PvPW1 and the encoding protein and application thereof

Abstract

The application relates to the field of genetic engineering, in particular to a bean beta-1,3-glucanase PvPW1, an encoding protein and application thereof. The application aims to provide the bean beta-1,3-glucanase PvPW1, the encoding protein and the application thereof, and provides a theoretical basis for bean biological breeding. The nucleotide sequence of the gene is shown in SEQ ID NO:1 in the sequence table, and the amino acid sequence of the encoding protein is shown in SEQ ID NO:9. Through the map-based cloning of a biparental genetic population and a gene haplotype analysis method, it is confirmed that the PvPW1 plays a role in regulating the width of a bean pod. The application is used for cultivating wide-pod or narrow-pod bean varieties through molecular assisted breeding and gene selection breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a bean β-1,3-glucanase PvPW1, its encoded protein, and its applications. Background Technology

[0002] Green beans are both a vegetable and a "minor grain," and when properly combined with other grain crops, they can balance the nutrients needed by the human body. Currently, high-quality green beans have become one of the most distinctive local agricultural products of Heilongjiang Province. With the promotion of Northeast China's culinary culture nationwide, green beans are gradually being accepted by consumers across the country, resulting in strong market demand. However, green bean gene cloning and functional analysis are still in their early stages; green bean breeding work is mainly based on conventional breeding, and a molecular design breeding module for green beans has not yet been established. Therefore, there has been no significant improvement in breeding efficiency or precise improvement of specific traits compared to 20 years ago. Thus, cloning and analyzing the regulatory genes related to the main agronomic traits of green beans, especially pod morphology and quality, will provide a theoretical basis for cultivating superior new green bean varieties through molecular breeding.

[0003] Common beans, cowpeas, and sword beans, among other legumes, are also crops whose fruits (fresh pods) are consumed. The pod morphology of common beans is one of the factors affecting their quality. Common bean pod morphology includes: pod color (pod skin color and color uniformity), pod characteristics, length, width, thickness, erectness, smoothness, cellulose content, seed growth and development rate, and harvesting point. Gene cloning and functional research on common beans is still in its early stages. Only four genes related to pod morphology have been clearly cloned: 1. Gene doubling and retrotransposon insertion leading to increased expression of the PvIND (Phvul.002G27100) gene result in the absence of sutures in common bean pods. 2. Mutations in the key enzyme gene pv-ye (Phvul.002G006200) involved in the chlorophyll synthesis pathway resulted in yellow pods (wax beans); 3. Upregulation of the expression level of the key enzyme gene F3'5'H (Phvul.006G018800) that regulates the conversion of dihydroflavanol to dihydromyricetin in the anthocyanin pathway resulted in purple pods; 4. The deletion of 2 bp of the PvGUX1_1 (Phvul.002G270800) gene affected the uprightness of the pods, and the regulatory genes of other traits were not cloned.

[0004] The width of bean pods varies by variety and is one of the important commercial attributes of beans. With pod length and thickness remaining constant, increasing pod width results in thicker, fleshier pods and higher fresh weight per pod. Therefore, pod width is not only a factor in the appearance quality of beans but also affects yield. Precisely pinpointing the major genes controlling bean pod width can provide a theoretical basis for accurately improving bean pod morphology through molecular design breeding and gene selection breeding methods, and can also enrich our understanding of the molecular mechanisms of fruit morphogenesis. Summary of the Invention

[0005] The purpose of this invention is to provide the bean β-1,3-glucanase PvPW1, its encoded protein, and its applications, thereby providing a theoretical basis for bean biobreeding.

[0006] The nucleotide sequence of the bean β-1,3-glucanase PvPW1 of the present invention is shown in SEQ ID NO: 1 in the sequence listing.

[0007] The amino acid sequence of the protein encoded by the bean β-1,3-glucanase PvPW1 of this invention is shown in SEQ ID NO: 9.

[0008] This invention relates to the application of bean β-1,3-glucanase PvPW1 in regulating the pod width of leguminous plants.

[0009] Furthermore, the legume in question is the common bean.

[0010] Furthermore, the PvPW1 regulates the size of bean pod pericarp cells.

[0011] The SNP marker primers used in this invention for detecting the width of common bean pods are:

[0012] Forward primer 28-3555SNP-F: 5'-GTCTCATCCTATCTTGGTTTGG-3'

[0013] Reverse primer 28-3555SNP-R: 5'-AATCATGTTCTGGAAATAGTAATCTC-3'.

[0014] The InDel molecular marker primers used in this invention for detecting the width of common bean pods are:

[0015] Forward primer PvM436-F: 5'-GTGCTGTAGTATCTAGTTCCTTC-3'

[0016] Reverse primer PvM436-R: 5'-CTTATGAAAAAGCTGAGATACATGAG-3'.

[0017] The beneficial effects of this invention are:

[0018] This invention marks the first successful cloning of bean β-1,3-glucanase PvPW1 at the molecular level.

[0019] This invention, through map-based cloning of parental genetic populations and haplotype analysis, confirms that PvPW1 plays a role in regulating the width of bean pods. The mutation at 3555 base of this gene, from C (cytosine) to G (guanine), is a genetic factor that increases the width of bean pods.

[0020] The discovery of the bean β-1,3-glucanase PvPW1 provides a theoretical basis for bean biobreeding and has important application value in the targeted breeding of broad-pod or narrow-pod bean varieties using molecular-assisted breeding and gene selection breeding methods. Attached Figure Description

[0021] Figure 1 A comparison image of PI60234 and Dalong No. 1 plants;

[0022] Figure 2 Statistical and comparative chart of pod width for PI60234 and Dalong No. 1;

[0023] Figure 3 Statistical and comparative graphs of pod width / thickness in the seed-bearing zone and interseedent zone of PI60234 and Dalong 1 pods;

[0024] Figure 4 Statistical and comparative graphs of pod width at different time points after flowering for PI60234 and Dalong 1;

[0025] Figure 5 Section images of the interspecies region of PI60234 and Dalong 1 (scale bar is 1 mm);

[0026] Figure 6 Comparison of pod pericarp cell size between PI60234 and Dalong No. 1 soybeans (scale bar is 1 mm);

[0027] Figure 7 Statistics on the pod width of 5 F1 generation plants;

[0028] Figure 8 The frequency of different pod widths in the F2 generation population;

[0029] Figure 9 To use the F2 generation population to initially locate the PvPW1 locus in the PvM114 to PvM115 segment of chromosome 6;

[0030] Figure 10 To validate PvPW1 using PvM230 and PvM235 in the remaining 162 F2 generation individuals;

[0031] Figure 11 For the first round of fine positioning, PvPW1 was positioned between PvM416 and PvM287;

[0032] Figure 12 For the second round of fine positioning, PvPW1 was located in a 42.3kb region between PvM440 and PvM439;

[0033] Figure 13 Candidate genes in the PvPW1 region;

[0034] Figure 14 Expression analysis of Phvul.006G072600, a candidate gene in the PvPW1 region, in PI60234 and Dalong 1;

[0035] Figure 15 Expression analysis of Phvul.006G072800, a candidate gene in the PvPW1 region, in PI60234 and Dalong 1;

[0036] Figure 16 Expression analysis of Phvul.006G072900, a candidate gene in the PvPW1 region, in PI60234 and Dalong 1;

[0037] Figure 17 Expression analysis of Phvul.006G073000, a candidate gene in the PvPW1 region, in PI60234 and Dalong 1;

[0038] Figure 18 Analysis of nucleotide polymorphisms in the coding regions of three candidate genes for the PvPW1 site in 11 common bean varieties;

[0039] Figure 19 Phylogenetic tree construction and structural analysis diagram for the betaGLU9 protein;

[0040] Figure 20 A sequence difference analysis diagram of five versions of the PvbetaGLU9 protein;

[0041] Figure 21 Structural analysis of five versions of the PvbetaGLU9 protein;

[0042] Figure 22 This is a haplotype analysis diagram of the PvPW1 gene;

[0043] Figure 23 Band size of the PvM436 molecular marker in 8 broad-pod and 4 narrow-pod varieties;

[0044] Figure 24A comparison of pod widths for different PvM436 molecular marker genotypes;

[0045] Figure 25 A graph showing the percentage of different PvM436 molecular marker genotypes in a population with a pod width ≥17 mm;

[0046] Figure 26 The graph shows the proportion of different PvM436 molecular marker genotypes in a population with a pod width ≤12mm.

[0047] Figure 27 Two haplotypes of the PvPW1 gene: PvPW1 G3555 and PvPW1 C3555 A comparison chart of pod widths;

[0048] Figure 28 For PvPW1 in populations with pod width ≥ 17 mm G3555 and PvPW1 C3555 Haplotype percentage chart;

[0049] Figure 29 For PvPW1 in populations with pod width ≤ 12 mm G3555 and PvPW1 C3555 Haplotype percentage chart. Detailed Implementation

[0050] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0051] Example 1: Positioning of PvPW1 in Bean

[0052] PI60234 is a broad-pod variety, while Dalong No. 1 is a narrow-pod variety. Figure 1 As shown. The pod width values ​​of 20 plants of PI60234 and Dalong 1 were statistically analyzed separately. The average pod width of PI60234 was 21 mm, while the average pod width of Dalong 1 was 12 mm. The former was approximately twice that of the latter. Figure 2 As shown. The pod of a common bean can be divided into two regions: the seed zone and the interseedental zone. Comparing the width-to-thickness ratios of the seed and interseedental zones of PI60234 and Dalong 1, it was found that the width / thickness ratios of both regions of PI60234 were greater than those of the Dalong 1 variety. Figure 3As shown, SCR refers to the seed-contained region, and IC refers to the interlocular cavitations between two seeds. This indicates that PI60234 is a flat-pod variety, while Dalong 1 is an oval-pod variety.

[0053] By statistically analyzing the pod width 6-18 days after flowering, it was found that the pod widths of PI60234 and Dalong 1 were similar in the first 8 days after flowering. The pod width of Dalong 1 reached its maximum on the 12th day after flowering, while the pod width of PI60234 reached its maximum on the 16th day after flowering. Figure 4 As shown. Using pods 16 days after flowering as material, paraffin section analysis was performed on the interspecific regions of PI60234 and Dalong 1 pods, as shown. Figure 5 As shown, this further proves that PI60234 is a flat-pod variety, while Dalong No. 1 is an oval-pod variety. Furthermore, it was found that the pod cells of PI60234 are larger than those of Dalong No. 1, especially the cells near the inner pod. Figure 6 The area within the square box indicates that increased cell volume is, at least partially, the cause of the increased pod width in PI60234. Genetic populations were created using these two varieties to clone the gene controlling the bean pod.

[0054] II. Using the common bean variety PI60234 as the male parent and Dalong 1 as the female parent, a cross was conducted to obtain 5 hybrid seeds. The pods of the F1 generation of hybrid plants all exhibited a narrow-pod phenotype similar to that of Dalong 1, indicating that the narrow-pod phenotype is dominant compared to the wide-pod phenotype. Figure 7 As shown.

[0055] After self-pollination of F1, an F2 population was generated. By statistically analyzing the frequency of different pod widths in the F2 population, a peak was found in both the narrow-pod and wide-pod regions. Figure 8 As shown in Table 1, the ratio of the peak value in the narrow pod region to the peak value in the wide pod region is approximately 3:1, indicating that there should be a major gene controlling the pod width of common bean in this population.

[0056] Table 1. Segregation ratio of F2 generation phenotypes

[0057]

[0058] III. The genomes of 94 individuals from both parents and the F2 population were extracted using the CTAB method. The extraction procedure was as follows: a. Approximately 200 mg of fresh, young leaves were placed in a labeled 2 mL EP tube. Two 2 mm diameter steel balls were added to the tube, and the mixture was ground using a cryogenic homogenizer (Shanghai Jingxin Tissuelyse-24) at 30 Hz for 30 seconds. b. 800 μL of preheated CTAB extraction buffer (20 g / L CTAB; 0.1 M Tris-HCl pH 8.0; 1.40 M NaCl; 2 mM EDTA pH 8.0; 10 g / L PVP40) was quickly added to the tube, and the mixture was inverted several times to mix the sample and the buffer. c. The sample was placed in a 56 °C water bath for 60 minutes, inverting the tube several times every 15 minutes. d. Add 800 μL of chloroform:isoamyl alcohol extraction buffer (chloroform:isoamyl alcohol ratio 24:1, v / v) to an EP tube, vortex at high speed for 30 minutes, centrifuge at 9,000 rpm for 30 minutes, and transfer 500 μL of the supernatant to a new labeled 1.5 mL EP tube. e. Add 50 μL of 10% CTAB (100 g / L CTAB) and 500 μL of chloroform:isoamyl alcohol extraction buffer (chloroform:isoamyl alcohol ratio 24:1, v / v), vortex at high speed for 30 minutes, centrifuge at 9,000 rpm for 15 minutes, and transfer 300 μL of the supernatant to a new labeled 1.5 mL EP tube. f. Add 450 μL of CTAB-PPT buffer (10 g / L CTAB, 50 mM Tris-HCl, 20 mM EDTA), mix well, and incubate at room temperature overnight. g. Centrifuge at 9,000 rpm for 10 minutes, discard the supernatant, add 200 μL NaCl-TE buffer (58.5 g NaCl, 10 mM Tris-HCl, 1 mM EDTA), and incubate at 56°C for 30 minutes until the precipitate is completely dissolved. h. Add 200 μL isopropanol, cool the precipitate at 4°C for at least 1 hour, centrifuge at 9,000 rpm for 15 minutes, and discard the supernatant. i. Wash the DNA once with 500 μL of 70% ethanol and once with 100% ethanol, centrifuge at 12,000 rpm for 2 minutes, discard the supernatant, and collect the DNA. j. Dissolve the DNA in 200 μL of ddH2O. Genotyping of the F2 population was performed using 124 molecular markers. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 35 cycles, followed by a final extension at 72℃ for 10 min. The PCR product size was distinguished using 5%-6% agarose gel electrophoresis, and the results were visualized using a gel imaging system. Genotyping results were analyzed using QTL IciMapping_4.0 software, which showed that candidate genes were located on the PvM114 and PvM115 gene segments on chromosome 6. Figure 9 As shown in Table 2, and named PvPW1.

[0059] Table 2 Initial Positioning of PvPW1

[0060]

[0061] IV. The genomes of the remaining 162 individuals in the F2 population were extracted using a simplified CTAB method. The extraction procedure was as follows: a. Approximately 200 mg of fresh, young leaves were placed in a labeled 2 mL EP tube. Two 5 mm diameter steel balls were added to the tube, and the mixture was ground using a cryogenic homogenizer (Shanghai Jingxin Tissuelyse-24) at 30 Hz for 30 seconds. b. 800 μL of preheated CTAB extraction buffer (20 g / L CTAB; 0.1 M Tris-HCl pH 8.0; 1.40 M NaCl; 2 mM EDTA pH 8.0; 10 g / L PVP40) was quickly added to the tube, and the mixture was inverted several times to mix the sample and the buffer. c. The sample was placed in a 56 °C water bath for 60 minutes, inverting the tube several times every 15 minutes. d. Add 800 μL of chloroform:isoamyl alcohol extraction buffer (chloroform:isoamyl alcohol ratio 24:1, v / v) to an EP tube, vortex at high speed for 30 minutes, and centrifuge at 9,000 rpm for 30 minutes. e. Transfer 60 μL of the supernatant to a new labeled 96-well plate, add 60 μL of isopropanol, cool and precipitate at 4°C for at least 1 hour, centrifuge at 4,000 rpm for 15 minutes, and discard the supernatant. f. Wash the DNA once with 200 μL of 70% ethanol and once with 100% ethanol, centrifuge at 4,000 rpm for 2 minutes, discard the supernatant, and collect the DNA. g. Dissolve the DNA in 100 μL of ddH2O. InDel molecular markers PvM230 and PvM235 were designed at both ends of the PvM114 and PvM115 gene segments, with sequences shown in Table 3. Validation of PvPW1 was performed, and the results showed that the pods of individuals with the same genotype as PI60234 were wider than those of individuals with the same genotype as Dalong 1. Figure 10 As shown, AA represents individuals with the same genotype as Dalong 1, and BB represents individuals with the same genotype as PI60234. This result demonstrates that PvPW1 is a candidate locus controlling pod width variation in this population.

[0062] Table 3 shows the two primers used for PvPW1 validation.

[0063]

[0064] V. Thirteen molecular markers were designed between PvM114 and PvM115 for the first round of fine mapping, as shown in Table 4. DNA was extracted from the progeny individuals of three heterozygous lines in the PvPW1 region using a simplified CTAB method. DNA was then analyzed in the F2 and F3 generations.2:3 Genotyping and phenotypic observation of the plants yielded 12 individuals suitable for fine gene mapping. PvPW1 was mapped to the region between PvM416 and PvM287. Figure 11 As shown.

[0065] Table 4 lists the 13 primers used for the first round of fine mapping.

[0066]

[0067] VI. Nine molecular markers were designed between PvM416 and PvM287 for the second round of fine mapping, as shown in Table 5. DNA was extracted from the progeny of Line 234, Line 253, R21-35, and R69-162 lines using a simplified CTAB method. DNA was then analyzed from the F... 2:3 generation and F 2:4 Genotyping and phenotypic observation of the plants yielded three individuals suitable for fine gene mapping. PvPW1 was mapped to a 42.3 kb region between PvM440 and PvM439. Figure 12 As shown.

[0068] Table 5 shows the nine primers used for the second round of fine mapping.

[0069]

[0070] Example 2: Identification of the PvPW1 gene

[0071] I. According to the annotation information of the reference genome, there are 5 annotated genes in the PvPW1 region, namely Phvul.006G072600, Phvul.006G072700, Phvul.006G072800, Phvul.006G072900, and Phvul.006G073000, as follows: Figure 13 As shown. The expression levels of these five candidate genes in PI60234 and Dalong 1 bean pods were detected using qPCR. The results showed that the Phvul.006G072700 gene was not expressed in the pods, while the other four genes were expressed in the pods, but there was no difference in expression levels between the parent lines (fold change less than 2). Figures 14 to 17 As shown in the figure. The results show that Phvul.006G072600, Phvul.006G072800, Phvul.006G072900 and Phvul.006G073000 are candidate genes for PvPW1, and the candidate variants occur in the coding region.

[0072] II. To further analyze the candidate genes of PvPW1, the variation of the CDS regions of the four candidate genes in PI60234 and DaLong 1 was manually examined using IGV software. Two, six, four, and two SNP variations were found in the CDS regions of the Phvul.006G072600, Phvul.006G072800, Phvul.006G072900, and Phvul.006G073000 genes, respectively, causing 0, 3, 2, and 2 amino acid sequence variations in the protein, as shown in Table 6. Based on these results, the Phvul.006G072800, Phvul.006G072900, and Phvul.006G073000 genes are considered candidate genes for PvPW1.

[0073] Table 6. Variation analysis of CDS sequences of candidate genes

[0074]

[0075]

[0076] 3. Using approximately 10mm pods from 8 broad-podded bean varieties (PI60234, Mengshiqidou No. 9, Jiulibai, Mengshiqidou 11, Mengshiqidou 13, Wujiaqidou, Baibulao, and Hongyundou) and 4 narrow-podded bean varieties (Dalong No. 1, Shengyou No. 1, Mengshiqidou 12, and Mengshiqidou 14), total RNA was extracted from the leaves using the instruction manual of the Omini Plant RNA Kit (CW25985) purchased from Comway Technology.

[0077] 1 μg of total RNA was used for cDNA synthesis. The cDNA synthesis procedure was performed according to the instruction manual of the TransScript One-Step gDNA Removal and cDNA Sythesis SuperMix Kit (AT311-03) purchased from TransGen Biotech.

[0078] Using the obtained cDNA as templates, the Phvul.006G072800 gene from the above 12 common bean varieties was amplified using forward primer 28F and reverse primer 28R as shown in Table 7; the Phvul.006G072900 gene from the above 12 common bean varieties was amplified using forward primer 29F and reverse primer 29R; and the Phvul.006G073000 gene from the above 12 common bean varieties was amplified using forward primer 30F and reverse primer 30R. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 38 cycles, followed by a final extension at 72℃ for 10 min. The PCR products were sequenced using an ABI 3130 sequencer (ABI Corporation). Sequencing results showed that among the seven DNA variants highly associated with pod width shown in Table 6, only the G3555C variant in the Phvul.006G072800 gene was completely associated with the pod width trait. Specifically, the genotype at this locus is G in wide-podded bean varieties and C in narrow-podded bean varieties. Figure 18 As shown in Table 8. These results indicate that the Phvul.006G072800 gene is the controlling gene for the width of common bean pods. Based on the homology annotation results, the Phvul.006G072800 gene encodes the common bean β-1,3-glucanase 9 protein, which is named PvbetaGLU9.

[0079] Table 7 Primers used for CDS cloning

[0080]

[0081]

[0082] Table 8 Genotypic analysis of three candidate genes at the PvPW1 locus in 11 common bean varieties.

[0083]

[0084] Bold letters indicate genotypes and phenotypes that do not match, while italic letters indicate loci where genotypes and phenotypes match perfectly.

[0085] IV. Using the common bean varieties in Table 8 as parent plants, 17 parent populations were constructed. The results of pod width measurements of the F1 generation showed that if either parent had individuals with narrow pods, the pod width of the F1 plants was similar to that of the narrow pods. This result further proves that narrow pods are a dominant trait and wide pods are a recessive trait, as shown in Table 9.

[0086] We used 28-3555SNP-F: 5'-GTCTCATCCTATCTTGGTTTGG-3' and 28-3555SNP-R: 5'-AATCATGTTCTGGAAATAGTAATCTC-3' as primers and the F2 generation genome as templates for PCR amplification. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 35 s, for a total of 35 cycles, followed by a final extension at 72℃ for 10 min. The amplified PCR products were sent to a sequencing company for sequencing. By comparing the sequenced products with the Phvul.006G072800 gene sequence, the SNP information at 3555 bases of the Phvul.006G072800 gene was determined. Based on this information, we screened for homozygous Phvul.006G072800 individuals in the F2 generation. G3555 Genotype and homozygous Phvul.006G072800 C3555 Genotype of individual.

[0087] Phvul.006G072800 homozygous in the F2 generation population was measured. G3555 Genotype and homozygous Phvul.006G072800 C3555 The average width of genotype individuals, as shown by Phvul.006G072800, was... G3555 The average width of genotype individuals is greater than Phvul.006G072800 C3555 The average width of individuals with the same genotype confirmed that the Phvul.006G072800 gene is the PvPW1 gene that controls the width of bean pods.

[0088] Table 9. Homozygous Phvul.006G072800 in F1 and F2 populations from 17 parental genetic populations. G3555 Genotype and homozygosity Phvul.006G072800 C3555 Statistics on pod width by genotype.

[0089]

[0090] In the table, - indicates that this type of genotype does not exist in the F2 generation.

[0091] Example 3: Analysis of the coding sequence and protein amino acid sequence of the PvPW1 gene

[0092] I. Using amino acid sequence alignment of the PvPW1 protein, identify the homologous proteins Vigun06g080900, Glyma.11G165100, Glyma.18G058300, Lj6g0008072, Lalb_Chr15g0083151, Ca_0900, Medtr3g031770, AT5G58480, Brara.J01233, LOC_Os03g18520, and Zm00001d047712 of this protein. The amino acid sequences were then downloaded from https: / / phytozome-next.jgi.doegov. Phylogenetic trees were constructed using Mega6 software. The results showed that the PvPW1 protein in common bean had the highest similarity to its homologous protein in cowpea; dicotyledons and monocotyledons could be divided into two independent branches; the distinction between legumes and cruciferous families within dicotyledons was clear, such as... Figure 19 As shown.

[0093] According to the annotation information, the PvPW1 gene encodes the bean β-1,3-glucanase PvbetaGLU9 protein, which contains three conserved domains: the N-terminal signal peptide (red box), the middle Glyco-hydro-17 domain (orange box), and the C-terminal X8 domain (blue box). The purple and green boxes represent the C-terminal low-repetition sequence region and the transmembrane region, respectively. Figure 19 As shown.

[0094] II. The PvPW1 gene has seven different versions of its coding sequence, encoding five different PvbetaGLU9 protein sequences, such as... Figure 20 As shown.

[0095] III. All five different PvbetaGLU9 protein sequences consist of 484 amino acids. Analysis of the structures of the five PvbetaGLU9 proteins using the protein structure analysis website http: / / smart.embl.de / revealed that PvbetaGLU9 v5 is equivalent to PvbetaGLU9. Dalong1 The C-terminal low-repetition sequence region is missing, such as Figure 21 As shown, this variation may be the reason for the reduced pod width.

[0096] Example 4: Visualized PvM436 molecular markers can be used for targeted molecular design breeding of pod morphology.

[0097] I. Using resequencing data from 59 common bean varieties, the haplotype distribution of the PvPW1 gene was analyzed. The results showed that the PvPW1 gene has 8 haplotypes, the original nucleotide sequence of which is shown in SEQ ID NO: 1 of the sequence listing, and the amino acid sequence encoding the protein is shown in SEQ ID NO: 9 of the sequence listing. The remaining 7 haplotypes are variant sequences from naturally inbred populations, with nucleotide sequences shown in SEQ ID NO: 2-8 of the sequence listing, encoding 4 different PvbetaGLU9 protein sequences, and amino acid sequences shown in SEQ ID NO: 10-13 of the sequence listing.

[0098] like Figure 22 As shown, the narrow-pod haplotype is PvPW1. C3555 Completely coupled with the 8-bp deletion at intron 1706, this 8-bp deletion was found to be located within the molecular marker PvM436 used for the second round of fine localization, as shown in the image. Figure 13 and Figure 22 As shown. It is believed that the visualizeable PvM436 molecular marker has the potential for application in molecular design breeding for pod width targeting in common bean. Furthermore, resequencing data from 59 common bean varieties were used to analyze the genotypes of the PvM436 molecular marker in common bean. It was found that the PvM436 molecular marker can be amplified into three bands (247, 227, and 219) that can be distinguished by 4% agarose gel electrophoresis. Therefore, the PvM436 molecular marker possesses the potential for pod width targeting. 247 PvM436 227 and PvM436 219 Three genotypes, such as Figure 22 .

[0099] II. Genotyping of the PvM436 molecular marker was performed on 8 broad-podded and 4 narrow-podded common bean varieties. The results showed that 4 out of the 8 broad-podded varieties were PvM436. 247 There are 4 varieties with the genotype PvM436. 227 Genotype: All four narrow-pod bean varieties are PvM436. 219 Genotypes, as shown in Table 8 and Figure 23 As shown.

[0100] III. Genotyping of the PvM436 molecular marker was performed on 317 common bean varieties, revealing that 168 varieties were PvM436. 247 Genotype, 50 of which are PvM436 227 Genotype, 99 of which are PvM436 219 Genotyping and statistical analysis of pod width in these 317 common bean varieties revealed that PvM436... 247 Genotype and PvM436 227The average pod width ratio of genotype individuals is PvM436. 219 The average pod width of individuals with different genotypes increased by 68.6% and 33.6%, respectively. Figure 24 As shown.

[0101] IV. The proportions of the three genotypes of the PvM436 molecular marker were analyzed in individuals with pod widths greater than 17 mm. The results showed that PvM436... 247 Genotype, PvM436 227 Genotype and PvM436 219 The proportions of genotypes were 85.7%, 12.0%, and 2.3%, respectively, with the first two accounting for a significantly larger proportion than the latter. Figure 25 As shown.

[0102] The analysis of the proportions of the three genotypes of the PvM436 molecular marker in individuals with pod widths less than 12 mm revealed that PvM436 247 Genotype, PvM436 227 Genotype and PvM436 219 The proportions of different genotypes were 5.1%, 12.8%, and 82.1%, respectively, with the latter being significantly higher than the former two. Figure 26 As shown in the figure. In summary, these results demonstrate that the visualized PvM436 molecular marker can be used for targeted molecular design breeding of pod morphology.

[0103] Example 5: The PvPW1 gene was artificially selected during the selective breeding of common bean.

[0104] I. Using resequencing data from 317 common bean varieties, the haplotype at position 3555 of the PvPW1 gene was analyzed. The results showed that, based on the nucleic acid polymorphism at position 3555, the PvPW1 gene can be divided into PvPW1... G3555 Haplotypes and PvPW1 C3555 Haplotypes. Analysis of PvPW1 G3555 Haplotypes and PvPW1 C3555 The average pod width of haploid individuals, as shown by PvPW1, indicates that... G3555 The average pod width of haploid individuals was greater than that of PvPW1 C3555 The average pod width of haplotype individuals increased by 60.6%, such as Figure 27 As shown.

[0105] II. Analysis of PvPW1 in individuals with pod width greater than 17 mm. G3555 Haplotypes and PvPW1 C3555 The proportion of haplotypes was found in PvPW1. G3555 The haplotype ratio was 97.7%, far exceeding that of PvPW1. C3555 2.3% of haplotypes, such as Figure 28 As shown.

[0106] Analysis was conducted on PvPW1 individuals with pod widths less than 12 mm. G3555 Haplotypes and PvPW1 C3555 The proportion of haplotypes was found in PvPW1. G3555 The haplotype ratio was 17.9%, far less than that of PvPW1. C3555 82.1% of haplotypes, such as Figure 29 As shown. Based on the above results, we believe that the PvPW1 gene was artificially selected during the selective breeding process of common bean, and PvPW1... G3555 Haplotypes are used in the selection and breeding of broad-podded bean varieties, while PvPW1 C3555 Haplotypes often appear in the selective breeding process of narrow-pod beans.

Claims

1. The application of an SNP molecular marker in identifying the width of common bean pods, wherein the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO: 1 in the sequence listing; a G / C mutation exists at the 3555th base position in SEQ ID NO:

1.

2. The application of primers for detecting SNP molecular markers in the detection of common bean pod width, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO: 1, and a G / C mutation exists at the 3555th base position in SEQ ID NO: 1; the primer is: Forward primer 28-3555SNP-F: 5'-GTCTCATCCTATCTTGGTTTGG-3', Reverse primer 28-3555SNP-R: 5'-AATCATGTTCTGGAAATAGTAATCTC-3'.

3. The InDel molecular marker primers used to detect the width of common bean pods are: Forward primer PvM436-F: 5'-GTGCTGTAGTATCTAGTTCCTTC-3', Reverse primer PvM436-R: 5'-CTTATGAAAAAGCTGAGATACATGAG-3'.

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