GENE LBA5 FOR REGULATING LATERAL SHOOT ANGLES, GROWTH HABITS, AND PLANT ARCHITECTURE OF ARACHIS HYPOGAEA L., AND USE THEREOF

Abstract

A gene LBA5 for regulating lateral shoot angles, growth habits, and a plant architecture of Arachis hypogaea L., and use thereof are provided. In the present disclosure, a major gene LBA5 for controlling lateral shoot angles, growth habits, and a plant architecture of Arachis hypogaea L. is mapped and cloned from Arachis hypogaea L., which includes two homologous genes LBA5b and LBA5a and promoters thereof. The allelic variations of the gene can be selected through crossbreeding and backcrossing to achieve the genetic improvement on an angle between an Arachis hypogaea L. lateral shoot and a main stem. Through a genetic engineering operation for the gene and a change for a promoter sequence of the gene, the function or expression level of this gene in a procumbent Arachis hypogaea L. variety can be adjusted to further regulate an angle between an Arachis hypogaea L. lateral shoot and a main stem.

Claims

1-5. (canceled)

6. Primers for cloning an Arachis hypogaea L. gene LBA5b, wherein the primers are LBA5b-F and LBA5b-R, sequences of the LBA5b-F and LBA5b-R are shown in SEQ ID NOs: 17-18; and the primers are used in cDNA of procumbent Arachis hypogaea L. to clone and amplify a complete coding frame of the Arachis hypogaea L. gene LBA5b by PCR.

7. Primers for constructing an overexpression transgenic vector of an Arachis hypogaea L. gene LBA5b, wherein the overexpression transgenic vector is pHB-LBA5b; the primers are LBA5b-OE-F and LBA5b-OE-R, sequences of the LBA5b-OE-F and the LBA5b-OE-R are shown in SEQ ID NOs: 19-20; and the primers are used in cDNA of procumbent Arachis hypogaea L. or a plasmid with the cDNA for amplification to obtain the Arachis hypogaea L. gene LBA5b, and an amplification product is digested with an enzyme and ligated into an overexpression vector pHB to construct the overexpression transgenic vector.

8. Sequences for constructing a target for gene editing on an Arachis hypogaea L. gene LBA5b, wherein the sequences comprise sgRNA1 and sgRNA2 shown in SEQ ID NOs: 21-22; and the sgRNA1 and the sgRNA2 are ligated into an sgRNA region of a CRISPR/Cas9 vector to construct a gene editing vector for a target gene LBA5, and then the gene editing vector is transformed into Arachis hypogaea L. to realize the gene editing for the Arachis hypogaea L. gene LBA5b.

9. Use of a gene LBA5 for regulating a lateral shoot angle, a growth habit, and a plant architecture of Arachis hypogaea L. in crop genetic improvement, wherein the use refers to use in an improvement of the lateral shoot angle, the growth habit, and the plant architecture of the Arachis hypogaea L.; the gene LBA5 comprises homologous genes LBA5b and LBA5a from two subgenomes of cultivated Arachis hypogaea L. and corresponding defunctionalized alleles lba5b and lba5a; the homologous gene LBA5b has a nucleotide sequence shown in SEQ ID NO: 1; the defunctionalized allele lba5b has at least three different allelic variations named as lba5b-1, lba5b-2, and lba5b-3, respectively; the lba5b-1 has a nucleotide sequence shown in SEQ ID NO: 2; the lba5b-2 has a nucleotide sequence shown in SEQ ID NO: 25; the lba5b-3 has a nucleotide sequence shown in SEQ ID NO: 26; the homologous gene LBA5a has a nucleotide sequence shown in SEQ ID NO: 3; and the defunctionalized allele lba5a has a nucleotide sequence shown in SEQ ID NO: 4.

10. The use of the gene LBA5 for regulating the lateral shoot angle, the growth habit, and the plant architecture of the Arachis hypogaea L. in crop genetic improvement according to claim 9, wherein the homologous genes LBA5b and LBA5a of the gene LBA5 are directly derived from the Arachis hypogaea L., or derived from Glycine max L., Brassica napus L., Gossypium spp., Oryza sativa L., Zea mays L., Triticum aestivum L., and other crops.

11. Use of mRNA or cDNA encoded by genes LBA5b and LBA5a for regulating a lateral shoot angle, a growth habit, and a plant architecture of Arachis hypogaea L. in crop genetic improvement, wherein the use refers to use in an improvement of the lateral shoot angle, the growth habit, and the plant architecture of the Arachis hypogaea L.; the cDNA encoded by the gene LBA5b has at least four forms shown in SEQ ID NOs: 5-8, respectively; and the cDNA encoded by the gene LBA5a has at least two forms shown in SEQ ID NOs: 9-10, respectively.

12. Use of amino acids, polypeptides, or proteins encoded by genes LBA5b and LBA5a for regulating a lateral shoot angle, a growth habit, and a plant architecture of Arachis hypogaea L. in crop genetic improvement, wherein the use refers to use in an improvement of the lateral shoot angle, the growth habit, and the plant architecture of the Arachis hypogaea L.; the proteins encoded by the gene LBA5b have amino acid sequences shown in SEQ ID NOs: 11-12; and the proteins encoded by the gene LBA5a have amino acid sequences shown in SEQ ID NOs: 13-14.

13. Use of promoters of genes LBA5b and LBA5a for regulating a lateral shoot angle, a growth habit, and a plant architecture of Arachis hypogaea L. in crop genetic improvement, wherein the use refers to use in an improvement of the lateral shoot angle, the growth habit, and the plant architecture of the Arachis hypogaea L.; the promoter of the gene LBA5b has a nucleotide sequence shown in SEQ ID NO: 15; and the promoter of the gene LBA5a has a nucleotide sequence shown in SEQ ID NO: 16.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 shows the BSA-seq genetic mapping of the gene LBA5b using an HN-F.sub.6 population according to the present disclosure.

[0021] FIG. 2 shows the BSR-seq genetic mapping of the gene LBA5b using a PF-F.sub.5 population according to the present disclosure.

[0022] FIG. 3 shows the expression level analysis of the genes LBA5a and LBA5b in stem ends of lateral shoots of three upright varieties and three procumbent varieties according to the present disclosure.

[0023] FIG. 4 shows the expression analysis of the gene LBA5b in different tissues of procumbent varieties and upright varieties according to the present disclosure.

[0024] FIG. 5 shows the phenotypes of upright varieties with transgenic overexpression of the gene LBA5b according to the present disclosure.

[0025] FIG. 6 shows the effect of gene editing on the gene LBA5b in procumbent varieties according to the present disclosure.

[0026] FIG. 7 shows the reduction of a lateral shoot angle of procumbent Arachis hypogaea L. varieties after shading and the change of an expression level of the gene LBA5b after shading according to the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The technology of the present disclosure is further illustrated through examples below. In the following examples, a method for mapping and cloning LBA5b and a homologous gene thereof, a method for verifying base differences among LBA5b alleles through sequencing, and use of a transgenic method to prove the function of the LBA5b gene are further described.

Example 1: Preliminary Mapping of an Arachis hypogaea L. Lateral Shoot Angle Using the BSA-Seq Technology

[0028] In this example, an Arachis hypogaea L. variety Xiaohongmao of a procumbent plant architecture that had a basal angle of 90° between a lateral shoot and a main stem and an Arachis hypogaea L. variety Henan Nanyang of an upright plant architecture were used to construct a hybrid combination, and a line breeding method was used to obtain a recombinant inbred line RIL-HN-F.sub.6 composed of 223 individuals; the lateral shoot angles, growth habits, and a plant architecture were investigated for the recombinant inbred line, and 30 of the individuals that had large lateral shoot angles and were more similar to the Xiaohongmao parent were selected and 26 of the individuals that had small lateral shoot angles and were more similar to the Henan Nanyang parent to constitute 2 DNA mixed pools P (procumbent) and Z (upright); sequencing was conducted on a machine, with a parental sequencing depth of >20× and a mixed pool sequencing depth of >30×, and a sequencing result was compared with a genome of the published cultivated Arachis hypogaea L. variety Tifrunner (a reference); with the upright plant architecture as a mutant plant architecture, SNP-index was calculated based on two progeny mixed pools, and a distribution map of the SNP-index of the two progeny mixed pools on each chromosome was plotted through a 2M sliding window; theoretically, a peak with an SNP-index value deviating from 0.5 was considered to be a result of trait selection, but due to the existence of distorted segregation, the presence of false QTL peaks could not be determined; and thus SNP-index of a wild plant architecture (a procumbent plant architecture) was subtracted from SNP-index of a mutant plant architecture (upright plant architecture) to obtain SNP-index (FIG. 1). In order to eliminate the interference of distorted segregation, the same sliding window was used to plot a ΔSNP-index trajectory map, which could accurately screen out a target interval. Calculated ΔSNP-index was subjected to 1,000 permutation tests, the 95% confidence interval and 99% confidence interval levels were used as thresholds for screening, and results were given.

[0029] It can be seen from FIG. 1 that SNPs beyond the 99% confidence level only existed on the three chromosomes of chromosomes 3, 6, and 15, where the chromosome 15 exceeded a threshold line very obviously and the highest ΔSNP-index was as high as 1.0; it could be preliminarily inferred that there was a major locus related to the lateral shoot angle at an end of the chromosome 15; and most SNP loci beyond the 99% confidence level appeared on the chromosome 15, the ΔSNP-index>0.5 accounted for 74.6% of all SNP loci beyond the 99% confidence level, and thus it could be inferred that there were a major locus at 153.8 M to 159.6 M on the chromosome 15.

[0030] According to a BSA-seq mapping result, 31 pairs of InDel markers between the two parents Xiaohongmao and Henan Nanyang on the chromosome 15 of Arachis hypogaea L. were developed and analyzed to obtain 19 pairs of InDel markers with prominent codominance and gel analysis effects between the two parents Xiaohongmao and Henan Nanyang, and the 19 pairs of InDel markers with prominent parental codominance were used to conduct genotyping for 192 members selected in the HN-F.sub.6 population; according to genotypes of InDel markers, a genetic map construction module in the software QTL IciMapping was used to conduct local genetic map construction, with LOD=3.0 as a threshold, a linkage group coverage genetic distance of 192.19 cM, and an average distance of 17.47 cM among markers; a value was assigned for a phenotype of the HN-F.sub.6 population: procumbent plant architecture: 90°, semi-procumbent plant architecture: 50°, and upright plant architecture: 10°; then in combination with a plant architecture of a population marker and the above genetic map, a mapping module in the software QTL IciMapping was used to conduct QTL mapping, and the inclusive composite interval mapping for additive and dominance (ICIM-ADD) was used to conduct iterative sampling 1,000 times to determine an LOD threshold, where the LOD threshold was 2.50, a major locus on the chromosome 15 was mapped between markers J15-11 and J15-12 with a contribution rate of 44.59%, and an enhanced locus was from Xiaohongmao, which could theoretically increase the lateral shoot angle by 25.91° C.; and InDel markers discovered by re-sequencing and the entire population of HN-F.sub.6 were used for linkage mapping to verify the BSA-seq mapping result and define the locus between 156859290 bp to 157561753 bp on the chromosome B05 (an interval of about 702.46 kb).

Example 2: Mapping of an Arachis hypogaea L. Lateral Shoot Angle Using the BSR-Seq Technology

[0031] In this example, another upright variety Pingdu 9616 and a procumbent variety Florunner were used to construct a recombinant inbred line PF-F.sub.5, and 30 procumbent members and 30 upright members were separately selected from the recombinant inbred line; transcriptome sequencing was conducted for the two parents, the 30 procumbent members, and the 30 upright members, where a sequencing output for each offspring generation was no less than 3 Gb Clean Date, and a sequencing output for each parental sample was no less than 6 Gb Clean Date; a sequencing result was compared with a genome of the published cultivated Arachis hypogaea L. variety Tifrunner (as a reference); and with 30 individuals as a mixed pool, SNP data obtained from the transcriptome sequencing were used to calculate ΔSNP-index for a procumbent pool and an upright pool (FIG. 2), and the loci related to the lateral shoot angles, growth habits, and a plant architecture of Arachis hypogaea L. were also mapped at an end of the chromosome B05, which coincided with a mapping result of the HN population. According to an SNP genotype of each individual, an interval range was defined between 153809051 bp to 158407822 bp on the chromosome B05. A gene expression level was analyzed for the 30 procumbent individuals and the 30 upright individuals, results showed that there was a differentially expressed gene (DEG) in a mapping interval, which strictly conformed to the rule that the gene was highly expressed in both the procumbent parents and individuals, but was almost not expressed in the upright parents and offsprings. The gene was initially determined as a candidate gene for LBA5b, and the gene from the chromosome B5 was named LBA5b (SEQ ID NO: 1), which had at least four different transcription modes (SEQ ID NOs: 5-8). A homologous gene derived from the chromosome A05 was named LBA5a (SEQ ID NO: 3), which had at least two transcription modes (SEQ ID NOs: 9-10). Then the expression levels of LBA5b in Tifrunner, Florunner, Monticola, Xiaohongmao, Pingdu 9616, and Henan Nanyang were analyzed (see FIG. 3), and it could be known that the LBA5b was not expressed or expressed at a low level in upright cultivated Arachis hypogaea L., and the LBA5b was expressed at a high level in procumbent cultivated Arachis hypogaea L. As a result, it can be concluded that the LBA5b gene is one of the key gene loci for controlling lateral shoot angles of Arachis hypogaea L.

Example 3: Gene Cloning and Structure and Function Prediction for LBA5b

[0032] (1) Gene Cloning for LBA5b:

[0033] Total RNA was extracted from a stem end of a lateral shoot of the procumbent Arachis hypogaea L. Tifrunner, and reverse-transcribed into cDNA. A cloning primer pair B5cd-F/R (with sequences of SEQ ID NO: 17/18) was used to conduct PCR amplification with the Tifrunner cDNA as a template. A PCR product was recovered and purified through gel, then ligated into a T vector, and transformed into Escherichia coli (E. coli), sequencing was conducted, and a strain with a correct sequence was reserved for later use. Specifically, in the PCR amplification, a PCR system (25 μl in total) included: 2×Gflex PCR Buffer (Mg.sup.2+, dNTP plus): 12.5 μl, Template: 1 μl, 10 μm upstream and downstream primers: 1 μl for each, Tks Gflex DNA Polymerase: 1 μl, and water: the balance; and a PCR procedure included: pre-denaturation at 94° C. for 1 min; denaturation at 98° C. for 10 s, annealing at 55° C. for 15 s, and extension at 68° C. for 30 s, with 35 cycles.

[0034] (2) Gene Structure Analysis for LBA5b:

[0035] It was found that a cloned LBA5b gene had 4 transcription modes, with two in the 5′UTR region and two in the coding region. In a first mode, 212 amino acids were encoded, and in a second mode, 227 amino acids were encoded. The first mode was dominant in transcription. Through domain analysis, it was found that an LBA5b protein was an MADS transcription factor with two protein domains of MADS and K-box for the MIKC plant architecture.

[0036] Genome sequencing and methylation analysis of this gene in multiple procumbent and upright varieties showed that a promoter of this gene (SEQ ID NO: 15) had no significant difference in sequence and methylation level among the varieties. However, in the upright variety shitouqi or Fu peanut, there was a double-base TA insertion in the first exon coding frame of the gene, such that a reading frame of the gene eventually underwent frameshift, a protein with a full function could not be formed, and thus the gene failed to be expressed; and the allelic variation was named lba5b-1 (SEQ ID NO: 2). Sequencing, enzyme digestion, or other means was used to determine whether there is the TA insertion mutation in Arachis hypogaea L. germplasm resources, and it was found that the TA insertion/deletion mutation was closely related to the upright/procumbent plant architecture of Arachis hypogaea L. In the upright varieties Luhua 11, Huayu 36, and Shanhua 11, there was a deletion of 1,870 bp in the first intron of the gene, and the gene was not transcribed, resulting in the upright plant architecture; and this allelic variation was named lba5b-2 (SEQ ID NO: 25). In the upright variety MJX7, there was a deletion of 985 bp that started from the 5′UTR region and included a first exon, and this gene was severely incomplete, resulting in the upright plant architecture; and this allelic variation was named lba5b-3 (SEQ ID NO: 26).

[0037] (3) Genetic Characteristics Analysis for LBA5b:

[0038] According to gene annotation, the Arachis hypogaea L. LBA5 encodes an MADS transcription factor for the MIKC plant architecture (MIKC MADS gene family). The most homologous gene in A. thaliana is AT2G45660 (AGAMOUS-like 42), and thus this gene is also called AhAGL42 (AGAMOUS-like 42 of Arachis hypogaea L., referred to as LBA5 in this example), which is also a member of the MIKC MADS gene family. AT2G45660 (AGAMOUS-like 42) plays an important role in the development and regulation of A. thaliana at a flowering phase, but plays no role in the regulation of a lateral shoot angle, and in this example, it was found for the first time that the gene has the function to regulate lateral shoot angles, growth habits, and a plant architecture of Arachis hypogaea L. Tissue transcriptome sequencing analysis and real-time fluorescence quantitative tissue expression profile analysis for the gene LBA5b showed that, in the procumbent varieties, LBA5b was predominantly expressed in the leaves, needle tips, and pistils of lateral shoots, but expressed at an extremely-low level in the main stem leaves (see FIG. 4). Shading can significantly reduce an expression level of the gene LBA5b, and can also significantly reduce an angle between a lateral shoot and a main stem (see FIG. 7). It is inferred that RNAi, antisense RNA, and other technologies can be used to regulate an expression level of this gene, thereby accurately regulating an angle between a lateral shoot and a main stem of Arachis hypogaea L.

[0039] Cloning and sequencing for the LBA5a gene in the procumbent and upright varieties of cultivated Arachis hypogaea L. showed that a coding region of this gene had no difference among the varieties. However, compared with a donor AA genome of the wild variety Arachis duranensis (A. duranensis), a coding frame of the gene had a C base deletion, resulting in the premature termination of an encoding protein; and the allelic variation was named lba5a (SEQ ID NO: 4). Through expression analysis by ordinary RT-PCR, it was found that, in both the upright variety and the procumbent variety, LBA5a was not expressed in the main stem (or expressed at a very low level), and expressed at a high level in lateral shoots; but the expression level in the procumbent variety was significantly higher than that in the upright variety. It was also found that, in the procumbent variety, the expression level of LBA5a was about ¼ lower than the expression level of LBA5b (see FIG. 2). The promoter sequences of LBA5b and LBA5a were analyzed, and many differences were discovered.

[0040] It could be inferred that the expression difference was caused by the differences in the promoter sequences of these two genes. Based on this, it was proposed that the promoter sequences of these two genes could be changed to change the expression levels of the two genes, thereby regulating lateral shoot angles, growth habits, and a plant architecture of Arachis hypogaea L.

[0041] Through continuous backcrossing, the TA insertion mutant allelic variation lba5b of LBA5b was introduced into the procumbent variety Tifrunner, and it was found that an angle between a lateral shoot and a main stem of Tifrunner.sup.lab5b was significantly reduced, and thus more individual plants could be planted in the same land, thereby increasing a yield per unit area of Arachis hypogaea L.

Example 4: LBA5b Overexpression Transgenesis can Increase an Angle Between a Lateral Shoot and a Main Stem of Arachis hypogaea L

[0042] In this example, 35S was used as a promoter to construct an overexpression vector, and the overexpression of LBA5b was achieved in an upright variety by the pollen tube introduction method. Specific steps were as follows: a T plasmid with the LBA5b gene and an overexpression vector plasmid pHB were separately digested with HindIII and pstl; a target fragment and a pHB plasmid vector backbone fragment obtained from enzyme digestion were recovered and purified through gel, and then ligated overnight by a T4 ligase; a ligation product was transformed into competent E. coli DH5a by heat shock, and then the competent E. coli was coated on a LB plate with kanamycin; single colonies were picked for PCR detection, positive colonies were sent to a biological company for sequencing, and correct strains were selected for shaking cultivation; a plasmid with the target fragment was extracted, which was an LBA5b overexpression plasmid: pHB-LBA5b; the LBA5b overexpression plasmid was transformed into competent Agrobacterium tumefaciens (A. tumefaciens), then the A. tumefaciens was coated on a YEB plate with kanamycin and rifampicin, and single colonies were picked for PCR detection to obtain positive colonies for later use, which were transgenic strain; and the overexpression vector with the LBA5b gene was transformed into the upright cultivated Arachis hypogaea L. Huayu 23, and positive individuals were screened out to observe the change in the lateral shoot angle.

[0043] The positive plants can be screened out by introducing a gene into a vector, and can also be determined by detecting an expression level of LBA5b. Phenotypic analysis of positive plants showed that the overexpression of the LBA5b gene in Huayu 23 can increase an angle between a lateral shoot and a main stem (see FIG. 5), proving that the LBA5b gene has the function of increasing a lateral shoot angle of Arachis hypogaea L.

Example 5: Knockout of LBA5b Through Gene Editing can Reduce an Angle Between a Lateral Shoot and a Main Stem of Arachis hypogaea L

[0044] In this example, the CRISPR/CAS9 system was used to conduct knockout through gene editing.

[0045] Specific construction steps were as follows: a gRNA target sequence was designed and generated online (http://www.biogle.cn/index/excrispr), and two targets sites sgRNA1 and sgRNA2 (SEQ ID NOs: 21-22) with the highest score were selected; a generated Oligo sequence was synthesized by a biological company, and synthesized Oligo was dissolved in water to 10 μM, and 18 μl of Buffer Anneal, 1 μl of Up Oligo, and 1 μl of Low Oligo were mixed in a 200 μl PCR tube, heated at 95° C. for 3 min, and then slowly cooled to 20° C. at a rate of about 0.2° C./s to prepare a Oligo dimer; the Oligo dimer was introduced into a CRISPR/Cas9 vector; 2 μl of CRISPR/Cas9 Vector, 1 μl of the Oligo dimer, 1 μl of Enzyme Mix, and 16 μl of ddH.sub.2O were thoroughly mixed in a 200 μl PCR tube to allow a reaction at room temperature (20° C.) for 1 h; a ligation product was transformed into competent E. coli DH5a by heat shock, and then the competent E. coli was coated on a LB plate with kanamycin; single colonies were picked for PCR detection, positive colonies were sent to a biological company for sequencing, and correct strains were selected for shaking cultivation; a plasmid with the target fragment was extracted, which was an AhLBA knockout plasmid: BGK041-AhLBA-½; the AhLBAb gene knockout plasmid BGK041-AhLBA-½ was transformed into competent A. tumefaciens, then the A. tumefaciens was coated on a YEB plate with kanamycin and rifampicin, and single colonies were picked and subjected to PCR detection with the primer pair CS4-F/R (SEQ ID NOs: 23-24); positive colonies were selected and transformed into Arachis hypogaea L. BGK041 was used as the CRISPR/Cas9 vector. The vector used the Glycine max L. U6 promoter to drive the SG sequence, which can be efficiently used for dicotyledonous plants. An enhanced CaMV promoter was used to achieve the efficient expression of the Cas9 protein.

[0046] Transgenic plants were subjected to PCR detection with the primer pair of CS4-F/R to determine whether the vector sequence was introduced. Genetic sequencing of the target gene LBA5b was conducted for positive transgenic plants, and offsprings whose target sequence LBA5b changed were detected (see FIG. 6). Individuals undergoing gene frameshift mutation or premature termination were selected, and it was found that a lateral shoot angle changed from procumbent to upright. That is, the knockout of the gene can significantly reduce an angle between a lateral shoot and a main stem of an offspring.

Example 6: Nucleic Acid and Protein Sequences

[0047]

TABLE-US-00001 TABLE 1 Sequence names and origins of SEQ ID Nos: 1-26 SEQ ID NO. Name Origin 1 LBA5b gDNA sequence Arachis hypogaea L. variety Tifrunner 2 lba5b-1 gDNA sequence Arachis hypogaea L. variety Shitouqi 3 LBA5a gDNA sequence Wild diploid A. duranensis 4 lba5a gDNA sequence Arachis hypogaea L. variety Tifrunner 5 LBA5b.1 cDNA sequence Arachis hypogaea L. variety Tifrunner 6 LBA5b.2 cDNA sequence Arachis hypogaea L. variety Tifrunner 7 LBA5b.3 cDNA sequence Arachis hypogaea L. variety Tifrunner 8 LBA5b.4 cDNA sequence Arachis hypogaea L. variety Tifrunner 9 LBA5a.1 cDNA sequence Wild diploid A. duranensis 10 LBA5a.2 cDNA sequence Wild diploid A. duranensis 11 LBA5b.1 protein sequence Arachis hypogaea L. variety Tifrunner 12 LBA5b.2 protein sequence Arachis hypogaea L. variety Tifrunner 13 LBA5a.1 protein sequence Wild diploid A. duranensis 14 LBA5a.2 protein sequence Wild diploid A. duranensis 15 LBA5b Promoter gDNA sequence Arachis hypogaea L. variety Tifrunner 16 LBA5a Promoter gDNA sequence Arachis hypogaea L. variety Tifrunner 17 LBA5b-F cloning primer sequence Artificial sequence 18 LBA5b-R cloning primer sequence Artificial sequence 19 LBA5b-OE-F overexpression Artificial sequence vector primer 20 LBA5b-OE-R overexpression Artificial sequence vector primer 21 sgRNA1 target sequence Artificial sequence 22 sgRNA2 target sequence Artificial sequence 23 CS4-F Cas9 detection primer Artificial sequence 24 CS4-R Cas9 detection primer Artificial sequence 25 lba5b-2 gDNA sequence Arachis hypogaea L. variety Luhua 11 26 lba5b-3 gDNA sequence Arachis hypogaea L. variety MJX7