Application of AIL1 gene in regulating soybean internode length and breeding of high-yield and high-density soybean varieties

Abstract

The present application belongs to the technical field of plant genetic engineering, and particularly relates to application of AIL1 gene in regulation of soybean internode spacing and cultivation of high-yield soybean varieties with high density planting. The present application finds that variation of the soybean AIL1 gene can change the length of internode cells and affect the internode spacing, and specifically, frame shift mutation of the AIL1 gene can lead to shortening of the soybean internode spacing. Therefore, the AIL1 soybean mutant is created by using gene editing technology, and a plurality of natural excellent allelic variation mutants are identified in a large number of soybean materials. After backcrossing the excellent allelic variation mutants to a high-yield soybean variety which is not resistant to high density planting, it is found that the backcrossed materials have stable internode spacing and small density impact on yield under different high density planting conditions, and it is confirmed that the AIL1 gene can be used for cultivation of high-yield soybean varieties with high density planting, and promote the development of the soybean industry.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the AIL1 gene in regulating internode spacing in soybeans and in the breeding of high-yield soybean varieties through dense planting. Background Technology

[0002] Soybeans (Glycine max) are a crucial source of plant protein for humans and animal feed protein, playing a vital role in food security. With rising living standards, the consumption of meat, poultry, eggs, and dairy products continues to grow, leading to a surge in demand for livestock feed and consequently, a rapid increase in the rigid demand for soybeans. However, a significant gap exists between total soybean production and market demand. Given limited arable land, simply expanding planting areas is insufficient to meet the demand. Therefore, increasing soybean yield per unit area has become a key approach to alleviating the supply-demand imbalance. This requires starting with variety selection and breeding, cultivating high-yield and high-quality varieties, and using scientific methods to tap the potential for increased soybean production, in order to better meet market demand and ensure a stable and sufficient food supply.

[0003] Internode spacing and number of nodes in soybeans are key agronomic traits that determine ideal plant architecture and yield per plant. Under conditions of relatively stable plant height, shorter internode spacing and more nodes are beneficial for increasing the number of pods per plant, thereby increasing yield per plant. However, currently, only a very small number of genes related to soybean internode spacing have been identified in natural populations, the molecular network regulating internode spacing is still unclear, and there is a lack of superior allelic variations in nature, which has prevented these genes from being applied in molecular breeding practices. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides an application of superior allelic variation of the key gene AIL1 for controlling internode spacing in soybean, which can provide an important theoretical basis for cultivating ideal soybean plant type and increasing soybean yield per plant, and has important scientific significance.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of this invention provides the application of the AIL1 gene mutant in the breeding of high-yield soybean varieties with dense planting, wherein the nucleotide sequence of the AIL1 gene is shown in SEQ ID NO:1.

[0007] The second aspect of this invention relates to the application of AIL1 gene mutants in regulating soybean internode spacing, wherein the nucleotide sequence of the AIL1 gene is shown in SEQ ID NO:1, and the regulation of soybean internode spacing is to shorten soybean internode spacing.

[0008] This invention, through research on the soybean AIL1 gene, discovered that its mutations can alter internode cell length and affect internode spacing. The developed molecular markers can precisely screen target plants. The created gene-edited mutants and identified superior natural allelic variants provide genetic resources for molecular design breeding. Backcross materials obtained using superior natural allelic variants or mutant materials obtained through gene editing exhibit stable internode spacing and minimal yield impact under different planting densities, making them suitable for breeding high-yielding, high-density soybean varieties and promoting the development of the soybean industry.

[0009] Preferably, the AIL1 gene has the amino acid sequence shown in SEQ ID NO:2.

[0010] Preferably, the AIL1 gene mutant includes an AIL1 gene-edited mutant and a naturally superior allelic variant mutant of the AIL1 gene.

[0011] More preferably, the AIL1 gene editing mutant includes the insertion of one base A between the 513th and 514th bases of the AIL1 gene; the insertion of one base C between the 513th and 514th bases; and the deletion of seven bases CCTTGTC between the 513th and 519th bases.

[0012] More preferably, the naturally superior allelic variant mutant of the AIL1 gene includes a 65 bp deletion at positions 1064-1128 of the AIL1 gene.

[0013] The third aspect of this invention provides a method for breeding high-yielding soybean varieties that are grown in densely. Specifically, soybean materials with naturally superior allelic mutations of the gene AIL1 are continuously backcrossed into high-yielding soybean varieties that are not tolerant of dense planting, so as to obtain backcross materials that retain the target mutation while restoring the superior traits of the recurrent parents.

[0014] Preferably, the naturally superior allelic mutant soybean material of the AIL1 gene includes Hap7 allelic mutant soybean, in which a 65bp deletion occurs at positions 1064-1128 of the AIL1 gene.

[0015] Preferably, the high-yielding but low-density soybean variety includes the Williams 82 soybean variety (W82).

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention focuses on the research of the soybean AIL1 gene. Firstly, through in-depth research on the AIL1 gene, it was discovered that it is closely related to the internode length of soybeans. Variations in this gene lead to changes in internode cell length, thereby affecting the internode spacing. Specifically, frameshift mutations in the AIL1 gene result in shortened internode spacing. Based on this, corresponding molecular markers were further developed, and AIL1 gene editing mutants were created. Several naturally occurring superior allelic mutants were identified in a large number of soybean materials. Finally, these superior allelic mutants were backcrossed into high-yielding but low-density planting soybean varieties. Field experiments showed that the internode spacing of the backcrossed materials remained stable under different dense planting conditions, and the yield per plant was minimally affected by density. This confirms that the AIL1 gene can be effectively applied to the breeding of high-yielding, high-density soybean varieties, providing a new approach to solving the problem of high yield in high-density soybean planting and playing a significant role in promoting the development of the soybean industry. Attached Figure Description

[0018] Figure 1 To identify AIL1, a key gene controlling internode spacing in soybean; A: Phenotypic images of W82 and DF02 plants; B: Statistical graph of internode spacing for W82 and DF02 plants; C: QTL mapping; DE: Fine mapping of AIL1.

[0019] Figure 2 The following data represent the statistical analysis of internode spacing phenotypes in near-isogenic AIL1 lines: A: Sequencing results of AIL1 in W82 and DF02 varieties; B: Statistical analysis of the average internode spacing phenotype of recombinant inbred lines; C: Phenotypic diagram of near-isogenic AIL1 lines in incubators; D: Statistical analysis of the average internode spacing of near-isogenic AIL1 lines in incubators; E: Cell length; F: Phenotypic diagram of near-isogenic AIL1 lines in the field; H: Statistical analysis of the average internode spacing of near-isogenic AIL1 lines in the field; I: Yield per plant in the field of near-isogenic AIL1 lines.

[0020] Figure 3 The gene editing mutation mode and the superior allelic mutation mode; A: AIL1 gene editing mutation mode; B: Identification of superior allelic variants of the AIL1 gene in 3921 soybean varieties.

[0021] Figure 4 For breeding applications of the AIL1 gene; A: field phenotype; B: average internode spacing; C: yield per plant. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0024] Example: Application of the AIL1 gene in regulating soybean internode spacing and in the breeding of high-yield soybean varieties through dense planting.

[0025] 1. Cloning of AIL1, a key gene controlling internode spacing in soybean

[0026] Phenotypic observations were conducted on soybean varieties W82 and DF02 (obtained from the Center for Molecular Genetics and Evolutionary Innovation, Guangzhou University) in an incubator based on their genome sequences. The W82 genome sequence is referenced in "Schmutz, J., Cannon, SB, Schlueter, J., Ma, JX, Mitros, T., Nelson, W., Hyten, DL, Song, QJ, Thelen, JJ, Cheng, JL, et al. (2010). Genome sequence of the palaeopolyploid soybean. Nature 463:178–183."; the DF02 genome sequence is referenced in "Lu, SJ, Dong, LD, Fang, C., Liu, SL, Kong, LP, Cheng, Q., Chen, LY, Su, T., Nan, HY, Zhang, D., et al. (2020). Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean." (domestication.Nat.Genet.52:428–436.) found that the average internode spacing of DF02 was significantly smaller than that of W82. Figure 1The mutation (AB) indicates that a key gene controlling soybean internode distance may have mutated in the DF02 variety, thus shortening the internode distance. To further investigate the key gene controlling soybean internode distance, F6 recombinant inbred lines (RILs) of the short-internode-distance local variety DF02 and the long-internode-distance cultivated soybean variety Williams82 (W82) were constructed. The F6 RILs population was then planted in a greenhouse (12 / 12h light / dark, 25℃), and the average internode distance phenotype was investigated 20 days after emergence. Two mixed pools were formed, with 30 individuals with long internode distances and 30 individuals with short internode distances respectively. Bulk segregant analysis (BSA) was used to locate the QTL controlling soybean internode distance. The results showed that a new QTL controlling the average internode distance in soybean was identified on chromosome 10. Figure 1 C), therefore it was named AIL1 (Average Internode Length 1). The AIL1 gene sequence and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0027] Using molecular markers (AIL1-IndelF: GCATAGGGCAGTGGTGAA (SEQ ID NO:3); AIL1-IndelR: TTCCTTTATCTGCCTTTGC (SEQ ID NO:4)), heterozygous individuals on chromosome 10 were selected from the F6 generation RILs population for fine mapping of the AIL1 locus. Through fine mapping analysis, the AIL1 gene was located within a 45kb physical region on chromosome 10. Figure 1 According to gene prediction results from the W82 reference genome (W82 genome sequence reference "Schmutz, J., Cannon, SB, Schlueter, J., Ma, JX, Mitros, T., Nelson, W., Hyten, DL, Song, QJ, Thelen, JJ, Cheng, JL, et al. (2010). Genome sequence of the palaeopolyploid soybean. Nature 463:178–183."), a gibberellin synthesis-related enzyme GA20OX was found in this region. It is speculated that this gene is a candidate coding gene. In the DF02 variety, this gene may have mutated, resulting in a reduction in endogenous gibberellin synthesis, which in turn shortens the internode spacing of soybean.

[0028] The nucleotide sequence (SEQ ID NO:1) of the coding region of the AIL1 gene (gene ID: Glyma.10G241100) is as follows:

[0029]

[0030] The amino acid sequence encoded by the AIL1 gene (gene ID: Glyma.10G241100) (SEQ ID NO:2) is as follows:

[0031] MAIDCITSIPMMPQPPTQETTKEQEQQQPLVFDASVLRHQLHIPSQFIWPDEEKACLDEPELFVPFIDLGGFLSGDPVAATEASRLAGEACQKHGFFLVVNHGIDQRLICDAHLYMEHFFGLPLSQKQRAQRKPGEHCGYASSFTGTDKNSSPALVKDYLCSKMGKEFEQFGKVYQDYCDAMS NLSLGIMELLGMSLGVGRACFREFFEENSSIMRLNYYPPCQKPDLTLGTGPHCDPTSLTILHQDQVGGLQVCVDNEWHSIKPDLNAFVVNVGDTFMALSNGRYKSCLHRAVVNSQTTRKSLAFFLCPRSDKVVSPPCELVDNLSPRLYPDFTWPMLLEFTQKHYRADMKTLEAFANWLRKSN.

[0032] 2. Significant differences in interstitial phenotypic differences between near-isogenic AIL1 lines.

[0033] To further confirm that the gibberellin synthesis-related enzyme GA20OX is the encoding gene for AIL1, the gene was sequenced in varieties W82 and DF02, respectively. The results showed that in DF02, a 65 bp nucleotide deletion was found in the fourth exon of GA20OX (TGCTGCTGGAGTTCACTCAAAAGCATTACAGAGCTGACATGAAAACCCTTGAGGCATTTGCCAAC, SEQ ID NO:5), resulting in a frameshift mutation of the amino acid following that position. Figure 2 A). Based on this 65bp difference, molecular markers were developed (AIL1-IndelF: GCATAGGGCAGTGGTGAA (SEQ ID NO:3); AIL1-IndelR: TTCCTTTATCTGCCTTTGC (SEQ ID NO:4)). These markers were used to divide the RIL population into AIL1 (RIL-AIL1, normal AIL1 gene) and ail1 (RIL-ail1, a 65bp deletion at positions 1064-1128 of the AIL1 gene). Internode spacing phenotypic results showed that the internode spacing of the RIL-ail1 population was significantly smaller than that of the RIL-AIL1 population. Figure 2B). Subsequently, using this molecular marker, near-isogenic lines of AIL1 (NIL-AIL1, with normal AIL1 gene; NIL-ail1, with a 65bp deletion at positions 1064-1128 of the AIL1 gene) were identified in F6 recombinant inbred lines. These lines were planted in incubators and in the field, and in both cases, the internode spacing of NIL-ail1 was found to be significantly smaller than that of NIL-AIL1. Figure 2 CD, 2F-I). Cell morphology in the stems of near-isogenic lines was examined (specific detection methods were described in the literature "Li, SC, Sun, ZH, Sang, Q., Qin, C., Kong, LP, Huang, X., et al. (2023). Soybean reduced internode 1 determines internode length and improves grain yield at dense planting. Nat. Commun. 14:7939"). It was found that the cell length of NIL-ail1 was significantly smaller than that of NIL-AIL1 (CD, 2F-I). Figure 2 E) suggests that AIL1 may affect intersegmental length by influencing cell length.

[0034] 3. Creation of AIL1 gene-editing mutants and identification of naturally occurring superior allelic variants

[0035] To further confirm the function of AIL1, three gene-edited mutants of AIL1 were created in the soybean variety Williams82 using gene editing and stable transgenic technology (the specific mutant methods are described in the literature "Cai, Y. Chen, L., Liu, X., Guo, C., Sun, S., Wu, C., Jiang, B., Han, T., Hou, W. (2018.) CRISPR / Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soybean. Plant Biotechnology Journal. 16, 176-185"). The first mutant inserts one base A between bases 513 and 514 (ail1). CR -1); The second method inserts one base C (ail1) between bases 513 and 514. CR -2); the third type has 7 bases missing at positions 513-519 (CCTTGTC, ail1) CR -3; Figure 3A). Subsequently, the natural allelic variation of AIL1 in 1295 soybean materials was further analyzed (specific analysis methods and references for 1295 soybean materials: "Lu, SJ, Dong, LD, Fang, C., Liu, SL, Kong, LP, Cheng, Q., Chen, LY, Su, T., Nan, HY, Zhang, D., et al. (2020). Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean"). The study, conducted in domestication (Nat. Genet. 52:428–436), identified seven allelic variants. The Hap1 variant corresponds to the W82 genotype in the reference genome. The Hap2 variant involves a base substitution at position 16 of the AIL1 gene, changing from A to T and from I to L. The Hap3 variant involves a base substitution at position 260, changing from C to T and from A to V, with a three-base ACT insertion at positions 500-502, resulting in an insertion of one amino acid Y at position 167. The ap4 type has a base deletion at positions 439-440, resulting in a frameshift mutation of the amino acid; the Hap5 type has a base substitution at position 802, changing from G to C, and the amino acid changes from G to R; the Hap6 type has a base substitution at position 944, changing from A to G, and the amino acid changes from Q to R; the Hap7 type has a 65bp base deletion at positions 1064-1128 (TGCTGCTGGAGTTCACTCAAAAGCATTACAGAGCTGACATGAAAACCCT TGAGGCATTTGCCAAC, SEQ ID NO:5), resulting in a frameshift mutation of the amino acid. Figure 3 B). The creation of the above gene-editing mutants and the screening of haplotypes will provide important genetic resource support for soybean molecular design breeding.

[0036] 4. Application of AIL1 gene editing mutants and superior allelic variants in high-density, high-yield soybean.

[0037] Soybean material DF02, containing the Hap7 allelic variant of the AIL1 gene, was backcrossed four times with W82 to obtain soybean material W82-ail1-BC3. W82, the backcross material W82-ail1-BC3, and the gene-edited mutant ail1 were then compared. CRSoybean varieties -1 were planted in a field in Gongzhuling City, Jilin Province, with planting densities of 100 plants / row, 80 plants / row, and 60 plants / row. The yield per plant was investigated at each planting density. The results showed that, for the control soybean material W82, internode lengthening and yield per plant decreased with increasing planting density, while W82-ail1-BC3 and ail1... CR Under different planting densities, the difference in internode spacing is not significant, and the difference in yield is also not significant. Figure 4 The same results were obtained by verifying five other superior allelic variants of the AIL1 gene (A, 4B), indicating that gene editing and natural variant mutants of AIL1 can be applied to the breeding of high-yield, densely planted soybean varieties.

[0038] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of AIL1 gene mutants in the breeding of high-yield, densely planted soybean varieties, characterized by, The nucleotide sequence of the AIL1 gene is shown in SEQ ID NO: 1; the AIL1 gene mutant is an AIL1 gene editing mutant or a naturally superior allelic variant mutant of the AIL1 gene; The AIL1 gene editing mutant is a gene editing mutant that inserts one base A between the 513th and 514th bases of the AIL1 gene; or a gene editing mutant that inserts one base C between the 513th and 514th bases of the AIL1 gene; or a gene editing mutant that deletes seven bases CCTTGTC between the 513th and 519th bases of the AIL1 gene. The naturally superior allelic variant of the AIL1 gene is a naturally superior allelic variant mutant with a 65 bp deletion at positions 1064-1128 of the AIL1 gene.

2. The application of AIL1 gene mutants in regulating soybean internode spacing, characterized in that, The nucleotide sequence of the AIL1 gene is shown in SEQ ID NO: 1, and the regulation of soybean internode spacing is to shorten soybean internode spacing; the AIL1 gene mutant is an AIL1 gene editing mutant or a naturally superior allelic variant mutant of the AIL1 gene; The AIL1 gene editing mutant is a gene editing mutant that inserts one base A between the 513th and 514th bases of the AIL1 gene; or a gene editing mutant that inserts one base C between the 513th and 514th bases of the AIL1 gene; or a gene editing mutant that deletes seven bases CCTTGTC between the 513th and 519th bases of the AIL1 gene. The naturally superior allelic variant of the AIL1 gene is a naturally superior allelic variant mutant with a 65 bp deletion at positions 1064-1128 of the AIL1 gene.

3. The application according to claim 1 or 2, characterized in that, The amino acid sequence of the AIL1 gene is shown in SEQ ID NO:

2.

4. A method for cultivating high-yield soybean varieties with dense planting, characterized in that, Soybean materials with naturally superior allelic variants of the AIL1 gene are repeatedly backcrossed into high-yielding but low-density-planting-tolerant soybean varieties to obtain backcross materials that retain the target variant while restoring the superior traits of the recurrent parents. The nucleotide sequence of the AIL1 gene is shown in SEQ ID NO:

1. The soybean materials with naturally superior allelic variants of the AIL1 gene are Hap7 allelic variant soybeans, which have a 65 bp deletion at positions 1064-1128 of the AIL1 gene. The high-yielding but low-density-planting-tolerant soybean variety is the Williams 82 soybean variety.

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