CN117587030A - Peanut pod size related gene AhPSW1 and application thereof - Google Patents

Abstract

The invention discloses a DNA sequence, a CDS sequence and a coding protein sequence of a peanut pod size related gene AhPSW1, and further discloses a method for utilizing the geneAhPSW1The method for carrying out fluorescence quantitative analysis lays a foundation for the application practice of peanut breeding,the molecular genetic foundation of the peanut pod size is revealed, and the molecular genetic foundation plays an important role in improving the peanut yield and cultivating high-yield peanut varieties rapidly and efficiently by utilizing the genetic engineering technology.

Description

Peanut pod size related gene AhPSW1 and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a peanut pod size trait related gene AhPSW1, a coding protein and application thereof.

Background

Peanuts are also called peanut, long peanut, and all of them are all Bao. The oil content of the peanut kernel is about 50 percent, the protein content is about 25 to 30 percent, which is an important source of vegetable oil and protein and provides 48 percent of high-quality edible oil and 25 percent of high-quality vegetable protein for the semiarid tropical region. In the peanut as a main oil crop, the planting area and the yield are in front, and the peanut has important significance for guaranteeing national grain safety. Meanwhile, the method is also one of the important targets for long-term pursuit of peanut breeders in China.

Plant seeds are one of the main factors affecting crop yield. Seed is an important source of human food and industrial raw materials, so seed size has long been one of the major targets for crop breeding improvement as an important yield component. Therefore, the genetic characteristics of the peanut seed size are explored, the molecular mechanism of the seed size regulation is analyzed, and important theoretical basis and gene resources are provided for high-yield breeding of crops.

Plant seeds are generally composed of three parts, embryo, endosperm and seed coat. Double fertilization of plants produces fertilized eggs and fertilized nuggets, which further develop into embryos and endosperm; the beads are surrounded by the bead core tissue and develop into seed coats; the ovary wall develops into the shell. At present, several functional genes controlling seed size have been discovered. These functional genes are involved in the development process of embryo, endosperm and integument, and the size of seeds is controlled by regulating the proliferation and elongation degree of cells. The mechanisms under which they are studied mainly involve the following pathways: the G protein signaling pathway (GPA 1, RGB1, GS3 and DEP 1), the mitogen-activated protein kinase signaling pathway (MKK 4), the transcriptional regulation-related pathway (GS 2/GL2/GLW2/PT2, ANT, AP2, TTG 2), the phytohormones (GS 5, GSE5/GW5/qSW5, osARF4 and ARF 18) and other pathways such as IKU2, MINI3, BZR1, TGW6, etc. by degradation by ubiquitin-mediated proteasomes (DA 1, DA2, GW2and GW5/qSW 5).

The pod related traits belong to typical quantitative traits, and in recent years, researchers have hybridized between different wild species and wild species, artificially created wild species and cultivars, cultivars and cultivars, and the pod length, pod width, shell thickness, pod area, kernel size, hundred fruit weight, hundred kernel weight, kernel yield, shan Zhuren weight and other traits are positioned in different offspring groups such as RIL, NAW and the like by using SSR, SNP, indel, SLAF, ahTE and other markers. Most QTL intervals are on chromosomes a05, a07, a09, B05, B06, and the like. With the continued advancement of technology, studies using BSA, BSR-seq and multiunit analysis have emerged.

The fine localization of new genes related to peanut pod size traits and the progress of gene mining work are slow, so far, cloning of new genes related to peanut pod size has been reported freshly. Thus, the isolation of new pod size related genes and the study of their function is of great importance for the resolution of the molecular mechanisms behind peanut pod sizes.

Disclosure of Invention

The invention aims to provide a peanut pod size trait related gene AhPSW1, a coded protein, a developed molecular marker and application thereof in breeding. Solves the problems of lack of new genes and lack of molecular markers related to peanut pod size traits, and provides more choices for peanut breeding.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the invention provides a peanut pod size trait related gene AhPSW1, the nucleotide sequence of the genome of which is shown as SEQ ID NO. 1.

As a further improvement of the invention, the invention provides a peanut pod size trait related gene AhPSW1, the full-length cDNA nucleotide sequence of which is as follows:

(1) A nucleic acid sequence as shown in SEQ ID NO. 2;

(2) A nucleic acid sequence of equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.2.

The invention also provides a protein AhPSW1 coded by the gene related to the pod size traits of the peanut, and the amino acid sequence of the protein AhPSW1 is as follows:

(1) An amino acid sequence as shown in SEQ ID NO. 3; or (b)

(2) A nucleic acid sequence of equivalent function derived from the nucleic acid sequence shown in SEQ ID NO. 3; addition, deletion or substitution of one or more amino acids based on the amino acid sequence shown in SEQ ID NO.3 to obtain the sequence of the active fragment or the conservative variant.

The invention also provides a sequence of 5'UTR and 3' UTR of peanut pod size trait related gene AhPSW 1:

(1) SEQ ID NO.4;

(2) A nucleic acid sequence of equivalent function derived from the nucleic acid sequence shown in SEQ ID NO. 4. That is, the sequence of the active fragment or the conservative variant is obtained by adding, deleting or substituting one or more amino acids based on the amino acid sequence shown in SEQ ID NO. 4.

Further, the invention provides a primer pair for amplifying the full length of the peanut pod size trait related gene AhPSW1 or any fragment thereof, wherein the primer pair is as follows:

AhPSW1-1F：5’-ATGTCTAGAAGCTTAAGAAACC-3’；

AhPSW1-1R：5’-TTAAAAACCTCCCTTGAACAC-3’；

furthermore, the invention provides a recombinant plasmid containing the peanut pod size trait related gene AhPSW1, which is formed by inserting CDS of the peanut pod size trait related gene AhPSW1 into pCAMBIA13001300-GFP plant over-expression vector.

Furthermore, the invention provides application of the peanut pod size trait related gene AhPSW1 or the recombinant plasmid in peanut yield breeding.

The invention further discloses a method for carrying out fluorescence quantitative analysis by utilizing the AhPSW1, which comprises the following steps:

1) The fluorescent quantitative specific primers were designed for the AhPSW1 gene sequence as follows:

AhPSW1-qF：5’-TCAAGAACTTCACAGATGCAG-3’；

AhPSW1-qR：5’-CCACTGAGAAGATTCTTTCCCAAG-3’；

the reference gene adopts ahacin 7, and the primer sequence is as follows:

Ahactin7-F：5’-GATTGGAATGGAAGCTGCTG；

Ahactin7-R：5’-CGGTCAGCAATACCAGGGAA；

2) UsingGreen qPCR SuperMix kit and Bio-Rad CFX96 Touch real-time PCR System, wherein 20. Mu.l of the reaction system, 1. Mu.l of cDNA, 0.5. Mu.l of each of the upstream and downstream primers, 2 x->Green qPCR SuperMix10 μl, nucleic-free water 8 μl; PCR procedure: 95 ℃ for 3min,95 ℃ for 5sec,55 ℃ for 30sec,72 ℃ for 30sec,40 cycles;

3) Taking 5 mu l of PCR reaction products, using 2.0% agarose gel electrophoresis, sequencing, and judging whether target fragments are identical;

4) By 2 ^-△△CT The relative expression level of the AhPSW1 gene was calculated by the method.

Compared with the prior art, the invention has the beneficial technical effects that:

1. the invention discloses and confirms a novel peanut pod size related gene named AhPSW1 for the first time, the gene is positioned on peanut chromosome 07, the CDS sequence length is 3201bp, the expressed protein length is 1066 amino acids, and the peanut pod length can be regulated and controlled.

2. The invention defines the DNA sequence, CDS sequence and coding protein sequence of peanut pod size related gene AhPSW1, which lays a foundation for the application practice of peanut breeding.

3. The invention is helpful for revealing molecular genetic basis of peanut pod size, and plays an important role in improving peanut yield and cultivating high-yield peanut varieties rapidly and efficiently by utilizing genetic engineering technology.

Drawings

FIG. 1 shows the analysis of the expression level of PSW1 gene in different tissues; and (3) injection: error bars represent standard deviation of 3 biological replicates. * P <0.05, < P <0.01 (two-tailed t-test), the same applies below.

FIG. 2 is a phenotype and plant height statistical analysis of PSW1 transgenic Arabidopsis; significant differences were assessed using one-way analysis of variance (ANOVA) followed by a multiple comparison test of Tukey's post hoc, and are indicated in lower case letters above each histogram, the same applies below.

FIG. 3 is a PSW1 expression analysis and phenotypic statistics of Arabidopsis transgenic lines; wherein A: wild-type and PSW1 Arabidopsis overexpression lines are in the seed phenotype of Arabidopsis. Scale bar = 500 μm. B: expression analysis of PSW1 in wild-type and transgenic Arabidopsis lines. C: seed area of the PSW1 overexpressed Arabidopsis line. D-E: statistical analysis of seed length (D) and width (E) of PSW1HapI and PSW1HapII overexpressing arabidopsis lines. Each blue circle represents a single data point (n= 103,60,91,129,187,203,221).

Fig. 4 is a population verification result.

Detailed Description

The following examples are given to illustrate the invention in detail, but are not intended to limit the scope of the invention in any way.

The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents on the market unless specified; the test methods are conventional, unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. Primer synthesis and sequencing work were all done by the biological engineering (Shanghai) Co., ltd.

Example 1: obtaining of peanut pod size and character related gene AhPSW1

The forward genetics combination is used for carrying out the gene excavation related to the peanut pod size traits, and the specific steps are as follows:

(1) Carrying out investigation on agronomic traits such as pod length, pod width, grain length, grain width and the like of a natural population constructed by 188 peanut resources from a laboratory; selecting two pods with different sizesObvious virginia-type peanut materials (nd_s and nd_l) served as parents. ND_S pods length/width (PL/W) of 2.5/1.1cm, kernel length/width (SL/W) of 1.2/0.8cm, a weight of hundred fruits (100-P-W) of 45g, a weight of hundred fruits (100-S-W) of 28g, a pod surface area (PS) of 2.1cm2, a Pod Volume (PV) of 1.5cm ³ As a female parent; the length/width of the pods of ND_L is 5.0/2.6cm, the length/width of the seeds is 2.2/1.2cm, the weight of the nuts is 180g, the weight of the nuts is 106g, and the surface area of the pods is 13.8cm ² Pod volume 17.6cm ³ As male parent, hybridization is carried out, and the identified 80 true hybrids are added to Hainan Ledong for generation, and then single plants are harvested. Spring sowing is carried out in Mao Zhuang science education park in the next year: harvesting all single plants to obtain F _2：3 The phenotypes of pods and seeds such as PL (pod length), PW (pod width), SL (seed length), SW (seed width), PC (pod perimeter), PS (pod surface area), 10-P-W (10 fruit weight), SC (seed perimeter), SS (seed surface area), 10-S-W (10 kernel weight) were measured, and genetic molecules were carried out. We can see that the offspring population has a rich variation in pod size and that superphilia occurs. Then, leaves of the 20 extreme individuals and parents with the pod size were selected for resequencing BSA analysis.

(2) In order to further fine position the target gene, we continued to develop more precise molecular markers InDel5, inDel8, SSR21, inDel15, inDel16, inDel19, inDel20, SSR133, inDel22, inDel27 and InDel28 within the candidate interval. In addition, we developed 4 pairs of KASP markers, KASP3, KASP6, KASP7 and KASP9 based on SNP mutation information, then used these molecular markers to correlate the F6:7 and F6:8 populations, and finally we reduced the candidate interval to a 20.4kb interval between KASP9 and C07_I15, the LOD value of this QTL reached 15.65, the additive effect was-4.0, accounting for the phenotype contribution rate of 40.22. Based on Tif1 reference genome assembly results, there are a total of 3 annotated genes within the localization: arahy. GJ51RN encodes ATP binding microtubule motor family protein; arahy.8KGR77 encodes regulatory protein RecX family protein; arahy.vaae0n encodes a serine threonine LRR receptor protein kinase. As a result of the re-sequencing, it was found that only the Arahy.VAAE0N gene had a deletion in the 5' UTR region and a non-synonymous mutation in the coding region 618 was observed, which was finally identified as the target gene AhPSW1.

(3) To verify whether the differences in ND_S and ND_L pod sizes described above are controlled by the gene AhPSW1, the gene was clone sequenced in ND_S and ND_L, respectively. The method comprises the following specific steps:

1.1 extraction of DNA

DNA of ND_S and ND_L was extracted by SLS method.

1.2 Total RNA extraction and Synthesis of first strand cDNA

RNA extraction of different peanut tissues Total RNA of ND_S and ND_L was extracted respectively using RNA room temperature extraction kit (RC 411, northenan, nanjing, china) from Nanjinothenan biotechnology Co., ltd. Reverse transcription of RNA with quality standard to cDNA using reverse transcription kitOne-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AT 311-02, full gold, beijing, china)

1.3 cloning and sequence analysis of AhPSW1 Gene

PCR primers were designed, which were designed at both ends of the initiation codon ATG and the termination codon TAA, as follows:

AhPSW1-1F：5’-ATGTCTAGAAGCTTAAGAAACC-3’；

AhPSW1-1R：5’-TTAAAAACCTCCCTTGAACAC-3’；

DNA of ND_S and ND_L is used as a template, ahPSW1-1F/R is used for PCR amplification, a PCR product is subjected to 1% agarose gel electrophoresis, recovered and connected to a 5x TA/Blunt-Zero Cloning Kit (C601-01, northenan, nanjing, china) for connection, and then correct monoclonal is selected for sequencing after bacterial inspection; the PCR reaction procedure was: pre-denaturing in a PCR instrument at 95 ℃ for 5min; then 30 cycles: denaturation at 95℃for 30sec, annealing at 55℃for 30sec, extension at 72℃for 90sec, extension at 72℃for 5min, and then storage at 4 ℃.

High-fidelity enzyme using cDNA of ND_S and ND_L as templateUc Super-Fidelity DNA Polymerase for Library Amplification (P507-01, northenan, nanjing, china) carrying out 1% agarose gel electrophoresis on the PCR product by AhPSW1-1F/R, connecting to 5xTA/Blunt-Zero Cloning Kit (C601-01, northenan, nanjing, china) after recovery, connecting, and then selecting the correct monoclonal for carrying out the sequencing by a worker after bacterial inspection, wherein the PCR reaction system and parameters are the same as the method.

The 5'/3' RACE specific primer of AhPSW1 was designed using the on-line RACE primer design software (http:// appbi. Vazyme. Com:8085 /) (tables 2-5). Then experiments were performed using HiScript-TS 5'/3' RACE (RA 101, northenan, nanjing, china) with the extracted high quality Total RNA as template. The primers shown in Table 1 were used to carry out 1min at 98℃for 10sec, 3min for 5 cycles at 72℃for 98 sec, 15sec at 70℃for 72℃for 5 cycles at 72℃for 68℃for 15sec, and 25 cycles at 72℃for 5min and 4℃for 3min in the drop PCR.

Mu.l of the previous round of PCR amplification product was added to 245. Mu.l of ddH2O to dilute the product, after which 5. Mu.l was pipetted as a template, 1. Mu.l of Nested GSP Primer (AhGSW1_GSP5 '/3' N) was added, and 1. Mu.l of Nested Primer 2 XPCR Mix 1. Mu.l, 25. Mu.l of 2 XPCR Mix were added, and the mixture was subjected to 30 cycles of 98℃1min,98℃10sec, 68℃15sec, 72℃3min, 72℃5min,4℃in a PCR apparatus, followed by gel recovery. Recovered and ligated to 5x TA/Blunt-Zero Cloning Kit (C601-01, northenzan, nanjing, china), followed by bacterial screening to select the correct monoclonal for sequencing.

TABLE 1 RACE cloning primers for AhPSW Gene

Meanwhile, the primer is designed to carry out PCR by taking DNA as a template, then sequencing is directly carried out, and the sequence of the promoter is verified to verify the mutation at the promoter.

AhPSW1-QF:5’-GATTGGAATTACACTCAGAC-3’；

AhPSW1-QR:5’-GAGAATCAATGTTATGGAAG-3’；

Sequencing results show that the DNA fragment obtained by PCR amplification by taking the DNA of peanut ND_L as a template is the genome level sequence of the AhPSW1 gene, and the length of the DNA fragment is 3347bp as shown in a sequence table SEQ ID NO. 1.

cDNA sequences of AhPSW1 genes were amplified by PCR using cDNA of peanut ND_S and ND_L as templates, and a cloning coding region sequence (CDS) of 3201bp SEQ ID NO.2 length was used. A non-synonymous mutation of the SNP of G/T occurs in its coding region 1853, resulting in a change of amino acid Ser/IIe at position 618 of SEQ ID NO.3. Further prediction of the structure of the PSW1 gene, we found that PSW1 amino acid was extracellular at the N-terminus, intracellular at the C-terminus, a signal peptide (1-35 aa) was found at the N-terminus followed by one LRR2 (37-70 aa) and two LRR8 (299-399 aa,615-669 aa) domains, possessing one transmembrane domain (Transmembrane domain, TM;711-733 aa) and one kinase domain (Pkinase, 772-1042 aa) at the C-terminus, and mutation of the coding region occurred in the second LRR8 domain.

Using the kit to amplify the 5'/3' RACE of the AhPSW1 gene, we found that the 5'UTR was 125bp in length and the 3' UTR was 255 bpSEQ ID No.4, while there was a total of 12 bases of 4 TAAs inserted into SEQ ID No.5 along with the PSW1 promoter region (Primer, -173 bp) at ND_L.

Example 2: fluorescent quantitative analysis of AhPSW1

(1) Fluorescence quantitative analysis is carried out on seeds and shells of different tissues of ND_S and ND_L and different periods of pod development; total RNA extraction and cDNA reverse transcription were performed as in example 1.

The fluorescent quantitative specific primers were designed for the AhPSW1 gene sequence as follows:

AhPSW1-qF：5’-TCAAGAACTTCACAGATGCAG-3’；

AhPSW1-qR：5’-CCACTGAGAAGATTCTTTCCCAAG-3’；

the reference gene is Ahactin 7 (XM_ 025826875), and the primer sequences are as follows:

Ahactin7-F：5’-GATTGGAATGGAAGCTGCTG；

Ahactin7-R：5’-CGGTCAGCAATACCAGGGAA；

usingGreen qPCR SuperMix kit (AQ 101-01, T)ransGen Biotech, china, beijing) and Bio-Rad CFX96 Touch real-time PCR System (Bio-Rad, hercules, calif., USA). 20 μl of the reaction system, 1 μl of cDNA, 0.5 μl of each of the upstream and downstream primers, 2x +.>Green qPCR SuperMix 10. Mu.l, nucleic-free water 8. Mu.l. PCR procedure: 95℃for 3min,95℃for 5sec,55℃for 30sec,72℃for 30sec,40 cycles. And 5 mu l of PCR reaction products are taken and subjected to 2.0% agarose gel electrophoresis, and simultaneously are sent to a biological engineering for sequencing, so as to judge whether the target fragments are identical. By 2 ^-△△CT The relative expression level of the AhPSW1 gene was calculated by the method, and the results are shown in FIG. 1. The expression pattern of the gene reflects the function of the gene to a certain extent, the relative expression quantity of the PSW1 gene is detected in different tissues of ND_S and ND_L and shells and seeds in different periods by a RT-qPCR method, and the result shows that the expression pattern of the PSW1 is constitutive expression and expression exists in all tissues of peanuts. The expression levels in the leaves, roots and stems of peanut are higher than other tissues, and the expression levels in the leaves reach significant difference levels, the expression levels in the needles, flowers, seeds and hulls are relatively low, but the expression levels in nd_l are higher than nd_s, and the difference levels are reached.

Example 3: acquisition and identification of AhPSW1 transgenic Arabidopsis thaliana

(1) Construction of plant overexpression vectors

Construction of an over-expression vector: ligation of AhPSW1 with sequencing correctness ^G And AhPSW1 ^T The T-carried plasmid of the gene is used as a template, and pCAMBIA1300-PSW1-GFP F/R is added with a homology arm primer for PCR amplification.

pCAMBIA1300-PSW1-GFP_F：

5’-AGAGAACACGGGGGACGAGCTCATGTCTAGAAGCTTAAGAAACC-3’；

pCAMBIA1300-PSW1-GFP_R：

5’-AGCTCCTCGCCCTTGCTCACCATAAAACCTCCCTTGAACAC-3’；

The overexpression vector pCAMBIA1300-GFP was digested with restriction enzymes KpnI-HF (star-selected enzyme) and SacI-HF (star-selected enzyme) at 37℃for 30min, heat-inactivated at 65℃for 20min to perform double digestion, and then the PCR product and the digested product were subjected to 1% agarose, after which the product was recovered. Ligation was performed according to the homologous recombination kit (C112, northenzan, nanjing, china) insert 4. Mu.l, linear vector 3. Mu.l, 5 XCE II Buffe 2. Mu.l, exnase I1. Mu.l metal bath at 37℃for 30 min. The ligated vector was transferred into DH 5. Alpha. And plated on a petri dish containing 50mg/L Kanamycin (Kanamycin, kan). Then, 1300_GFP-F/R primer is used for bacterial detection, and the correct monoclonal bacterial liquid is sent to Shanghai biological company for sequencing.

1300_GFP-F:5’-ATGGTGAGCAAGGGCGAGGAGCT-3’；

1300_GFP-R:5’-ACCGATGATACGAACGAAAG-3’；

Finally construct pCAMBIA1300, pCAMBIA1300-AhPSW1 ^G -GFP

And pCAMBIA1300-AhPSW1 ^T GFP vector was transferred into Agrobacterium tumefaciens GV3101, spread on YEP solid medium containing 50mg/L Kan and 25mg/L Rifampin (Rif) resistance, cultured at 28℃for 2-3 d, and selected for monoclonal bacterial examination.

(2) Acquisition of AhPSW1 transgenic Arabidopsis thaliana

pCAMBIA1300, pCAMBIA1300-AhPSW1 ^G -GFP

And pCAMBIA1300-AhPSW1 ^T GFP was transformed into Arabidopsis by Agrobacterium infection.

(3) PCR identification of transgenic peanuts

Screening was performed on 30mg/L hygromycin (Hyg) screening medium using hygromycin tag (Hyg) with Hyg-1F/R primer pair T ₁ The generation transgenic plants are identified positively, and the primer sequences are as follows:

Hyg-F：5’-CGAGTACTTCTACACAGCCATC-3’；

Hyg-R：5’-TCCACTATCGGCGAGTACTTCT-3’；

obtaining 7 positive transgenic plants in total, and obtaining T after generation ₃ The generation homozygous transgenic lines are respectively: OE-S-2, OE-S-3, OE-S-6; the phenotype of the plants OE-L-3, OE-L-4, OE-L-7 is shown in FIG. 2.

Example 4: phenotypic identification of AhPSW1 transgenic Arabidopsis thaliana

Phenotype observation and statistical analysis were performed on mature seeds of the resulting arabidopsis lines, in which seed sizes of both PSW1 genotype transgenic lines were significantly increased compared to wild type (Col-0) (fig. 3A-E) while expression level analysis was performed on transgenic arabidopsis using fluorescent quantitative primers for AhPSW1-qF/R, with atacin 2 (U37281.2) as an internal reference (fig. 3B).

Atactin 2F:5’-GTCGTACAACCGGTATTGTGCT-3’；

Atactin 2R:5’-TGTCTCTTACAATTTCCCGCTCT-3’；

AhPSW1 ^G OE-S-2, OE-S-3, OE-S-6 strains and AhPSW1 ^T The seed length of OE-L-3, OE-L-4, OE-L-7 lines was increased by 2.6%, 5%, 8%10%, 8% and 20%, respectively (FIG. 3D). AhPSW1 ^T Seed length ratio AhPSW1 of transgenic lines OE-L-3 and OE-L-4 ^G The strain OE-L-3 of (C) was high but its expression level was low (FIG. 3B), and the result showed that overexpression of AhPSW1 increased the seed size of Arabidopsis thaliana, and AhPSW1 ^T Has stronger effect.

Example 5: marker development group verification

KASP and InDel markers (ahfsw 1-QF/R) were developed based on the difference between SNP and InDel of the PSW1 gene between the two materials:

KASP10_F1:5’-GAAGGTGACCAAGTTCATGCTTTTCACCATAAAACTG GTTATAGC-3’；

KASP10_F2:5’-GAAGGTCGGAGTCAACGGATTATTTCACCATAAAACT GGTTATAGA-3’；

KASP10_R:5’-CAAAGGAAATTGGTCAAATTCCTTCGCTT-3’；

afterwards we divided RIL population and natural population into 4 haplotypes, respectively: hapI: (TAA) _0/1/2 –G(ND_S)；HapII：(TAA) _3/4 –T(ND_L)；HapIII：(TAA) _0/1/2 –T；HapIV：(TAA) _3/4 -G. Of the 88 natural populations there were 5 HapI, 45 HapII, 4 HapIII and 34 HapIV. The pod phenotype data associated with each material was further analyzed for pods with greater HapII than HapI in the RIL population, pods with greater HapII and HapIV than HapI and HapIII in the natural population and reaching significanceWhile HapII and HapIII, although larger than HapIV and HapI pods, respectively, did not reach significant differences (fig. 4). From this we can conclude that both the TAA repeat insertion of the promoter region of the candidate gene PSW1 and the SNP variation of the coding region have a strong correlation with the pod size.

In conclusion, the peanut pod size trait related gene AhPSW1 can regulate and control the peanut pod length, can be used for researching and controlling molecular mechanism research of the peanut pod size trait related, and has important application value in the aspect of peanut breeding.

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments may be changed without departing from the spirit of the invention, and a plurality of specific embodiments are common variation ranges of the present invention, and will not be described in detail herein.

Claims (8)

1. The peanut pod size trait related gene AhPSW1 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. The gene AhPSW1 related to the peanut pod size trait of claim 1, wherein the full-length cDNA nucleotide sequence is:

(1) A nucleic acid sequence as shown in SEQ ID NO. 2; or (b)

(2) A nucleic acid sequence of equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.2.

3. The peanut pod size trait related gene ahfsw 1 of claim 2, wherein the CDS nucleotide sequence is:

(1) A nucleic acid sequence as shown in SEQ ID NO. 3; or (b)

(2) A nucleic acid sequence of equivalent function derived from the nucleic acid sequence shown in SEQ ID NO.3.

4. The peanut pod size trait related gene ahfsw 1 of claim 2, wherein the 5'utr and 3' utr sequences of gene ahfsw 1:

(1) As shown in SEQ ID NO.4; or (b)

(2) A nucleic acid sequence derived from the nucleic acid sequence shown in SEQ ID NO.4 and having an equivalent function.

5. A primer pair for amplifying the full length of the peanut pod size trait related gene ahfsw 1 or any fragment thereof of claim 1, said primer pair being as follows:

AhPSW1-1F：5’-ATGTCTAGAAGCTTAAGAAACC-3’；

AhPSW1-1R：5’-TTAAAAACCTCCCTTGAACAC-3’。

6. a recombinant plasmid comprising the peanut pod size trait related gene ahfsw 1 of claim 1, wherein the recombinant plasmid is produced by inserting the CDS of the peanut pod size trait related gene ahfsw 1 into a pCAMBIA13001300-GFP plant over-expression vector.

7. Use of the peanut pod size trait related gene ahfsw 1 of claim 1 or the recombinant plasmid of claim 6 in peanut yield breeding.

8. A method for performing fluorescent quantitative analysis using ahfsw 1 as claimed in claim 1, comprising the steps of:

1) The fluorescent quantitative specific primers were designed for the AhPSW1 gene sequence as follows:

AhPSW1-qF：5’-TCAAGAACTTCACAGATGCAG-3’；

AhPSW1-qR：5’-CCACTGAGAAGATTCTTTCCCAAG-3’；

the reference gene adopts ahacin 7, and the primer sequence is as follows:

Ahactin7-F：5’-GATTGGAATGGAAGCTGCTG；

Ahactin7-R：5’-CGGTCAGCAATACCAGGGAA；

2) UsingGreen qPCR SuperMix kit and Bio-Rad CFX96 Touch real-time PCR System, wherein 20. Mu.l of the reaction system, 1. Mu.l of cDNA, 0.5. Mu.l of each of the upstream and downstream primers, 2 x->Green qPCR SuperMix10 μl, nucleic-free water 8 μl; PCR procedure: 95 ℃ for 3min,95 ℃ for 5sec,55 ℃ for 30sec,72 ℃ for 30sec,40 cycles;

3) Taking 5 mu l of PCR reaction products, using 2.0% agarose gel electrophoresis, sequencing, and judging whether target fragments are identical;

4) By 2 ^-△△CT The relative expression level of the AhPSW1 gene was calculated by the method.