CN107400703B - Molecular marker linked with major QTL qFSW-2 and qFSW-5 of corn seed storage tolerance and application thereof - Google Patents

Abstract

The invention relates to a molecular marker linked with major QTL qFSW-2 and qFSW-5 in corn seed storage tolerance and application thereof. The major QTL for the storability of the corn seeds is qFSW-2 positioned in the Bin 2.06 region of the No. 2 chromosome of the corn and qFSW-5 positioned in the Bin5.03 region of the No.5 chromosome of the corn. Wherein, the molecular markers closely linked with qFSW-2 comprise markers umc1079 and umc 1028; molecular markers closely linked to qFSW-5 include markers umc2063 and umc 2400. According to the invention, through positioning 2 major QTL sites qFSW-2 and qFSW-5 of the corn seed storage tolerance, 4 SSR molecular markers closely linked with the 2 major QTLs are discovered, and a feasible technical approach is provided for corn seed storage tolerance molecular breeding.

Description

Molecular marker linked with major QTL qFSW-2 and qFSW-5 of corn seed storage tolerance and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a molecular marker linked with major QTL qFSW-2 and qFSW-5 in corn seed storage tolerance and application thereof.

Background

The storage resistance of corn seeds has important value for safe storage of the seeds, the annual demand of the corn seeds in China is more than 157 ten thousand tons, but the actual production per year is far greater than the consumption, a certain amount of the prepared barren seeds must be stored, and a large amount of the seeds need to be stored for a short time or a long time. At present, a low-temperature storage method is generally adopted, but even if the seeds are stored at low temperature, the seed vigor is also reduced year by year, and finally the seed use value is lost.

The method for measuring the storage stability of the seeds mainly comprises a natural aging method and an artificial accelerated aging method. Because the natural aging method has long detection period of the storage-resistant phenotype index, the artificial accelerated aging test is generally adopted to measure the main indexes of the aged seeds, such as germination vigor, germination rate, vitality index, life index and the like, so as to evaluate and detect the storage resistance of the seeds. This method was first proposed by Delouche to predict seed longevity. The current common artificial accelerated aging methods mainly comprise a high-temperature high-wet method, a hot water bath method, a chemical reagent method and the like. The method for measuring the storage stability of the corn mainly comprises a high-temperature high-wet method and a hot water bath method, and the methods have the advantages and the disadvantages and have different standards. Because the corn seed storage-resistant character genetic basis is complex and is seriously influenced by factors such as temperature, humidity, harvesting time and the like, the results obtained by different aging methods and materials are different, and a stable and efficient corn seed storage-resistant identification method is not established.

With the development of molecular assisted breeding technology, genetic research and gene mapping of seed storage tolerance become possible, and great progress has been made in recent years. So far, researches on seed storage resistance and seed vigor are mainly focused on model plants such as rice, wheat and arabidopsis thaliana, but with the improvement of technology, researches on crop species such as corn, tomato and cabbage are increasing.

Miura et al found 3 gene loci associated with storage endurance on chromosomes 2, 4, and 9, respectively, by performing potassium dichromate-soaking artificial aging on 98 backcross populations of indica rice and japonica rice, and then performing QTL mapping. The contribution rate of the gene locus on chromosome 9 is as high as 59.5%, and the contribution rates of qLG-2 and qLG-4 are respectively 13.4% and 11.6%. Emile utilizes the recombination inbred line of arabidopsis thaliana as a material, detects a series of QTLs for controlling seed storage tolerance by measuring the sugar content of the seeds, the germination rate of the aged seeds, ABA and the adversity stress, and finds that all characters have one or more common QTL sites. After Sasaki and the like store the recombinant inbred lines of indica-type rice and japonica rice for 1 year, 2 years and 3 years respectively, QTL analysis is carried out, 12 QTL sites for controlling germination rate and seedling growth are found and are respectively positioned on chromosome 7 and chromosome 9, the contribution rate is between 6.7% and 17.3%, and the contribution rate of the growth site RC9-2 of the seedlings stored for two years is the highest.

And S, Landjeva and the like carry out QTL analysis on the germination rate, the seed vigor, the seed life and the seedling growth vigor of the recombinant inbred line of the wheat containing the D genome, and the results of 20 QTLs are detected in total to indicate that the gene controlling the seed life is positioned on 1D or 5D, the contribution rate is between 15.3 and 31.3 percent, and the related loci on the 1D chromosome are relatively rich. QTL analysis by Fujino et al using 122 abundant inbred lines found 3 QTLs associated with low temperature germination (qLTG-3-1, qLTG-3-2 and qLTG-4) and qLTG-3-1 had been cloned.

the research is mainly carried out aiming at morphological indexes related to germination, QTLs (quantitative trait loci) analysis on physiological indexes of seed storage tolerance are less, Cui and the like utilize a recombinant inbred line of indica rice and japonica rice to detect QTLs (quantitative trait loci) for controlling seedling vitality indexes such as total amylase activity, soluble sugar content, and α -amylase activity and the like, 31 QTLs are detected in total, 5 different indexes are respectively controlled and are mainly positioned on chromosomes 3, 5 and 6, and the QTLs for controlling the characters are found to be positioned in similar areas.

Disclosure of Invention

The invention aims to provide a molecular marker linked with major QTL qFSW-2 and qFSW-5 in corn seed storage endurance and application thereof.

To achieve the object of the present invention, the present invention is a molecular marker closely linked to a major QTL for maize seed storability, which is qFSW-2 located within the region of chromosome 2, Bin 2.06, and qFSW-5 located within the region of chromosome 5, Bin5.03, of maize.

Wherein, the molecular markers closely linked with qFSW-2 comprise 2SSR markers umc1079 and umc 1028; molecular markers that are closely linked to qFSW-5 include the 2SSR markers umc2063 and umc 2400. Primers for amplifying each molecular marker were as follows:

the sequences of the forward primer and the reverse primer of umc1079 are SEQ ID No.1 and 2 respectively;

the sequences of the forward primer and the reverse primer of umc1028 are SEQ ID No.3 and 4, respectively;

the sequences of the forward primer and the reverse primer of umc2063 are SEQ ID NO.5 and 6, respectively;

the forward and reverse primer sequences of umc2400 are SEQ ID NO.7 and 8, respectively.

Characteristic bands with the size of 121bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.1 and SEQ ID NO. 2;

characteristic bands with the size of 138bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.3 and SEQ ID NO. 4;

characteristic bands with the size of 91bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.5 and SEQ ID NO. 6;

the characteristic band with the size of 114bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.7 and SEQ ID NO. 8.

The invention also provides application of the molecular marker in identification of maize seed storage-resistant major QTL sites qFSW-2 and qFSW-5.

The invention also provides application of the molecular marker in screening or identifying maize varieties with strong storage resistance.

The application comprises the following steps:

1) extracting the genome DNA of a plant to be detected;

2) taking the genome DNA of a plant to be detected as a template, and carrying out PCR amplification reaction by using a primer for amplifying the molecular marker;

3) and detecting the PCR amplification product.

The invention also provides application of the molecular marker in maize molecular marker assisted breeding.

The invention also provides a PCR detection kit for identifying the maize variety with strong storage resistance, and the kit comprises a primer for amplifying the molecular marker.

The invention further provides a molecular marker which is developed according to the molecular marker and closely linked with the maize seed storage-resistant major QTL.

According to the invention, through positioning 2 major QTL sites qFSW-2 and qFSW-5 of the corn seed storage tolerance, 4 SSR molecular markers closely linked with the 2 major QTLs are discovered, and a feasible technical approach is provided for corn seed storage tolerance molecular breeding.

Drawings

FIG. 1 shows the result of detecting polymorphism of maize parent by agarose electrophoresis in example 1 of the present invention.

FIG. 2 shows the result of polyacrylamide electrophoresis for detecting polymorphism of maize parent in example 1 of the present invention.

FIG. 3 shows the primer pairs of bnlg1265 and F in example 1 of the present invention_2：3Separating agarose gel electrophoresis patterns of population genotype detection.

FIG. 4 shows primers phi027 to F in example 1 of the present invention_2：3Separating polyacrylamide gel electrophoresis pattern of group genotype detection.

FIG. 5 shows (east 156X east 237) F in example 2 of the present invention_2：3And (5) distributing the germination rate of the seeds after artificial accelerated aging treatment of the population.

FIG. 6 shows (east 156X east 237) F in example 2 of the present invention_2：3And (5) distributing the germination potential of the seeds after the artificial accelerated aging treatment of the population.

FIG. 7 shows (east 156X east 237) F in example 2 of the present invention_2：3And (5) distributing the fresh weight of the seedlings after the group artificial accelerated aging treatment.

FIG. 8 shows (east 156X east 237) F in example 2 of the present invention_2：3And (4) distributing the germination index of the seeds after the artificial accelerated aging treatment of the population.

FIG. 9 shows (east 156X east 237) F in example 2 of the present invention_2：3And (5) distributing the seed vitality index after the artificial accelerated ageing treatment of the population.

FIG. 10 shows a graph based on F in example 3 of the present invention₂the maize genetic linkage map constructed by the colony comprises a QTL position ◆ which is positioned by the fresh weight of seedlings, a QTL position ◆ which is positioned by vigor index, and a QTL position ◆ which is positioned by the germination vigor.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

Example 1 establishment and genotyping of corn seed storage tolerance-related trait genetic study population

1. Test materials

2 parts of maize inbred lines (table 1) with obvious seed storage resistance difference are bred according to the early stage, wherein the seeds of the middle east 156 are stored durably, and the germination rate is still kept above 90% in 8 years under the natural storage condition; east 237 is not storable, and the germination rate is reduced to about 80 percent after 1-year storage under natural conditions. The test material seeds were provided by the corn research institute of northeast university of agriculture.

TABLE 1 relevant information of two maize inbred lines with significant differences in storability

2 test method

2.1F₂Population construction and planting of materials

Dong 156X Dong 237 made in Harbin in spring 2011, and F planted in Hainan in autumn 2011₁Selfing to obtain F₂And (4) seeds. Dong 156, Dong 237 and F were planted on Heilongjiang Harbin fragrance farm in spring 2012₁、F₂. Planting the single plants, wherein the row spacing is 0.70m, the plant spacing is 0.3m, and the row length is 6.0 m. The parents are planted with 3 rows of plants each, F ₁3 lines of planting, F₂And planting 25 rows.

2.2 genotype analysis

2.2.1 extraction of maize leaf DNA

Selecting parent strain F of corn leaves growing to 3 leaves in 1-heart stage from corn seedlings in 2012₁And F₂DNA extraction from individual leaves F₂The individual plants are numbered in order. The DNA was extracted by CTAB method. Adding about 1.0g of liquid nitrogen into young leaves, quickly grinding into powder,transfer to a 10mL centrifuge tube. Adding 7.5mL of 1mol/L Tris-HCl with pH8.0 in 100mL of buffer solution of CTAB extraction buffer solution preheated at 65 ℃; 0.5mol/L EDTA3.0m with pH8.0; 6.2g of NaCl; CTAB 2.0 g; 0.2% mercaptoethanol (added when used) is fully shaken and mixed evenly. And (3) preserving the heat in a water bath at 65 ℃ for 50-60 minutes (taking out and shaking for several times), taking out, cooling to room temperature, adding 4mL of chloroform: isopentanol (24: 1) was placed on a shaker and shaken slowly for 10 minutes. Centrifuging at 4 deg.C for 10 min with a 12000r/min centrifuge, and transferring the supernatant to another clean centrifuge tube. Then 4mL of chloroform: the shaking and the centrifugation of isoamyl alcohol (24: 1) are repeated, the supernatant is taken to another clean centrifugal tube, the equal volume of isopropanol is added, the mixture is gently shaken up and is kept stand in a refrigerator at the temperature of 4 ℃ for a period of time, and a DNA precipitate is hooked by a crochet hook. Washing the DNA precipitate with 70% ethanol for three times, drying, adding appropriate ddH₂And (3) fully and uniformly mixing the O solution and the RNAase solution (10mg/ml) in a blow-dried centrifugal tube, carrying out warm bath at 37 ℃ for 1h, measuring the concentration of the extracted DNA by using an ultraviolet spectrophotometer, diluting to 25ng/ul, and storing at-20 ℃ for SSR marker analysis.

2.2.2 screening of SSR primers

949 pairs of SSR marker loci distributed on 10 chromosomes of corn are selected, and the primer sequences are derived from the MaizeGDB genome database (http:// www.maizegdb.org /). PCR amplification was performed using genomic DNA of east 156 and east 237 as templates, and primers having polymorphism between east 156 and east 237 were selected for F₂And (4) detecting the genotype of the individual plant. The PCR products were electrophoresed on 3% agarose or 8.0% polyacrylamide gels, and the silver staining procedure was performed according to the method of Bassam et al. The specific operation process is as follows: after all the reactants were mixed uniformly according to the following system, 18uL of mineral oil was added to cover the mixture, and amplification was performed on a PCR instrument.

TABLE 2SSR reaction System

TABLE 3 touchdown PCR amplification reaction procedure

2.2.3 detection of amplification products

According to the size of the PCR amplification product fragment, 3% agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis is used for detection, and primers with polymorphism among parents are selected.

preparation of agarose gel electrophoresis

BIO RAD Power pac model 300, electrophoresis tank DYCP-34A.

Buffer solution: 0.5 XTBE (45mmol/L Tris-borate, 1mmol/L EDTA), and the specific glue making process and the notice are referred to the molecular biology experimental guidance.

② polyacrylamide gel electrophoresis

Gel 8% polyacrylamide non-denaturing gel was used, and the apparatus BIO RAD Power pac model 3000 electrophoresis apparatus was used.

③ electrophoresis process:

assembling the glass plates: and a parting bead is added between the glass plate and the rubber plate, the bottom ends of the glass plate and the rubber plate are aligned, clamped and mounted with a glue pouring base for standby. Pouring electrophoresis gel: and (3) fully shaking the glue solution to obtain a polyacrylamide gel solution, lightly filling the prepared glue between the two glass plates by using a suction bottle, inserting a comb at a glue filling opening, and paying attention to prevent bubbles from being generated at the bottom of the comb. The mixture was left at room temperature to polymerize for more than half an hour.

Electrophoresis conditions: the loading amount was 1.0uL of PCR amplification product, PBR322 was used as molecular weight Marker, electrophoresis buffer was 1 XTBE, and constant power 85W was used for about 60 minutes of pre-electrophoresis.

silver staining procedure

Fixing: the film was drawn down with a syringe needle into a plastic box of approximately 70cm by 50cm by 15cm, and 1.8L of glacial acetic acid solution (10%) was added, gently shaken for 10 minutes.

Rinsing: rinse 3 times with 2L of ultrapure water for 15s each time.

Silver staining: adding 1.5L of newly prepared staining solution (0.1% AgNO)₃) Gently shake for 10 minutes.

Rinsing: rinsing with 2L of ultrapure water for no more than 10 seconds.

And (3) developing: the gel plate was placed in a fresh 1.5L developer (22.5g NaOH, 1mL formaldehyde) and gently shaken until the streaks were clear.

2.2.4 genotype statistics

F₂There were three banding patterns at each site in the population samples: the band derived from east 156 was designated as 2, the band derived from east 237 was designated as 0, the heterozygous was designated as 1, and the deletion was designated as-1.

3 results and analysis

3.1 parent polymorphism assay

949 pairs of SSR primers are uniformly selected on the MaizeGDB, and polymorphism between the east 156 (storage-tolerant) parent and the east 237 (non-storage-tolerant) parent is detected by using 3% agarose gel electrophoresis and polyacrylamide gel electrophoresis to obtain 223 pairs of polymorphism primers, wherein the polymorphism proportion is 23.49%. To F₂The population is subjected to genotype analysis, 192 pairs of SSR primer amplification bands are clear in pattern and good in repeatability, and the population can be used for constructing a genetic linkage map, wherein 21, 23, 21, 24, 20, 18, 16, 13, 17 and 19 markers are respectively arranged on 1-10 chromosomes (Table 4). The results of screening the parent polymorphic primers with partial primers are shown in FIGS. 1 and 2, and F is simultaneously selected₂The population families were tested for polymorphisms and the results of partial primer detection are shown in FIGS. 3 and 4.

TABLE 4 polymorphic SSR primers between east 156 and east 237

3.2 population genotype composition and marker site segregation

Analysis of 267F by 192 pairs of SSR markers₂Genotype composition of individuals, with the east 156 gene accounting for 47.71% and the east 237 gene accounting for 52.28% of the genotype composition of the entire population. Of the 192 sites, 58 sites deviated from the theoretical separation ratio of 1:2:1, accounting for 30.2% of the total sites (Table 5). Further examination of the segregation ratio of genotypes, 29 of the 58 sites significantly deviated from the theoretical segregation ratio of 1:2:1, accounting for 50.00%. These deviated sites are mainly concentrated on the 1 st, 2 nd, 7 th and 9 th chromosomes, and the specificity of a certain segment of the structure of the chromosomes can cause site deviation, and when the site deviation belongs to the normal condition, the site deviation is still used for constructing the linkage map.

TABLE 5 marker site frequency distribution and chi-square detection

Example 2 phenotypic assay of maize seed storage-resistant related traits

1 test Material

Prepared east 156 × east 237F₂The population had 267 individuals.

2 test method

2.1 Artificial aging treatment

Randomly taking out 150 seeds from each individual plant, repeating for 3 times, repeating for 50 seeds each time, sterilizing with 1% sodium hypochlorite disinfectant for 30min, placing into a small mesh bag, adjusting the temperature of a water bath kettle to 58 deg.C, stabilizing for 10 min, and aging in a water bath kettle for 60 min. After aging, the small mesh bag was taken out of the water bath, washed 2 times with tap water and 2 times with distilled water. And (3) placing the washed seeds in room temperature for airing, and measuring the related indexes of the seed vitality after 2-3 balance of water.

2.2 Standard Germination test

Standard germination tests are carried out according to the germination method specified in GB/T3543.4-1995 in national seed test regulations. Mixing the sterilized sand with distilled water to make the water content consistent, placing in a germination box with thickness of about 3cm, uniformly placing seeds, and covering with 2cm wet sand. The germination boxes were placed in a 25 ℃ light incubator with 50 seeds per box, repeated every second box, 4 times per treatment. During the germination process, counting the number of normal germination seeds day by day, counting the germination vigor at 4 days, counting the germination rate at 7 days, taking out the seedlings after 7 days, cleaning the seedlings with clear water, and measuring the fresh weight of the seedlings. And calculating the germination index and the vitality index.

Germination index Gi ═ Sigma (Gt/Dt) (Gt: number of sprouts on t days, Dt: number of sprouts on days)

Vitality index Vi ═ Gi × St (Gi: germination index, St: fresh weight of seedling)

2.3 Normal distribution test of traits

And analyzing 5 results such as germination rate, germination vigor, germination index, vitality index, fresh weight of seedlings and the like by using SPSS software, analyzing skewness, kurtosis, mean value and the like of the results, and detecting whether the results are normally distributed.

3 results and analysis

The early-stage research shows that standard germination tests are carried out after the treatment of hot water bath aging at 58 +/-1 ℃, and the germination vigor, the germination rate, the germination index, the vigor index, the relative germination vigor, the relative germination rate, the relative germination index, the relative vigor index and the like are used as the storage-resistant evaluation and identification indexes, so that the method is a suitable method for evaluating the storage resistance of the corn seeds. Due to the limited test materials, only 5 indexes such as germination rate, germination vigor, germination index, vitality index, fresh weight of seedlings and the like of aged seeds can be detected.

Using SPSS16.0 software for F_2：3The normal distribution detection is carried out on the seeds storage-resistant related indexes after the group aging, the result is shown in table 6, the difference of each index between the parents after the hot water bath aging is extremely obvious, the germination potentials are 84.00 percent and 7.00 percent respectively, the germination rates are 90.00 percent and 8.00 percent respectively, the germination indexes are 32.80 percent and 2.68 percent respectively, the vitality indexes are 13.54 percent and 1.23 percent respectively, and the fresh weight difference of the seedlings is relatively small and is 0.41g and 0.45g respectively. The normal distribution diagrams of the five properties are shown in figures 5-9, and as can be seen from figures 5 and 6, the germination percentage variation of the seeds is 0-98.66%, the average value is 62.39%, the skewness and the kurtosis are between-1 and 1, two obvious peaks exist, the difference between families is obvious, the super-parent phenomenon exists, and the distribution is basically in accordance with the positive and Taiji distribution. As can be seen from FIG. 6 and Table 6, the variation of the germination potential is 0-98%, the mean value is 55.66%, the skewness and the kurtosis are between-1 and 1, no obvious peak exists, the difference between families is obvious, the super-parent phenomenon exists, and the distribution is basically consistent with the positive distribution. From the figure 7 and the table 6, the fresh weight amplitude of the seedling is 0-1.84 g, the average value is 0.78g, the skewness is between-1 and 1, a very obvious peak is formed, the difference between parents is small, but the difference between families is obvious, the super-parent phenomenon exists, and the distribution basically accords with the positive-Taiji distribution. As can be seen from FIG. 8 and Table 6, the germination index variation is 0-69.07, the mean value is 23, the skewness and the kurtosis are between-1 and 1, an obvious peak exists, the family-to-family difference is obvious, the super-parent phenomenon exists, and the distribution is basically consistent with the regular distribution. From fig. 9 and table 6, it can be seen that the average value of activity index variation is 0-67.98, the average value is 19.07, the skewness and the kurtosis are between-1 and 1, an obvious peak exists, the inter-family difference is obvious, the super-parent phenomenon does not exist, and the distribution is basically consistent with the positive distribution.

Table 6 parent and F2: 3 group seed storage-resistant related index statistical parameter

Example 3 maize seed storage tolerance QTL analysis and development of molecular markers

1 test Material

Respectively establishing an SSR marker genotype database of 267 individuals of a mapping population on Microsoft excel, and F_2：3The phenotype database of the related indexes of the storage resistance of the seeds comprises the germination rate, the germination vigor, the fresh weight of seedlings, the germination index and the vitality index (each index takes the average value of 3 times of repetition).

2 method

2.1 construction of SSR map

The SSR marker loci of the F2 population are constructed by utilizing Isimulping 4.0 software (2014), partial markers are grouped by using a "group" command, and the arrangement order (LOD is 3.0) of the markers of each linkage group is determined by using an "order" command. The recombination values were converted to graph distances (cM) using a "Kosambi" function. And (3) constructing a genetic linkage map by referring to a corn SSR Bin map and using a map instruction.

2.2QTL localization and Effect analysis

By Chi²the test method detects whether the separation ratio of each marker genotype is consistent with 1:2:1, normal distribution test is carried out on the group gene frequency and the field character distribution, Isimiping 4.0 software is operated, QTL analysis is carried out on the storage-resistant related characters by adopting a composite interval mapping method, corresponding operation parameters are Window size which is 5.00cM, Model which is ICIMADD and LOD which is 3.0, 1000 times of repeated replacement test is carried out, the LOD threshold value on the level of α which is 0.05 in the genome range is estimated, and LOD is used>3.0 is the threshold for detecting the presence of a QTL site, while analyzing the QTL for interpretable phenotypic variation rate, additive effects (A) and dominant effects (D).

3 results and analysis

3.1 construction of linkage map

A genetic map was constructed using icimapping4.0 based on 192 primer amplification information (LOD 3.0). And (3) converting the recombination value into map distance (cM) by selecting a Kosambi function, constructing an SSR marker genetic linkage map (figure 10), wherein 192 SSR loci are fitted on the map 10 in total, 2204.3cM covering the maize genome is covered, and the average distance between markers is 11.48 cM. Covering 10 chromosomes of the corn, the number of markers is 21, 23, 21, 24, 20, 18, 16, 13, 17 and 19 respectively.

3.2 preliminary analysis of maize seed storage-tolerant related traits by QTL

And carrying out QTL preliminary analysis and gene effect analysis on the genetic linkage map and the population storage-resistant related traits by adopting a composite interval mapping method according to the constructed genetic linkage map and the population storage-resistant related traits, wherein the QTL is not detected by taking the germination rate and the germination index as indexes.

3.2.1 QTL analysis for storage durability using vitality index as indicator

QTL analysis is carried out by taking the vitality index as an index, 4 QTLs (table 7) for controlling the vitality of seeds are detected in total and are respectively positioned on 5 chromosomes, 7 chromosomes and 10 chromosomes, qSVI-5 positioned on a 5.02 locus on the 5 th chromosome is positioned between umc2167 and umc1597, and the contribution rate is 6.51 percent; two QTL sites are detected on the 7 th chromosome, and the contribution rates are 9.16% and 20.87% respectively; the 10 th chromosome detected 1 QTL with a contribution rate of up to 24.35%, between umc2043 and umc 1367. All QTL additive effects were positive, indicating that the role of parental east 156 at these sites was to enhance shelf-life.

TABLE 7 QTL analysis of maize viability index using composite interval mapping method

3.2.2 storage tolerance QTL analysis Using fresh weight of seedlings as an indicator

QTL analysis is carried out by taking the fresh weight of seedlings as an index, 5 QTLs (table 8) of the QTLs for controlling the fresh weight of the seedlings are detected together, qFSW-1 positioned on the 1.08 locus of the 1 st chromosome is positioned between markers bnlg1671 and bnlg1643, and the contribution rate is 9.75 percent; qFSW-2 at position 2.06 on chromosome 2 was located between umc1079 and umc1028 with a contribution of 17.09%; qFSW-5 at position 5.03 on chromosome 5 was located on umc2063 and umc2400 with a contribution rate of 22.68%; qFSW-7 at chromosome 7.05 position 7 at a contribution rate of 4.37% between markers umc1545 and umc 2333; qFSW-10 at position 10.03 on chromosome 10 was located between umc2043 and umc1367 with a contribution rate of 10.83; all QTL additive effects were positive, indicating that the role of parental east 156 at these sites was to enhance shelf-life.

TABLE 8 QTL analysis of fresh weight of maize seedlings using composite interval mapping

3.2.3 storage durability QTL analysis Using germinating potential as an indicator

QTL analysis is carried out by taking the germination potential as an index, 1 QTL (Table 9) for controlling the fresh weight of seedlings is detected together, the qGE-8 locus at the 8.02 position of chromosome 8 has the contribution rate of 5.58 percent between bnlg2235 and a primer bnlg1194, and the additive effect is a positive value, which indicates that the parents have an enhancement effect on the storage resistance of the locus.

TABLE 9 QTL analysis of maize germination vigor using composite interval mapping method

The invention co-locates 4 corn seed storage-resistant main effect QTLs, wherein qSVI-7-2 and qSVI-10 are the corn seed storage-resistant main effect QTLs obtained by using vitality indexes as indexes, and qFSW-2 and qFSW-5 are the corn seed storage-resistant main effect QTLs obtained by using fresh weights of seedlings as indexes.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

1、Delouche J.Accelerated aging techniques for predicting the relativestorability of seed lots[J].Seed Science and Technology,1973,2:427-452.

2. Dongcha, He Nanfeng, Ztong, et al, research on peroxidation of lipid of artificially aged soybean seed membranes, proceedings of Henan university, 1995,25(3):91- -95

3. The principle and the method of the vitality determination of the seeds [ M ] of Beijing, China agricultural Press 2006,5-126.

4. Comparison of several testing methods for vigor of corn seeds [ J ]. proceedings of Beijing college of agriculture, 1995,6:2-3.

5. Xubenmei for brightness increase, artificial aged seed experiment with methanol [ J ] seed, 1985,5:14-15.

6. Experimental study on accelerated aging of radish seeds by methanol and hot water [ J ] seed science and technology, 2000.

7、Miura K,Lin SH,Yano M,Nagamine T(2002)Mapping quantitative traitloci controlling seed longevity in rice(Oryza sativa L.)[J].Theor Appl Genet104:981–986

8、Emile J M,Hetty B V,Gerda J R,Steven P C G,Maarten K.Geneticdifferences in seed longevity of various Arabidopsis mutants[J].PhysiologicalPlant arum,2004,121:448-461.

9、Sasaki K,Fukuta Y,Sato T.Mapping of quantitative trait locicontrolling seed longevity of rice(Oryza sativa L.)after various periods ofseed storage[J].Plant breeding,2005,124:361-366.52

10、S.Landjeva.Genetic mapping within thewheat D genome reveals QTLfor germination,seed vigor and longevity,and early seedling growth[J].Euphytica,2010,171:129–143.

11、Fujino.K,Sekiguchi.H,Sato.T,Kiuchi.H,Nonoue.Y,Takeuchi.Y,Ando.T,Lin.S.Y,Yano.M,2004.Mapping of quantitative trait loci controlling low-temperature germinability in rice[J].Theor.Appl.Genet.,2008,108(5):794-799.

12、Cui K H,Peng S B,Xing Y Z,Xu C G,Yu S B,Zhang Q.Moleculardissecting of seedling-vigor and associated physiological traits in rice[J].Theoretical and Applied Genetics,2002,105:745-753.

13. National technical supervision agency, crop seed test protocol [ M ]. Beijing, published by the Chinese Standard Press, 1995, 40.

Claims (4)

1. Identification of major QTL site of maize seed storage tolerance by molecular marker closely linked with major QTL of maize seed storage toleranceqFSW-2AndqFSW-5the use of (1);

the major QTL of the maize seed storage tolerance is positioned in the region of the No. 2 chromosome Bin 2.06 of maizeqFSW-2And located within the region of maize chromosome 5 Bin5.03qFSW-5；

Wherein, withqFSW-2Closely linked molecular markers include 2SSR markers umc1079 and umc 1028; andqFSW-5closely linked molecular markers include 2SSR markers umc2063 and umc 2400; primers for amplifying each molecular marker were as follows:

the sequences of the forward primer and the reverse primer of umc1079 are SEQ ID No.1 and 2 respectively;

the sequences of the forward primer and the reverse primer of umc1028 are SEQ ID No.3 and 4, respectively;

the sequences of the forward primer and the reverse primer of umc2063 are SEQ ID NO.5 and 6, respectively;

the forward and reverse primer sequences of umc2400 are SEQ ID NO.7 and 8, respectively.

2. The use of claim 1, wherein the characteristic bands with the size of 121bp can be amplified in the maize inbred line east 156 with strong seed storability by using SEQ ID No.1 and 2;

characteristic bands with the size of 138bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.3 and SEQ ID NO. 4;

characteristic bands with the size of 91bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.5 and SEQ ID NO. 6;

the characteristic band with the size of 114bp can be amplified in a maize inbred line east 156 with strong seed storage resistance by utilizing SEQ ID NO.7 and SEQ ID NO. 8.

3. The application of the molecular marker closely linked with the major QTL of the storage tolerance of the corn seeds in screening or identifying corn varieties with strong storage tolerance,

the major QTL of the maize seed storage tolerance is positioned in the region of the No. 2 chromosome Bin 2.06 of maizeqFSW-2And located within the region of maize chromosome 5 Bin5.03qFSW-5；

Wherein, withqFSW-2Closely linked molecular markers include 2SSR markers umc1079 and umc 1028; andqFSW-5closely linked molecular markers include 2SSR markers umc2063 and umc 2400; primers for amplifying each molecular marker were as follows:

the sequences of the forward primer and the reverse primer of umc1079 are SEQ ID No.1 and 2 respectively;

the sequences of the forward primer and the reverse primer of umc1028 are SEQ ID No.3 and 4, respectively;

the sequences of the forward primer and the reverse primer of umc2063 are SEQ ID NO.5 and 6, respectively;

the forward and reverse primer sequences of umc2400 are SEQ ID NO.7 and 8, respectively.

4. Use according to claim 3, characterized in that it comprises the following steps:

1) extracting the genome DNA of a plant to be detected;

2) taking the genome DNA of a plant to be detected as a template, and carrying out PCR amplification reaction by using a primer for amplifying the molecular marker;

3) and detecting the PCR amplification product.

Images