CN110241125B

CN110241125B - Soybean hundred-grain weight synergistic gene and molecular marker and application thereof

Info

Publication number: CN110241125B
Application number: CN201910702026.3A
Authority: CN
Inventors: 盖钧镒; 陈先连; 王吴彬; 刘成
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2021-03-30
Anticipated expiration: 2039-07-31
Also published as: CN110241125A

Abstract

The invention discloses a soybean hundred-grain weight synergistic gene, a molecular marker and application thereof, wherein the base sequence of the gene is shown as SEQ ID No.2, and the gene has synergistic effect on soybean hundred-grain weight; the invention identifies a gene GmSLK which is positioned on a soybean Gm16 chromosome and used for regulating the soybean hundred grain weight, and the grain size of the gene can be obviously improved by overexpression in arabidopsis thaliana. Meanwhile, a marker InDelSLK for identifying the gene is provided. The marker provides convenience for identifying whether the GmSLK is a synergistic gene or a reduced gene in germplasm resources and filial generations, and provides important judgment value for soybean breeding and material selection.

Description

Soybean hundred-grain weight synergistic gene and molecular marker and application thereof

Technical Field

The invention belongs to the field of breeding, and particularly relates to excavation and application of a gene GmSLK for regulating and controlling the hundred grain weight of soybean grains.

Background

Soybeans are crops of great economic significance and provide edible oil, protein and the like for human beings and animals. With the continuous improvement of the quality of life of human beings, the demand of soybeans is more and more, so that the improvement of the yield of the soybeans is always a main target of soybean breeding at home and abroad. However, the hundred grain weight, an important determinant of soybean yield, is also classified as one of the major breeding traits by breeders. However, the soybean hundredth trait is a quantitative trait controlled by multiple genes and is susceptible to the environment, which brings great hindrance to researchers.

Aiming at the soybean hundred-grain weight trait, many scholars at home and abroad position a large number of QTL (quantitative trail loci) by using different primary mapping populations and based on different marker maps. However, the primary mapping population can cause the QTL positioning interval to be overlarge, and a large number of gene loci are separated simultaneously, thereby causing genetic background effect and increasing the difficulty of fine positioning of the main QTL. An ideal approach is found for solving the problem by utilizing a secondary mapping population, such as the application of a Chromosome Segment Substitution Line (CSSL). Ideally, individual genomes of chromosome fragment replacement lines carry a small number of fragments of the donor parent chromosome, and the genetic background of the individual genomes is highly consistent, which is undoubtedly ideal material for QTL positioning. Theoretically, the property value of the chromosome fragment substitution line can be compared with the property value of the recurrent parent, and the property difference between two lines is caused by the difference QTL of the introgression fragment of the introduced line, so that the QTL analysis by using the homologous chromosome fragment substitution line can improve the positioning accuracy.

In order to better realize the fine positioning of QTL and molecular assisted breeding, the detected substitution line with important target characters can be purposefully subjected to marker-assisted backcross according to the positioning result of the chromosome fragment substitution line, secondary segregation populations are constructed, the important sites are dispersed in different individuals, the effect of each candidate site is evaluated one by one under the highly consistent genetic background, and the effective means of the positioning of candidate genes, the molecular assisted breeding and the gene function research can be realized.

Although a large number of QTLs related to soybean hundred-grain weight are positioned at present, few genes which directly regulate and control the soybean hundred-grain weight are determined, so that the research on candidate genes and functions of the genes which regulate and control the soybean hundred-grain weight can provide useful information and important breeding materials for molecular assisted breeding of the soybean hundred-grain weight.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a key candidate gene GmSLK for regulating and controlling the soybean hundredfold and application thereof, and provides useful information and important breeding materials for the molecular assisted breeding of the soybean hundredfold.

The technical scheme of the invention is as follows:

the soybean hundred-grain weight synergistic gene has a base sequence shown in SEQ ID No. 2.

The amino acid sequence of the protein coded by the soybean hundred-grain weight synergistic gene is shown in SEQ ID No. 4.

A polynucleotide fragment encoding the amino acid sequence shown in SEQ ID No. 4.

A recombinant vector comprising the polynucleotide fragment; preferably, the sequence of the polynucleotide fragment is shown in SEQ ID No. 2.

The specific primer for amplifying the soybean hundred-grain weight-enhanced gene has an upstream primer sequence of SEQ ID No.5 and a downstream primer sequence of SEQ ID No. 6.

The molecular marker of soybean hundredfold weight synergistic gene is located in the position of chromosome 16 Chr16:35923082 and 35923257bp, and the primer of the molecular marker is shown as SEQ ID No.7 and SEQ ID No. 8.

The method for identifying whether the soybean contains the soybean hundred-grain weight synergistic gene or not takes the genomic DNA of the soybean to be identified as a template and carries out PCR amplification by using primers shown in SEQ ID No.7 and SEQ ID No.8, and if 175bp of amplification product can be obtained, the soybean contains the soybean hundred-grain weight synergistic gene.

The soybean hundred-grain weight synergistic gene or protein or recombinant vector or specific primer or molecular marker is applied to soybean breeding.

The invention discovers a soybean hundred-grain weight regulation gene GmSLK which is located in chromosome 16 Chr16:35920962-35928190bp, a whole genome length of 7229bp and a whole coding sequence length of 2640bp, and the analysis and comparison finds that the GmSLK gene sequence from wild bean N24852 has one more glutamine in the glutamic acid tandem repeat region of a first exon compared with the GmSLK gene sequence of cultivated bean NN 1138-2. And the GmSLK gene from the wild beans has a reducing effect on the soybean hundred grain weight, and the GmSLK gene from the cultivated beans has a synergistic effect on the soybean hundred grain weight. Through further positioning, a molecular marker InDelSLK is found in the difference interval of the GmSLK gene between the wild bean N24852 and the cultivated bean NN1138-2, so that whether the GmSLK gene in different soybean varieties is a synergistic gene or a reduced gene can be judged through the marker, and the marker has important value in soybean breeding and material selection.

Compared with the prior art, the invention has the following beneficial effects:

the invention identifies a gene GmSLK which is positioned on a soybean Gm16 chromosome and used for regulating the soybean hundred grain weight, and the over-expression of the gene can obviously improve the soybean hundred grain weight property. Meanwhile, a marker InDelSLK for identifying the gene is provided. The marker provides convenience for identifying whether the GmSLK is a synergistic gene or a reduced gene in soybean seed resources and filial generations, and provides important judgment value for soybean breeding and material selection.

Drawings

FIG. 1: a schematic diagram of population construction; a chromosome fragment substitution line (constructed in 2013 by Wubin et al) is constructed by taking wild bean N24852 as a male parent and cultivated bean NN1138-2 as a recurrent parent, and CSSL3068 is one family line in the substitution line. The secondary population takes CSSL3068 as female parent and NN1138-2 as male parent, 294 offspring are obtained by hybridization and selfing, and F of 294 family lines is obtained by single plant planting and harvesting₂The population is then planted in one row per family, selfed and harvested in rows to yield 294F plants_2:3Family, in the same way, obtain F_2:4And F_2:5Family members.

FIG. 2: (iii) a hundred-grain phenotype of the parent and the location population; (A) a seed size schematic for the secondary population parents NN1138-2 and CSSL 3068; (B) comparison of the hundred particle weight for the Secondary population parents NN1138-2 and CSSL3068 (. about.P)<0.01); (C) location group F_2:3Planting Jiangsu Nanjing (2015NJ), F in 2015_2:4The population was planted in 2016 in Jiangsu Nanjing (2016NJ) and F_2:5The number of times of the colony planted with Bai Lian weight phenotype of Jiangsu Nanjing (2017NJ) and Anhui Dangdui (2017AH) in 2017 respectively.

FIG. 3: fine positioning process; (A) primary physical map of chromosome 16; (B) a local high-density physical map of chromosome 16; (C) analysis of the Secondary population F Using Single-marker analysis (IciMapping2.0) in combination with high Density mapping_2:3、F_2:4And F_2:5Phenotypic data in four environments; (D) analyzing phenotype and genotype positioning candidate genes of 11 recombinant individuals by using a displacement mapping method; (E) schematic structural diagram of GmSLK gene.

FIG. 4: GmSLK amplification banding pattern; GmSLK-N24852 represents that GmSLK comes from wild bean N24852, GmSLK-NN1138-2 represents that GmSLK comes from cultivated bean NN1138-2, GmSLK-CSSL3068 represents that GmSLK comes from substitution line CSSL3068, Marker is 5000bp, and a band indicated by a red arrow is a 3000bp band.

FIG. 5: comparing the sizes and fresh weights of the seeds of the parents at different periods of development. (A) Comparing the sizes of the parent NN1138-2 and the parent CSSL3068 in different periods of grain development; (B) comparing the parent NN1138-2 and CSSL3068 in terms of fresh weight of grains at different stages of grain development. Statistical analysis of 3 biological replicates and 3 technical replicates per sample (. p <0.05,. p < 0.01).

FIG. 6: relative expression of GmSLK in different stages of the development of parent NN1138-2 and CSSL3068 grains. The reference gene was UBI3 gene, and 3 biological and 3 technical replicates were analyzed statistically per sample (. p <0.05,. p < 0.01).

FIG. 7: transgenic Arabidopsis thaliana was analyzed for GmSLK-NN1138-2 (left) and GmSLK-CSSL3068 (right) gene function. (A) Analyzing the relative expression quantity of the homozygous transgenic line, wherein the reference gene is an Actin gene; (B) comparing the shape and size of the transgenic plant with that of the wild type 3-week plant; (C) transgenic and wild-type plants were compared for 1000 grain weight and 3 biological and 3 technical replicates were statistically analyzed per sample (. p <0.05,. p < 0.001).

Detailed Description

Example 1 acquisition of Soybean hundred grain weight major site qSW16.1

(1) Positioning group construction and planting

Wubin et al, Wang in 2013, constructed a set of chromosome fragment replacement line population consisting of 151 families by using wild bean N24852 and cultivated bean NN1138-2, found that the hundred weight of one family CSSL3068 is 14.68g according to the hundred weight phenotype information of the population family, and obviously reduced compared with the recurrent parent NN1138-2 hundred weight (18.7g), and found that the CSSL3068 contains one hundred weight wild site Sat _224 according to the marker information of the population. In 2014, a secondary population is constructed by hybridizing a family CSSL3068 with a recurrent parent NN1138-2, and F of 294 lines is obtained₂And (5) positioning the population in generation and level. In 2015, F was extracted from Jiangsu Nanjing (2015NJ)₂The secondary population is planted with 294 plant rows, 3 repeats, then each row is harvested separately, and 294 families of F are obtained_2:3Population, assay F_2:3The hundred-grain phenotype of a population's pedigree. In 2016, F was collected from Nanjing, Jiangsu (2016NJ)_2:3294 plant rows are planted in a colony, 3 are repeated, and then each row is harvested separately to obtain F of 294 families_2:4Population, assay F_2:4The hundred-grain phenotype of a population's pedigree. 2017F is to be_2:4294 plant rows of the population are respectively planted in Jiangsu Nanjing (2017NJ) and Anhui Dangdui (2017AH), 3 times of the plant rows are repeated, and then each row is independently harvested to obtain 294 family F_2:5Population, assay F_2:5The hundred-grain phenotype of the population pedigree (figure 1).

(2) Determination and analysis of phenotype of hundred grain weights of parents and positioning groups

Planting in rows, harvesting, baking at 30 deg.C until the weight of seeds is constant, and selecting 100 full seeds with good development for each parent and family.

First, seed size between the two parents was characterized and analyzed (fig. 2A and B), and as a result, the hundred weight of parent NN1138-2 was found to be significantly greater than the hundred weight of parent CSSL3068 under four different circumstances (see table 1). In addition, the frequency distribution graph of the soybean weight phenotype of the secondary population in four environments was statistically analyzed by using SPSS 2.0 (fig. 2C), and the results showed that the secondary population was continuously distributed, and the average value of the soybean weight phenotype in the F2:5 population was 16.94g at the maximum in 2017AH environment, and the average value of the soybean weight in the 2015NJ environment was 13.88g at the minimum (as shown in table 1). In addition, the location population has a lower phenotypic Coefficient of Variation (CV) and a higher genetic rate (h) under different environments²) This indicates that the major variation of the soybean hundredth phenotype is genetically controlled.

TABLE 1 hundred particle weight of parent NN1138-2 and CSSL3068 under different environments

CV, coefficient of variation; h is²And the genetic rate.

(3) Group genotype identification

Extraction of the Secondary population F by CTAB method₂The genomic DNA of 294 lines of soybean leaf of (2) above, the primers used were synthesized by the EnxWeijie (Shanghai) trade company. PCR reaction system 10ul, which contained 3ul DNA (15ul), 1.5 ul each of upstream and downstream primers (0.2mmol/L), 1.2 ul Mgcl₂(2.5mmol/L)、0.24mu.L dNTP (10mmol/L, N ═ A, C, G, T), 0.12. mu.L Taq enzyme (5U/ul) and 1.4. mu.L ddH₂And O. PCR reaction procedure: denaturation at 94 deg.C for 5 min; followed by 33 cycles of denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 40 s; then completely extending for 10min at 72 ℃; finally, the mixture is stored at 4 ℃. And (3) carrying out electrophoresis on the PCR amplification product by using 8% polyacrylamide gel, and carrying out silver staining and color development to identify the polymorphism of the band.

(4) Construction of genetic linkage map

Two parents are analyzed by using 49 SSR molecular markers on

chromosome

16, 7 sites with polymorphism between the parents are statistically analyzed, the genetic types with the polymorphism markers between the parents are identified in secondary population F2 strains, and then linkage analysis is carried out on the genetic types to construct a genetic linkage map of a secondary population (FIG. 3A).

(5) Development of molecular marker

By utilizing SSR marker information of soybean genome published by Song et al and InDel information of wild bean N24852 and cultivated bean NN1138-2 on chromosome Gm16, a new molecular marker is designed, 3 InDel markers and 16 InDel markers with polymorphism between parents are statistically analyzed (Table 2), the genetic type with the polymorphism markers between the parents is identified in a secondary population F2, and then linkage analysis is carried out on the genetic type to construct a high-density map of the secondary population (figure 3B).

TABLE 2 molecular marker information related to the present invention

(6) Analysis of the results

The primary mapping results using SAS9.4 in combination with secondary population phenotypic data and molecular data showed that the soybean kernel weight major locus qswp β 16.1 is located between chromosomes 16 Sat _224 and Satt 431.

Combining high density profiles with Secondary population F_2:3、F_2:4And F_2:5Phenotypic data in four environments, fine localization using single marker analysis (IcMapping 2.0) resulted in localization of soybean percent weight major site qSW16.1 to InDel16_01 and BARCSYSSR 16_1215 between two markers (fig. 3C).

Example 2 Regulation of acquisition of Soybean hundred-grain weight Gene GmSLK

Determination of the Gene GmSLK

The soybean hundred weight major site qSW16.1 was located between two markers Indel16_01 and BARCSYSSR 16_1215 by fine localization, two candidate genes were found in common between these two markers by alignment of soybean reference gene sequences, and to further determine candidate genes, from F ₂11 families with recombination in the 35889420bp-35952811bp region of the Gm16 chromosome were selected from the population, and the 11 recombinant families were further finely mapped by comparing and analyzing the genotype and the phenotype data of the two parents through a displacement mapping method, and no recombination site was found between the two markers InDel16_03 and BARCSYSSR 16_1215, and the genes Glyma.16g198300 were co-isolated (FIGS. 3D and E). The gene Glyma.16g198300 was found by gene annotation analysis to belong to the LIM domain, and its function was annotated as a Transcriptional Regulator (SLK 2) and was therefore named GmSLK.

Example 3 application of soybean hundred-grain weight gene GmSLK in soybean breeding

The specific amplification upstream primer sequence of the gene for regulating the soybean hundred-grain weight trait is SEQ ID No.5, and the specific amplification downstream primer sequence is SEQ ID No.6 (Table 3).

TABLE 3 specific primer sequences for amplifying the GmSLK gene

The coding region (CDS) sequence of GmSLK was amplified using the primer sequences SEQ ID No.5 and SEQ ID No.6, using RNA of wild bean N24852 and two parents (NN1138-2 and CSSL3068) as templates, respectively. For amplification, high Fidelity enzyme (phase Super-Fidelity DNA polymerase, Vazyme) from Takara was used, PCR reaction systems and procedures are shown in tables 4 and 5, and the amplified band sizes are shown in FIG. 4:

TABLE 4 PCR reaction System

TABLE 5 PCR reaction procedure

The CDS sequence difference of the gene between two parents and wild bean N24852 is compared through sequencing amplification product analysis, and the gene GmSLK is found to be consistent in the sequences of the parents CSSL3068 and the wild bean N24852, the nucleotide sequence is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 3. Indicating that the GmSLK of the parent CSSL3068 is from wild beans. The nucleotide sequence of the GmSLK gene of the cultivated bean NN1138-2 is shown as SEQ ID No.2, and the coded amino acid sequence is shown as SEQ ID No. 4. The GmSLK gene of wild bean had one more glutamine in the glutamic acid tandem repeat region of the first exon than that of cultivated bean NN1138-2 (FIG. 3E). And the GmSLK gene from the wild beans has a reducing effect on the weight per hundred grains of the soybeans, and the GmSLK gene from the cultivated beans (NN1138-2) has a synergistic effect on the weight per hundred grains of the soybeans.

InDel16_03 was found to be located in the difference region between wild bean N24852 and cultivated bean NN1138-2 of GmSLK gene by sequence alignment, and InDel16_03 was named InDelSLK. Therefore, whether the GmSLK gene in different soybean varieties is a synergistic gene or a reduced gene can be judged through the marker, and the marker has important value in soybean breeding and material selection.

Example 4 functional verification of GmSLK Gene

In order to rapidly identify the influence of the GmSLK gene on the grain weight, the experiment selects a mode crop Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 wild type as a transgenic receptor material, respectively overexpresses the GmSLK (GmSLK-CSSL3068) of a parent CSSL3068 and the GmSLK (GmSLK-NN1138-2) of a cultivated bean NN1138-2, analyzes and compares 1000 grain weights of a single-copy homozygote, and thereby verifies the influence of the GmSLK gene on the grain weight.

(1) GmSLK gene expression level analysis

To better understand the effect of the GmSLK gene on the soybean grain development period, the experiment analyzes and compares the expression levels of the GmSLK gene in different grain development periods of parents (NN1138-2 and CSSL 3068). Seeds of 7_ DAF, 14_ DAF, 21_ DAF,28_ DAF, 35_ DAF and 42_ DAF (DAF: day after flower) of 8 individuals NN1138-2 and CSSL3068 were taken, respectively, and 3 biological replicates of each sample were used to determine the phenotype (FIG. 5) and extract RNA for Real-time RT-PCR analysis (FIG. 6).

Observing and comparing the sizes of the seeds of the parents NN1138-2 and CSSL3068 in different development periods of the seeds, the seeds of the NN1138-2 are found to be larger in the different development periods of the seeds than the seeds of the CSSL3068 (figure 5A), and determining the fresh weight of the seeds shows that the fresh weight of the seeds of the NN1138-2 is obviously larger than that of the seeds of the CSSL3068 in the different development periods (figure 5B).

The relative expression levels of the GmSLK gene in different stages of the parental grain development are analyzed and compared, and the UBI3(ribosomal-ubiquitin fusion protein) gene of soybean is selected as an internal reference in the experiment. The result analysis shows that the expression abundance of the GmSLK gene in parents is increased along with the development of grains, and the expression amount reaches the maximum in the 42_ DAF period. However, the expression level of the GmSLK gene is very similar in the prophase and metaphase (14_ DAF-35_ DAF) of the parental kernel, but the relative expression level of the GmSLK gene in the parental NN1138-2 is obviously higher than that of the parental CSSL3068 in the 42_ DAF period.

(2) GmSLK gene transgenic Arabidopsis thaliana validation

In the research, a pCAMBIA 3301 vector is selected for transformation of Arabidopsis, the restriction enzyme sites are (Bst EII and NcoI), and upstream and downstream primers of GmSLK gene amplification are respectively SEQ ID No.3 and SEQ ID No.4 (Table 6). The recombination reaction was performed using a cloning recombination kit (CloneExperss II). Then the constructed over-expression vectors pCAMBIA 3301-GmSLK-NN1138-2 and pCAMBIA 3301-GmSLK-CSSL30068 are respectively transformed into DH5a competence for plasmid propagation, and then the plasmids are extracted through cloning identification and then are transformed into agrobacterium EHA 105. Respectively transferring the constructed overexpression vectors into the wild type of Arabidopsis thaliana Columbia-0 by a pollen-soaking method.

TABLE 6GmSLK Gene construction vector primer design

The over-expression vector pCAMBIA 3301 has glufosinate resistance, and positive seedlings are screened by 1/2MSA +10mg/l glufosinate culture medium. Harvesting T1 generation seeds of infected Arabidopsis plants, firstly screening T2 generation positive seedlings on a resistance culture medium, then planting and harvesting T2 seeds, planting the T2 seeds on the resistance culture medium and statistically analyzing survival/death ratio, selecting survival/death to accord with 3:1 through chi-square detection, indicating that the transgenic plants belong to single copy plants, then planting and harvesting T3 generation seeds, then planting the T4 generation seeds on the resistance culture medium and selecting homozygous plants with 100% survival rate, planting and harvesting T4 seeds and statistically calculating 1000 grain phenotype. 3 single copy homozygous lines were selected for the GmSLK-NN1138-2 and GmSLK-CSSL30068 overexpressing plants, 3 plants were grown for each line, then leaves were taken for RT-PCR analysis, the Actin gene of Arabidopsis was selected as the reference gene, and the relative expression of the exogenous gene of the transgenic plants was detected (FIG. 7A). The transgenic plants were also observed and analyzed for plant size at 3 weeks, and no difference was found between the overexpressed GmSLK-NN1138-2 and GmSLK-CSSL30068 plants compared to the wild type plant size (FIG. 7B). Harvest T5 seeds were statistically analyzed for a 1000 grain weight phenotype (fig. 7C), and the results analysis showed that over-expressed GmSLK-NN1138-2 significantly increased grain weight compared to wild type, whereas over-expressed GmSLK-CSSL30068 significantly decreased grain weight. Therefore, the GmSLK genotype from the wild bean or the parent CSSL3068 can be proved to belong to the anti-aging genes and can reduce the grain weight, while the GmSLK genotype from the cultivated bean NN1138-2 belongs to the synergistic genes and can increase the grain weight, and powerful evidence is provided for yield breeding of soybeans.

Sequence listing

<110> Nanjing university of agriculture

<120> soybean hundred-grain weight synergistic gene and molecular marker and application thereof

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<170> SIPOSequenceListing 1.0

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gattccatgc aattccaggg tcgtaatccc cagttacagg ctttccttca gcagcagcag 720

cagagactga gacaacaaca aatgtttcag caaatgccac aattacaccg agcacacttg 780

cagcagcagc aacaacaaca acaaatgcaa ttgaggcagc agcagcagca gcagcaacaa 840

caacaacaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacagcaa 900

cagcaagtga tgcagccctc ttctgtggtc aagcgtccat atgacagtag tgttagtggg 960

gtatgtgccc gtcgattgat gcagtatctc tatcatcaaa ggcaacgacc aaatgataat 1020

agtattgcct attggagaaa atttgtggct gaatattact ctcctcgtgc aaagaaacgg 1080

tggtgcttgt cattatatag taatgttggg catcacgcac ttggtgtttt tccccaagca 1140

tctatggatg catggcactg tgacatatgt ggttctaaat ctggaagggg atttgaggca 1200

acttatgaag tattacctag acttaatgaa atcaaatttg gcagtggagt aattgatgaa 1260

ctattgtttc tggacatgcc acgtgaaatg agattcgctt ctggtgcgat gatgttagaa 1320

tatggaaaag cagttcaaga gagtgtatat gagcagcttc gtgttgttcg tgaaggtcaa 1380

cttcgtatca tattcactca agacttgaag atattatctt gggagttctg tgcaaggtgc 1440

catgaggaac ttcttcctcg aaggttggtt gcaccacagg tcaaccagtt agttcaggta 1500

gctaaaaaat gtcaaagtac aattgctgaa agtggttctg atggggtttc tcaacaagac 1560

atccaaacaa acagcaacat gttgttgaca gctgggggtc agcttgcgaa gattttggag 1620

atgcaatcgc taaatgagtt gggcttttct aaaagatacg tgagatgttt gcaaatttcg 1680

gaggttgtca atagcatgaa agacctaata gatatctgtg cagatcacaa aattggtgcc 1740

attgagagtt tgaaaaattt tcctcgtcta gcaacagcct caaaggtcca gatgcagaag 1800

atgcaggaaa tggaacagct agcaaatgtt caaggtctgc caactgatcg aaacacactc 1860

aataagctaa tggcactgaa tcctggattg aacaaccata taaacaaccc tcataatatg 1920

gttaatcgtg gtgctttgag tgggtcagcc caagctgctt tagcactgaa caactaccaa 1980

aatcttctca tgaggcaaaa ttcaatgaac tctagccctg gctcacttca gcgcgaaggg 2040

tcctctttca ataattcaaa ccagagtcca tcttcagctt tgcaaggagc tggtcctgct 2100

ttaattccag gcccaatgca gaattcatct gttagtggtt tcccaagccc ccgtctaccc 2160

ccacagcagc agcaacacca cctacaacag ccctcattaa gtgcaaatgc tttactgcaa 2220

caaaatcatt cacagggttc ccaaggaaat caagctctgc agcagcagat gatccatcaa 2280

ctactgcagg agatgtcaaa taacaacggg ggagtgcaac cacagtctct tggtggaccc 2340

agtgcaaata tggcaaagaa tgcactgggg tttgggggcc attatccatc cttaagtgga 2400

ggttctgcca atgttacagg aaacaatgga cctatgtcaa ggaataatag cttcaaaaca 2460

actgcaaata gtgattcttc tgctgctggt ggcaacaatg gattaaacca gagaacatct 2520

gagatgccac aaaatctaca tttgcaagat gtggttcagg atattggcaa tgaattcacg 2580

gataatccct tccttaacag tgatcttgat gataacatgg gttttggctg gaaggca 2637

<210> 3

<211> 880

<212> PRT

<213> Soybean (Glycine max)

<400> 3

Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn

1 5 10 15

Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn

20 25 30

Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly

35 40 45

Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala

50 55 60

Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser

65 70 75 80

Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln

85 90 95

Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser

100 105 110

Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val

115 120 125

Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn

130 135 140

Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser

145 150 155 160

Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met

165 170 175

Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser

180 185 190

His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met

195 200 205

Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln

210 215 220

Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln

225 230 235 240

Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His

245 250 255

Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg

260 265 270

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

275 280 285

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val

290 295 300

Met Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser

305 310 315 320

Gly Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln

325 330 335

Arg Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu

340 345 350

Tyr Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser

355 360 365

Asn Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp

370 375 380

Ala Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu

385 390 395 400

Ala Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser

405 410 415

Gly Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg

420 425 430

Phe Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu

435 440 445

Ser Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile

450 455 460

Ile Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg

465 470 475 480

Cys His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn

485 490 495

Gln Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser

500 505 510

Gly Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met

515 520 525

Leu Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser

530 535 540

Leu Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile

545 550 555 560

Ser Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp

565 570 575

His Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala

580 585 590

Thr Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu

595 600 605

Ala Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu

610 615 620

Met Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn

625 630 635 640

Met Val Asn Arg Gly Ala Leu Ser Gly Ser Ala Gln Ala Ala Leu Ala

645 650 655

Leu Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser

660 665 670

Ser Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn

675 680 685

Gln Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro

690 695 700

Gly Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu

705 710 715 720

Pro Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala

725 730 735

Asn Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln

740 745 750

Ala Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn

755 760 765

Asn Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn

770 775 780

Met Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser

785 790 795 800

Gly Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn

805 810 815

Asn Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly

820 825 830

Asn Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His

835 840 845

Leu Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro

850 855 860

Phe Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala

865 870 875 880

<210> 4

<211> 879

<212> PRT

<213> Soybean (Glycine max)

<400> 4

Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn

1 5 10 15

Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn

20 25 30

Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly

35 40 45

Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala

50 55 60

Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser

65 70 75 80

Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln

85 90 95

Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser

100 105 110

Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val

115 120 125

Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn

130 135 140

Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser

145 150 155 160

Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met

165 170 175

Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser

180 185 190

His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met

195 200 205

Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln

210 215 220

Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln

225 230 235 240

Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His

245 250 255

Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg

260 265 270

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

275 280 285

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val Met

290 295 300

Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser Gly

305 310 315 320

Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln Arg

325 330 335

Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu Tyr

340 345 350

Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser Asn

355 360 365

Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp Ala

370 375 380

Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu Ala

385 390 395 400

Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser Gly

405 410 415

Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg Phe

420 425 430

Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu Ser

435 440 445

Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile Ile

450 455 460

Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg Cys

465 470 475 480

His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn Gln

485 490 495

Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser Gly

500 505 510

Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met Leu

515 520 525

Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser Leu

530 535 540

Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile Ser

545 550 555 560

Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp His

565 570 575

Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala Thr

580 585 590

Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu Ala

595 600 605

Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu Met

610 615 620

Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn Met

625 630 635 640

Val Asn Arg Gly Ala Leu Ser Gly Ser Ala Gln Ala Ala Leu Ala Leu

645 650 655

Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser Ser

660 665 670

Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn Gln

675 680 685

Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro Gly

690 695 700

Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu Pro

705 710 715 720

Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala Asn

725 730 735

Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln Ala

740 745 750

Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn Asn

755 760 765

Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn Met

770 775 780

Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser Gly

785 790 795 800

Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn Asn

805 810 815

Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly Asn

820 825 830

Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His Leu

835 840 845

Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro Phe

850 855 860

Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala

865 870 875

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgacacctt tgcgagtggc 20

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tcatgccttc cagccaaaac cc 22

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acaattacac cgagcaca 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttgaccaca gaagaggg 18

Claims

1. The molecular marker of soybean hundredth synergistic gene with the nucleotide sequence as shown in SEQ ID No.2 is located in chromosome 16 of soybean Chr16:35923082 and 35923257bp, and the primer of the molecular marker is shown in SEQ ID No.7 and SEQ ID No. 8.

2. The method for identifying whether soybean contains soybean hundred grain weight synergistic gene with nucleotide sequence shown as SEQ ID No.2 is characterized in that genomic DNA of the soybean to be identified is taken as a template, primers shown as SEQ ID No.7 and SEQ ID No.8 are used for PCR amplification, and if 175bp of amplification product can be obtained, the soybean contains the soybean hundred grain weight synergistic gene.

3. The soybean hundred-grain weight synergistic gene with a nucleotide sequence shown as SEQ ID No.2, or a recombinant vector containing the nucleotide sequence shown as SEQ ID No.2, or a primer pair shown as SEQ ID No.5 and SEQ ID No.6, or the application of the molecular marker in soybean hundred-grain weight trait breeding, wherein the primer pair is shown as in claim 1.