoresis; when the amplification product fragment is a small fragment, the soybean to be identified is judged to be a soybean variety with a large number of pods and a high yield; when the amplification product fragment is a large fragment, the soybean to be identified is judged to be a soybean variety with a small number of pods and a low yield.

[0021] As a preferred embodiment, the length of the small fragment is 301 bp; the length of the large fragment is 390 bp.

[0022] As a preferred embodiment, in step (2), the PCR amplification system is: a total volume of 10 μL, including 1 μL of 30 ng template DNA, 5 μL of nucleic acid dye, 2 μL of ddH2O, and 2 μL of 2 mM amplification primer.

[0023] As a preferred embodiment, in step (2), the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses an Indel molecular marker located on soybean chromosome 6 that is closely related to the soybean pod number and single plant yield traits, and establishes a method for efficiently identifying and breeding multiple pod number and high-yield soybean lines. By detecting the size of the amplified fragment of the molecular marker Indel 6-19.85 site, materials carrying allelic variation of the pod number and single plant yield can be detected, avoiding time-consuming and labor-intensive phenotypic identification, costly and complex experimental process identification methods, greatly reducing the cost of detection and breeding, and increasing the selection time of multiple pod number and high single-yield varieties in the breeding process, improving the breeding effect of high single-yield soybean varieties, accelerating the process of soybean yield improvement, and shortening the soybean breeding time.

[0026] The Indel molecular marker provided by the present invention can be widely used to simultaneously screen the number of pods and the yield per plant in soybean materials with polymorphism at the molecular marker, serving the rapid genetic improvement of soybean yield, and providing a more feasible and practical solution for the efficient selection and breeding practice of the number of pods and the yield per plant traits of soybean. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The amplification results of the molecular marker Indel 6-19.85 in the two parents. In the figure, M is a DNA molecular weight standard marker.

[0028] Figure 2 The amplification results of the molecular marker Indel 6-19.85 in some offspring of the population. In the figure, 1-96 are 96 individual plants in the F2 test population.

[0029] Figure 3 It is a display of the significance of phenotypic differences predicted for two traits related to molecular markers. DETAILED DESCRIPTION

[0030] The first object of the present invention is to provide an Indel site that is simultaneously related to soybean pod number and single plant yield. The Indel site is located at 19,855,388 bp on soybean chromosome 6, and the polymorphism is an 89 bp difference in the amplified fragment.

[0031] The second object of the present invention is to provide the application of the above-mentioned Indel site simultaneously associated with soybean pod number and single plant yield in the breeding of soybean multiple pods and high yield traits and the early prediction of soybean pod number and single plant yield traits.

[0032] The third object of the present invention is to provide a molecular marker Indel 6-19.85 containing the above-mentioned Indel site that is simultaneously related to soybean pod number and single plant yield.

[0033] The fourth object of the present invention is to provide the application of the above-mentioned molecular marker Indel 6-19.85 in the breeding of soybean with multiple pods and high yield and the early prediction of soybean pod number and yield per plant.

[0034] A fifth object of the present invention is to provide primers for amplifying the above-mentioned molecular marker Indel 6-19.85, comprising two primers:

[0035] F: 5'-CCCAATTCTCTGTTTGCACC-3';

[0036] R: 5'-TTCGGAAGCAGTTTAGTGGAG-3'.

[0037] The sixth object of the present invention is to provide the application of the above-mentioned amplification primers in the breeding of soybean with multiple pods and high yield and the early prediction of soybean pod number and single plant yield.

[0038] The seventh object of the present invention is to provide a method for detecting soybean pod number and single plant yield using the above-mentioned molecular marker Indel 6-19.85, which mainly comprises the following steps:

[0039] (1) extracting genomic DNA of soybean to be identified;

[0040] (2) Using the extracted genomic DNA as a template, PCR amplification is performed using amplification primers to obtain an amplification product; the sequence information of the amplification primers is as follows:

[0041] F: 5'-CCCAATTCTCTGTTTGCACC-3';

[0042] R: 5'-TTCGGAAGCAGTTTAGTGGAG-3';

[0043] (3) The size of the PCR amplification product fragment is detected by 1% agarose gel electrophoresis; when the amplification product fragment is a small fragment, the soybean to be identified is judged to be a soybean variety with a large number of pods and a high yield; when the amplification product fragment is a large fragment, the soybean to be identified is judged to be a soybean variety with a small number of pods and a low yield.

[0044] Preferably, the length of the small fragment is 301 bp; the length of the large fragment is 390 bp.

[0045] Preferably, in step (2), the PCR amplification system is: a total volume of 10 μL, including 1 μL of 30 ng template DNA, 5 μL of nucleic acid dye, 2 μL of ddH2O, and 2 μL of 2 mM amplification primer.

[0046] Preferably, in step (2), the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0047] 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 creative work are within the scope of protection of the present invention.

[0048] Example 1 QTL (Quantitative Trait Locus) Positioning Analysis and Development of Indel Molecular Markers

[0049] Test Materials:

[0050] Female parent: soybean variety Jiyu 101;

[0051] Male parent: soybean line GB222;

[0052] F2 generation segregating population of the hybrid of “Jiyu 101” and “GB222”.

[0053] 1. Parental genome resequencing and sequence difference analysis between parents

[0054] DNA libraries were constructed and sequenced for the parental materials "Jiyu 101" and "GB222", with a sequencing data volume of ~10 Gb / sample. The sequencing platform was DNBSEQ-T7, and the sequencing strategy was double-end 150bp (PE150).

[0055] The high-quality sequences of the two parents were aligned to the soybean reference genome (Glycine max cv.Wm82.v4) using BWA (v0.7.17-r1188) software. BCFtools (v1.3.1) was used for variation detection to mine SNPs and small Indels between parents. Manta was used to detect large Indels between parents, and sites with Indel sizes of 50-800bp were screened for designing molecular markers.

[0056] 2. Analysis of extreme traits of progeny population by pooled resequencing (BSA-seq)

[0057] According to the test data of the target traits (pod number and single plant yield) of the population, the extreme phenotypic individuals of these two traits in the F2 population were screened to construct DNA pools. Each extreme pool contained 30 plants. The library was constructed and sequenced for the DNA in the pool. The sequencing data volume of each pool was ~30 Gb / pool. The sequencing platform was DNBSEQ-T7, and the sequencing strategy was double-end 150bp (PE150).

[0058] BWA (v0.7.17-r1188) software was used to filter the sequencing data of each extreme pool 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 program package QTLseqr (v0.7.5.2) was used to analyze the ΔSNP index and G'value of each trait. In this study, the areas with G' values ​​exceeding the threshold (adjusted p-value < 0.05) and ΔSNP index > 0 and confidence interval > 95% were defined as the QTL loci for the pod number and single plant yield traits, and 5-10 Indel molecular markers larger than 50bp were obtained during the screening.

[0059] 3. Screening of Indel molecular markers between parents

[0060] Extract the upstream and downstream sequence information of the above Indel molecular markers, use Primer5.0 software to design the amplification primers of the Indel molecular markers, the primer annealing temperature is 58-61°C, the primer length range is 20-30bp, and the product length range is 150-800bp. Perform PCR amplification and 1% agarose gel electrophoresis on the DNA of the two parents to screen out the Indel molecular markers that effectively distinguish the two parents.

[0061] Among them, the molecular marker Indel 6-19.85 can effectively distinguish the two parental genotypes, amplifying a small fragment of 301 bp in Jiyu 101 and a large fragment of 390 bp in GB222 ( Figure 1 ).

[0062] 4. Genotype identification of offspring population

[0063] 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 that was simultaneously related to soybean pod number and single plant yield was detected between 19,855,388bp-19,855,477bp on soybean chromosome 6 (see Table 1), with a 95% confidence interval of less than 1cM, and the phenotypic contribution rates to soybean pod number and single plant yield were 12.5% ​​and 12.8%, respectively. The Indel molecular marker closely linked to this site can be used as an effective marker to assist in the selection of soybean pod number and single plant yield traits, namely the molecular marker Indel 6-19.85.

[0064] Table 1 Molecular marker site information

[0065]

[0066] Therefore, when the amplified product fragment is a small fragment, the soybean to be identified is judged to be a soybean variety with a large number of pods and a high yield. When the amplified product fragment is a large fragment, the soybean to be identified is judged to be a soybean variety with a small number of pods and a low yield.

[0067] Example 2: Establishment of a method for detecting soybean pod number and single plant yield using molecular marker Indel 6-19.85 using soybean materials with known phenotypic data

[0068] Test samples: 280 individual plants of the F2 generation of hybrids of “Jiyu 101” and “GB222”

[0069] (1) Extracting genomic DNA;

[0070] The genomic DNA of soybean leaves to be identified was extracted according to the operating instructions of TIANGEN Plant Genomic DNA Extraction Kit (DP305);

[0071] (2) PCR amplification;

[0072] The extracted genomic DNA was used as a template and PCR amplification was performed using amplification primers to obtain the amplified product, which was stored at 4°C.

[0073] The above amplification primer sequence information is as follows:

[0074] F: 5'-CCCAATTCTCTGTTTGCACC-3';

[0075] R: 5'-TTCGGAAGCAGTTTAGTGGAG-3';

[0076] The PCR reaction system is as follows: the total volume is 10 μL, including 1 μL of 30 ng template DNA, 5 μL of nucleic acid dye (UElandy, Super GelBlueTM nucleic acid dye, S2019S), 2 μL of ddH2O, and 2 μL of 2 mM amplification primer.

[0077] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0078] The above PCR reaction was amplified on a PCR amplification thermal cycler from ABI (Applied Biosystems, USA).

[0079] (3) The above amplified products were detected by 1% agarose gel electrophoresis, and the results of the size fragment differences obtained from 280 hybrid F2 generation segregation population individual materials are shown as follows Figure 2 As shown, the length of the large fragment is 390 bp, and the length of the small fragment is 301 bp.

[0080] The above results are statistically analyzed, and the results are as follows Figure 3 As shown, in the prediction of soybean pod number, the average number of pods of the soybean population to be identified with the large fragment (390bp) and the small fragment (301bp) amplified by the marker segment were 72 and 124, respectively, and there was a very significant difference between the two; in the prediction of soybean single-plant yield, the average single-plant yield of the soybean population to be identified with the large fragment (390bp) and the small fragment (301bp) amplified by the marker segment were 19.5 grams and 33.8 grams, respectively, and there was also a very significant difference between the two.

[0081] In summary, the Indel site, molecular marker Indel 6-19.85 and amplification primers provided by the present invention, which are simultaneously related to the number of soybean pods and the yield per plant, can be applied to the breeding of soybean pods and high yield traits and the early prediction technology of the number of soybean pods and the yield per plant. Among them, the 19,855,388±50kb interval of chromosome 6 of the present invention is an ideal marker interval for regulating the number of soybean pods and the yield per plant, and the genetic contribution rate of the number of pods and the yield per plant of the 19,855,388bp position Indel is 12.5% ​​and 12.8% respectively.

[0082] The present invention discloses an Indel site, a molecular marker and its application that are simultaneously related to the number of soybean pods and the yield per plant. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out 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 that is simultaneously associated with soybean pod number and single plant yield, characterized in that: The large fragment Indel site is located at 19,855,388 bp on soybean chromosome 6, and the polymorphism is a difference of 89 bp in the amplified fragment.

2. Use of an Indel site simultaneously associated with soybean pod number and single plant yield as claimed in claim 1 in soybean breeding for multiple pods and high yield traits and early prediction of soybean pod number and single plant yield traits.

3. A molecular marker Indel 6-19.85 comprising the Indel site associated with soybean pod number and single plant yield as claimed in claim 1.

4. Use of the molecular marker Indel 6-19.85 as claimed in claim 3 in the breeding of soybean with multiple pods and high yield and in the early prediction of soybean pod number and yield per plant.

5. The amplification primer for the molecular marker Indel 6-19.85 according to claim 3, characterized in that: Includes two primers: F: 5'-CCCAATTCTCTGTTTGCACC-3'; R: 5'-TTCGGAAGCAGTTTAGTGGAG-3'.

6. Use of the amplification primers as claimed in claim 5 in the breeding of soybean with multiple pods and high yield per unit area and in the early prediction of soybean pod number and yield per plant.

7. A method for detecting soybean pod number and single plant yield using the molecular marker Indel 6-19.85 according to claim 3, characterized in that: The following steps are involved: (1) extracting genomic DNA of soybean to be identified; (2) Using the extracted genomic DNA as a template, PCR amplification is performed using amplification primers to obtain an amplification product; the sequence information of the amplification primers is as follows: F: 5'-CCCAATTCTCTGTTTGCACC-3'; R: 5'-TTCGGAAGCAGTTTAGTGGAG-3'; (3) The size of the PCR amplification product fragment is detected by 1% agarose gel electrophoresis; when the amplification product fragment is a small fragment, the soybean to be identified is judged to be a soybean variety with a large number of pods and a high yield; when the amplification product fragment is a large fragment, the soybean to be identified is judged to be a soybean variety with a small number of pods and a low yield.

8. The method according to claim 7, characterized in that The length of the small fragment is 301 bp; the length of the large fragment is 390 bp.

9. The method according to claim 7, characterized in that: In step (2), the PCR amplification system is: a total volume of 10 μL, including 1 μL of 30 ng template DNA, 5 μL of nucleic acid dye, 2 μL of ddH2O, and 2 μL of 2 mM amplification primer.

10. The method according to claim 7, characterized in that In step (2), the PCR amplification program is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 40 s, 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

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