CN110777214B

CN110777214B - SSR (simple sequence repeat) marker closely linked with corn seed storage resistance and application thereof in molecular marker-assisted breeding

Info

Publication number: CN110777214B
Application number: CN201910625889.5A
Authority: CN
Inventors: 邸宏; 张�林; 王振华; 封陈晨; 周羽; 曾兴
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-07-11
Filing date: 2019-07-11
Publication date: 2022-05-27
Anticipated expiration: 2039-07-11
Also published as: CN110777214A

Abstract

The invention discloses a molecular marker closely linked with a major QTL (quantitative trait locus) segment related to the storage tolerance of corn seeds and application thereof; the molecular marker is closely linked with a maize seed storage-resistant related major QTL segment cQTL-10 in the Bin10.03 region of the maize chromosome 10, and the molecular marker is phi050 and umc 1648. According to the invention, 2 SSR molecular markers phi050 and umc1648 which are closely linked with the storage tolerance of the corn seeds are discovered by positioning 1 main effect QTL section of the storage tolerance of the corn seeds, wherein the section comprises 2 QTL qRVI-10 and qRSVI-10 and respectively influence the relative vitality index and the relative simple vitality index, and the SSR molecular markers provided by the invention can be applied to molecular assisted breeding for improving the storage tolerance of the corn seeds.

Description

SSR (simple sequence repeat) marker closely linked with corn seed storage resistance and application thereof in molecular marker-assisted breeding

Technical Field

The invention relates to SSR molecular markers related to the storage tolerance of corn seeds, in particular to molecular markers closely linked with cQTL-10 located in a main effect QTL section related to the storage tolerance of corn seeds, and further relates to application of the molecular markers in molecular assisted breeding, belonging to the field of the molecular markers and application thereof.

Background

The storage tolerance of the seeds is the capacity of the seeds for enduring storage, and different varieties show different storage physiological characteristics due to different genetic bases, and are comprehensively expressed on the requirements and adaptability of the seeds to the environment before and during storage. The seed vigor refers to the potential germination capacity of the seed or the vitality of the embryo, and is one of the important characteristics for determining whether the seed is storage-resistant. Seeds having normal physiological activity under dry conditions can maintain their vitality for a certain period of time, and there are differences in the vitality maintaining time of different types of seeds, and deterioration and aging of seeds inevitably occur with the increase of storage time, improper storage conditions, and stress of internal and external environments. The seed vitality is reduced due to aging, which mostly shows that the germination capacity is reduced or does not germinate, the growth of seedlings is slow or stops, abnormal seedlings appear, the grown seedlings have poor development of roots and stems and leaves, the fertility of mature plants is reduced, and the like, so that the seed value is obviously reduced, and serious loss is caused to a user.

In most cases, the lower the temperature and the water content, the longer the seed can keep alive, and the decrease of the seed vitality can be properly delayed and the storage time can be prolonged by controlling the storage condition of the seed. The method is a fundamental way for solving the problems that the self genetic factors of the seeds are utilized, the storage resistance of the seeds is improved by combining the molecular breeding technology, and a new variety with high self activity and storage resistance is bred.

Corn is the first crop of feed, grain and industrial raw material in China, the annual seed demand is about 9 hundred million kilograms, the actual annual seed production is about 10-11 hundred million kilograms, and in addition, the germplasm resources of barren seeds and breeding are added, so that a large amount of seeds for use need to be stored every year, and therefore, the improvement of the seed storage capacity has important value on corn genetic breeding and agricultural production. Data of national agricultural technology promotion service centers (https:// www.natesc.org.cn) show that the effective stock of corn seeds in 2016 is about 8 hundred million kilograms, the newly-produced seeds in 2017 are 10.58 hundred million kilograms, the seed demand in 2018 is estimated to be about 11 hundred million kilograms, and 7-8 million kilograms of seeds are required to be stored. The germination rate is reduced and the amount of seeds losing the planting value reaches 3-5 million jin each year due to the storage conditions and the self reasons of the seeds, thereby causing serious loss to the planting industry and the corn production. Although the traditional modes of low temperature, medicament, sealing, drying and the like can prolong the service life of seeds, the traditional modes have limited effectiveness, and have the problems of high storage cost, medicament residue and the like, and the key for solving the problem is to improve the self storability of the corn and breed a new corn variety with storability.

With the development of molecular biology, researchers have been exploring the genetic mechanism of seed storability by using methods such as molecular marker technology, omics sequencing technology, and related gene cloning and transformation.

In the middle of Zhuyu (2018), sweet corn varieties 'Dongsan 88' and 'nongsuan 99' are used as test materials, and SRAP (sequence related amplified polymorphism) marker detection shows that the genetic diversity of aged seeds is reduced, and the genetic diversity of seeds with good storability is reduced a little. Li Chunlei (2015) takes Zhengdan 958 and Xiyu 335 seeds as test materials, and related researches are carried out on the corn seed storability from the aspect of epigenetic DNA methylation by utilizing an MSAP molecular marker technology, and the correlation between the seed storability and the DNA methylation of CG level is found to be high.

Li et al (Li T, Zhang Y, Wang D, et al. Regulation of Seed Vigor by management of Raffinose Family Oligosaccharides in Maize and Arabidopsis thaliana [ J ]. Molecular Plant, 2017, 10(12):1540.) found that over-expressing ZmGOLS2 and ZmRS gene simultaneously and over-expressing ZmGOLS2 gene separately improves Arabidopsis thaliana Seed Vigor, but over-expressing ZmRS gene alone reduces Arabidopsis thaliana Seed Vigor.

QTL positioning related to the corn seed storability is also reported, the utilized population materials comprise a temporary segregation population F2, a permanent segregation population RIL, a backcross population and a haploid population, the phenotype detection method comprises a high-temperature high-humidity aging method, a hot-water bath aging method and the like, and the genotype detection method comprises an SNP marker, an SSR marker and the like. Cheng et al (Cheng X, Geng G. QTL Analysis of gain Storage Dual for size Under Controlled conversion Using SSR Markers [ J ]Agricultural science and technology (english edition), 2012) locates 3 storability-related QTLs on chromosomes 1, 6, and 9, respectively, with contribution rates of 8.1%, 23.0%, and 10.1%, respectively; lonicera japonica et al (Lonicera japonica, Lixinhai, Wangfengge, et al. maize seed dormancy QTL location [ J)]Crop science, 2007, 33(9): 1474-1478) detects 7 QTLs with contribution rate of 2.45% -26.09%, which are located on

chromosomes

1, 3, 5 and 10, respectively, wherein the contribution rate of the major QTL located on chromosome 1 reaches 26.09%; lorenting (Lorenting, artificial accelerated ageing identification of corn seed storability method comparison and QTL preliminary analysis of related characters [ D]The harbin: northeast university of agriculture, 2015) detected 10 QTLs at 1, 2, 5, 7, 8 and 10 chromosomes respectively, and the contribution rate of the phenotypeThe range is between 4.37% and 24.35%; hund et al (Hund A, Frachboud Y, Soldai A, et al. QTL controlling root and shoot tracks of mail threads under Cold stress [ J]Theanalytical and Applied Genetics, 2004, 109(3):618-629) found 20 QTLs, of which the major QTL associated with the germination index at chromosome 5 contributed 12% and the QTL associated with the primary lateral root length 14%; QTL positioning and genetic effect analysis of Liuhai English (Liuhai English. corn seed vitality related characters [ D) ]The university of agriculture, henan, 2012) detected 30 storability-related QTLs, distributed on

chromosomes

1, 2, 3, 4, 5, 8, 9, and 10, with a contribution rate between 6.3% and 12.6%; liu et al (2011) found 16 QTLs with 4 related traits such as germination rate, germination vigor, germination index and vitality index; 172 QTLs related to seed vigor are detected by Hanzang (2014), are distributed on 9 chromosomes except No. 6, and the explained trait genetic variation is between 5.39 and 12.11 percent; QTL (quantitative trait Locus) positioning analysis of corn seed vigor related characters under different conditions [ D ]]The university of agriculture, heinan, 2012)) located 9 QTLs associated with maize seed vigor, distributed on

chromosomes

1, 4, 5, and 10, with the locus located on chromosome 10 being the major locus; wang et al (2015) detected 49 QTLs associated with seed vigor and storability, of which qGP5 was detected in both location populations^[53]. Ningqia (Ningqia. corn seed vigor related trait research and germination vigor and germination rate QTL analysis based on SNP marker [ D)]Jilin university of agriculture, 2017) under standard germination conditions, 4 germination potential-related QTLs were detected as qsgp2, qsgp1, qsgp3 and qsgp4, respectively, distributed on chromosomes 1, 6 and 7, accounting for phenotypic variation rates of 17.25%, 16.04%, 8.65% and 10.86%; under the condition of low-temperature stress, a qtlgp1 which is located on chromosome 10 and related to the germination potential and explains the phenotypic variation of 6.67 percent, and a qtlgr1 which is located on chromosome 6 and explains the phenotypic variation of 7.75 percent is detected.

Disclosure of Invention

One of the objectives of the present invention is to provide molecular markers closely linked to major QTL segments associated with maize seed storage tolerance;

the second purpose of the invention is to apply the molecular marker obtained by screening to the molecular assisted breeding of the corn seed storage-tolerant new variety.

The above purpose of the invention is realized by the following technical scheme:

the invention firstly provides an SSR molecular marker closely linked with a main effect QTL section related to the storage tolerance of corn seeds, wherein the SSR molecular marker is closely linked with a cQTL-10 of the main effect QTL section related to the storage tolerance of the corn seeds in a No. 10 chromosome Bin10.03 region; preferably, the SSR molecules are labeled phi050 and umc 1648.

The invention discovers that 5 storability-related main effect QTLs with relative germination rate, relative germination vigor, relative germination index, relative vigor index and relative simple vigor index as indexes exist between markers phi050 and umc2043 on chromosome 10 through construction of a genetic linkage map and QTL analysis related to corn seed storability, wherein the QTLs are qRGP-10, qRGE-10, qRGI-10, qRVI-10 and qRSVI-10 respectively, the phenotype contribution rates are 12.43%, 20.11%, 14.33%, 10.08% and 10.85 respectively, and the section is determined to be a storability-related main effect QTL section.

The invention further relocates the QTL with the F_2:3Carrying out consistency analysis again on the population positioning result, determining 2 consistency QTL sections related to the corn seed storability, and reducing the sections compared with the previous sections; chromosome 10 is located in the consensus major QTL segment cQTL-10 between markers umc1648 and phi050, and comprises 2 QTL qRVI-10 and qRSVI-10, which respectively affect the relative viability index and the relative ease viability index, the phenotype contribution rate is 18.30% and 17.91%, respectively, and the segment size is about 39.15 Mb.

The invention further selects the family DNA which is extremely storable in 30 RIL groups to be equivalently mixed to form an anti-pool, selects the family DNA which is extremely non-storable in 30 RIL groups to be equivalently mixed to form a sensing pool, and utilizes the BSA method to build the pool to screen the molecular marker. SSR markers near and inside the consistency main effect QTL section are selected to detect resistance pool and sensing pool genotypes, the genotype separation condition is subjected to X2 detection, and SSR markers which have polymorphism between parents and between resistance pool and are closely linked with the QTL are screened out. And (3) carrying out genotype detection on the screened molecular markers in 85 storability families of the RIL population, and screening out the storability related linkage markers according to the coincidence degree between the genotype detection result and the storability phenotype detection and the chi 2 test result.

Near and inside the boundaries of 2 storability-related consistency QTL sections cQTL-7 and cQTL-10, 9 markers such as umc1295, umc1671, phi328175, umc1367, phi054, umc1648 and phi050 are selected for detection of selection efficiency. As a result, the molecular markers umc1295, phi082, umc1367, phi054 and umc2043 are only polymorphic in the amphiphilic sample, and are not polymorphic in the influenza resistance pool; and umc1671, phi328175, phi050 and umc1648 have polymorphism between parents and between influenza resistant pools, and the effective transmission rate of the polymorphic markers between the parents is 44.44%. The invention utilizes the single plants in the resistant pool and the sensitive pool to carry out single plant genotype analysis on the screened differential markers, and uses the Chi 2 fitness test to evaluate the relevance of the markers and the corn seed storability sites. The suitability detection analysis of chi 2 shows that the markers umc1671, phi328175, phi050 and umc1648 have obvious correlation with the main effect sites of the corn seed storability, and chi 2 detection values are 0.862, 1.690, 0.133 and 1.286 respectively, are less than 3.84(p is 0.05 level, n is 1), and can be used for screening the main effect sites of the corn seed storability; and the other 5 markers χ 2 all detected values greater than 3.84(p ═ 0.05 levels). And (3) combining the genotype detection results of the 4 markers in 85 storage-tolerant families and the storage-tolerant phenotype detection results to perform coincidence rate analysis. The genotype and storability phenotype concordance rates for the 4 markers were 83.12%, 80.82%, 88.89% and 82.93%, respectively, with an average of 83.94% (see tables 3-8), with the highest concordance rate for marker umc 1648. Finally, 4 markers umc1671, phi328175, phi050 and umc1648 from the parent east 156 are determined to be related linkage markers of maize seed storability.

The invention also provides a primer for amplifying the molecular marker, wherein the nucleotide sequence of the primer for amplifying the molecular marker umc1648 is shown as SEQ ID No. 1 and SEQ ID No. 2; the nucleotide sequences of the primers for amplifying the molecular marker phi050 are shown as SEQ ID No. 3 and SEQ ID No. 4.

The invention further uses 2 developed storability related linkage markers umc1648 and phi050 to carry out genotype detection on 141 American corn inbred lines. The 2 markers were individually tested as 8, 13, 10 and 9 parts of selfed lines for tolerance. Inbred lines identified as containing qRGE-7 major QTL in the storability linked marker assay were ND248, ND252, ND246, SD65, N532, N209, Tx 110; the ND248, ND252, ND246 and SD65 are identified as inbred lines with the strongest storability in the detection of the storability phenotype, and the N532, N209 and Tx110 are identified as stronger in the detection of the storability phenotype.

The test results prove that the molecular markers phi050 and umc1648 which are screened out by the invention and closely linked with the main effect QTL section cQTL-10 positioned in the Bin10.03 region of the maize seed storage tolerance can be used for breeding new maize seed storage-tolerant varieties.

According to the invention, 2 molecular markers closely linked with the storage tolerance of the corn seeds are discovered by positioning 1 main effect QTL section of the storage tolerance of the corn seeds, wherein the section comprises 3 QTL qRGP-7, qRGE-7 and qRGI-7, and respectively influences the relative germination rate, relative germination potential and relative germination index, so that the molecular markers can be used for screening the storage tolerance molecular markers of the corn seeds to assist breeding.

Detailed description of the overall solution of the invention

Analysis and positioning of maize seed storage tolerance related QTL

The invention takes the corn tolerance inbred line east 156 and the maize intolerance inbred line east 237 as parents, obtains the RIL group containing 288 families by selfing for 7 generations through a single seed transmission method, and is used for the QTL analysis and the marker development of seed tolerance.

The genetic linkage map is constructed on the SSR genotype database by utilizing Ichimping 4.1 software, partial markers are grouped by using a group command, the sequence of the markers of each linkage group is determined by using an order command (LOD is 3.0), and a Kosambi function is selected to convert a recombination value into a graph distance (cM). And (3) constructing a genetic linkage map by using a map instruction by referring to a corn SSR Bin map.

And (3) operating Isimulping 4.1 software by combining a phenotype database and a genotype database, and carrying out QTL analysis on the storage-resistant related characters by adopting a composite interval mapping method. The corresponding operating parameters were Window size 5.00cM, Model: ICIMADD, LOD: 3.0. LOD >3.0 is used as a threshold for the presence of detectable QTL sites.

The located major QTL locus and the topic group are utilized at the early stage by F_2:3And comparing the group positioning results to determine the consistency main effect QTL section. And adding SSR marks according to the sections where the consistent main effect QTL is located, and repositioning the corn seed storage-tolerance related QTL by adopting a composite interval mapping method to reduce the interval where the storage-tolerance related main effect QTL is located.

Construction of genetic linkage map

1349 SSR markers uniformly distributed on 10 chromosomes of corn are selected in a MaizeGDB genome database, polymorphism detection is carried out between two parents of east 156 and east 237, and 226 primers with polymorphism and clear bands among the parents are screened out for constructing a genetic linkage map.

According to a genotype database for detecting 226 SSR markers to RIL populations, a genetic map (LOD is 3.0) is constructed by means of Ichimpaging 4.1 software to fit 226 SSR marker loci, 10 chromosomes of corn are covered, the total length is 3467.4cM, the average marker interval is 15.34cM, and the marker numbers of chromosomes 1-10 are 28, 30, 28, 20, 16, 20 and 16 respectively.

QTL analysis related to maize seed storability

Combining the RIL group genotype detection result and the seed storability phenotype detection result, carrying out corn seed storability related QTL analysis by adopting a composite interval mapping method, wherein 17 QTLs influencing 6 corn seed storability related indexes such as relative germination rate, relative germination potential and relative germination index are detected together and distributed on

chromosomes

1, 2, 7, 9 and 10 of the corn, and the phenotypic variation explained by the single QTL is from the lowest 2.33 percent to the highest 20.11 percent; the additive effect values of 9 QTLs are positive values, and the allele of east 156 has synergistic effect on the loci, accounting for 52.94% of the number of the QTLs; while the east 237 allele has a synergistic effect on the remaining 8 QTL sites, accounting for 47.06% of the total number of QTLs.

5 main QTLs related to storability and taking relative germination rate, relative germination potential, relative germination index, relative vitality index and relative simple vitality index as indexes exist between the markers phi050 and umc2043 on the chromosome 10, wherein the QTLs are qRGP-10, qRGE-10, qRGI-10, qRVI-10 and qRSVI-10 respectively, and the phenotype contribution rates are 12.43%, 20.11%, 14.33%, 10.08% and 10.85% respectively; the synergistic effect of both storability-related major QTL segments is from east 156.

Maize seed storability-related major QTL relocation

QTL mapping results using RIL populations with previous F_2:3The results of population mapping were compared and 2 consensus major QTL segments were found, located on cQTL-7 on chromosome 7.05 and cQTL-10 on chromosome 10.03, respectively. The consensus QTL sections cQTL-7 and cQTL-10 are located between chromosome 7, umc1295 and umc2333, approximately 9.73Mb in size and between chromosome 10, phi054 and umc2043, approximately 93.13Mb in size, respectively. The encryption of SSR markers is carried out near and inside the boundary of 2 consistency major QTL sections cQTL-7 and cQTL-10, wherein 9 markers are added to the cQTL-7 section of the chromosome 7, 22 markers are added to the cQTL-10 section of the chromosome 10, and the construction of genetic linkage maps and the QTL analysis are carried out again. The total number of the encrypted SSR markers is increased to 257, the genotype detection result and the storability phenotype detection result are combined, a composite interval mapping method is adopted to analyze the storage-tolerance related QTL again, and the positioning result is changed on the No. 7 chromosome and the No. 10 chromosome.

9 QTLs are detected on chromosome 10, namely qRGP-10 influencing relative germination rate, qRGE-10 influencing relative germination potential and qRGI-10 influencing relative germination index, which are respectively positioned between phi054 and umc2043, the contribution rates are 14.19%, 17.99% and 18.26%, and the additive effect values are 0.1466, 0.1786 and 0.1142; 3 qRVI-10-1, qRVI-10-2 and qRVI-10-3 influencing the relative vitality index are respectively positioned between markers phi050 and phi054, phi054 and umc2043 and umc1930 and umc1648, the contribution rates are respectively 22.39%, 12.80% and 19.71%, and the additive effect values are respectively 0.1418, 0.1425 and 0.1381; 3 qRSVI-10-1, qRSVI-10-2 and qRSVI-10-3 affecting the relative ease vitality index, respectively located between markers phi050 and phi054, phi054 and umc2043, and umc1930 and umc1648, with contributions of 19.59%, 19.78% and 14.36%, respectively, and additive effect values of 0.1567, 0.1571 and 0.1495, respectively. The 9 sites all received synergy from east 156.

Wherein 3 major QTLs qRGP-10, qRGE-10 and qRGI-10, each with a contribution rate of greater than 10% on chromosome 10, remain localized to the phi054-umc2043 segment; 2 major QTL qRVI-10 and qRSVI-10, each with a contribution rate greater than 15%, were mapped to the umc1648-phi050 segment, which was determined to be a storability-related major QTL segment, with synergism from east 156.

The invention further combines the QTL relocation result with the F_2:3Carrying out consistency analysis again on the population positioning result, determining 2 consistency QTL sections related to corn seed storability in total, and reducing the sections compared with the previous sections; chromosome 10 is located in the cQTL-10 of the consensus major QTL segment between the markers umc1648 and phi050, comprises 2 QTL qRVI-10 and qRSVI-10, and respectively influences the relative vitality index and the relative ease vitality index, the phenotype contribution rate is respectively 18.30 percent and 17.91 percent, and the segment size is about 39.15 Mb.

Development and application of linkage markers

Selecting the family DNA which is extremely storable in 30 RIL groups to be equivalently mixed to form an anti-pool, selecting the family DNA which is extremely non-storable in 30 RIL groups to be equivalently mixed to form a sensing pool, and building the pool by using a BSA method for screening the marker.

SSR markers near and inside the consistency main effect QTL section are selected to detect resistance pool and sensing pool genotypes, the genotype separation condition is subjected to X2 detection, and SSR markers which have polymorphism between parents and between resistance pool and are closely linked with the QTL are screened out.

And (3) carrying out genotype detection on the screened molecular markers in 85 storability families of the RIL population, and screening out the storability related linkage markers according to the coincidence degree between the genotype detection result and the storability phenotype detection and the chi 2 test result.

Near and inside the boundaries of 2 storability-related consistency QTL sections cQTL-7 and cQTL-10, 9 markers such as umc1295, umc1671, phi328175, umc1367, phi054, umc1648 and phi050 are selected for detection of selection efficiency. Constructing an anti-infection pool formed by mixing equal amounts of DNA of extremely-resistant families in 30 RIL populations, mixing equal amounts of DNA of extremely-non-resistant families in 30 RIL populations, and performing genotype detection among the anti-infection pools by using the 9 SSR markers. The results are shown in FIGS. 3-4, where the markers umc1295, phi082, umc1367, phi054 and umc2043 are polymorphic only between the amphiphiles, and are not polymorphic in the influenza pool; and umc1671, phi328175, phi050 and umc1648 have polymorphism between parents and between influenza resistant pools, and the effective transmission rate of the polymorphic markers between the parents is 44.44%. And (3) carrying out individual plant genotype analysis on the screened differential markers by using individual plants in the resistant pool and the sensitive pool, and evaluating the relevance of the markers and the corn seed storability sites by using a Chi 2 fitness test.

As shown by suitability detection analysis of chi 2, the markers umc1671, phi328175, phi050 and umc1648 have remarkable correlation with the main effect sites of the corn seed storage tolerance (see tables 3-7), and chi 2 detection values are 0.862, 1.690, 0.133 and 1.286 respectively, are less than 3.84(p is 0.05 level, n is 1), and can be used for screening the main effect sites of the corn seed storage tolerance; and the other 5 markers χ 2 all detected values greater than 3.84(p ═ 0.05 levels).

And (4) combining the genotype detection results of the 4 markers in 85 storage-tolerant families and the storage-tolerant phenotype detection results to perform coincidence rate analysis. The genotype and storage phenotype concordance rates for the 4 markers were 83.12%, 80.82%, 88.89% and 82.93%, respectively, with an average of 83.94%, with the highest concordance rate for marker umc 1648. Thus, the invention finally determines 4 markers umc1671, phi328175, phi050 and umc1648 from the parent east 156 as related linked markers of maize seed storability.

141 American maize inbred lines were genotyped with 4 developed storability-associated linkage markers umc1671, phi328175, umc1648 and phi 050. The inbred lines tested individually for tolerance by the 4 markers were 8, 13, 10 and 9 parts, respectively.

Inbred lines containing qRGE-7 with chromosome 7 affecting relative germination vigor are ND248, ND252, ND246, SD65, N532, N209, Tx110, LH181, PHPR5, ICI 581; the inbred lines qRVI-10 containing chromosome 10 affecting relative viability index are ND248, ND252, ND246, SD65, N532, N209, Tx110, LH192, PHJ90, 3IIH6, OQ 403; the inbred line containing 2 major QTLs comprises ND248, ND252, ND246, SD65, N532, N209 and Tx 110.

141 American corn inbred line seeds were subjected to artificial aging treatment and then tested for phenotypic data related to seed storability. The reference material shows larger variation amplitude in 6 storage-resistance related indexes, and the difference is extremely obvious (P < 0.01); the coefficient of variation of other storage related indexes except relative seedling length is more than 35%, wherein the coefficient of variation of relative germination potential is the largest and is as high as 68.63%.

According to the relative values of 6 storability related character indexes, adopting a system clustering method to divide the seeds of 141 parts of corn inbred lines into 5 classes according to the storability, and according to the clustering analysis result, the inbred line with the strongest I class of storability comprises 23 parts of N193 parts, ND250 parts, ND252 parts and the like; the II type selfing line with strong storability comprises 35 parts of 912 parts, W8555 parts, N209 parts and the like; class III self-bred line with moderate storability comprises 32 parts of N544, 3IIH6, N543 and the like; the IV self-line with weak storability comprises 33 parts of Mo48, RS710, MBWZ and the like; the selfing lines with the weakest storability in class V include 20 parts of OQ403, CQ702RC, MM402A, etc.

Inbred lines identified as containing qRGE-7 and qRVI-102 major QTLs in the storability linked marker test are ND248, ND252, ND246, SD65, N532, N209 and Tx 110; the inbred lines with the strongest storability are identified as ND248, ND252, ND246 and SD65 in the storability phenotype test, and the inbred lines with the stronger storability are identified as N532, N209 and Tx110 in the storability phenotype test.

The test results prove that the SSR molecular marker which is screened out by the invention and is closely linked with the cQTL-10 of the main effect QTL section which is positioned in the Bin10.03 region of the No. 10 chromosome of the corn and is related to the storage resistance of the corn seeds can be used for breeding new corn seed storage-resistant varieties.

Drawings

FIG. 1 is based on F_7:8A corn genetic linkage map constructed by the RIL group; relative germination percentage: :; relative germination vigor:

relative germination index: a tangle-solidup; relative viability index: ●, respectively; relative seedling length: ■, respectively; relative simple vitality index: solid content;

RGP:★；RGE

RGI:▲；RVI:●；RSL:■；RSVI:◆

FIG. 2 is a histogram of the relative values of corn seed storability related indicators; (a) relative germination percentage RGP; (b) relative germination potential RGE; (c) relative germination index RGI; (d) relative viability index RVI; (e) relative seedling length RSL; (f) relative simple viability index RSVI

FIG. 3 reconstructed genetic linkage maps of chromosomes 7 and 10; relative germination percentage: :; relative germination vigor:

relative germination index: a tangle-solidup; relative viability index: ●, respectively; relative seedling length: ■, respectively;

relative simple vitality index: solid top of

RGP:★；RGE:

；RGI:▲；RVI:●；RSL:■；RSVI:◆

FIG. 4 screening for storability-linked markers; the lower case letters (a-i) represent the labels: (a) the method comprises the following steps umc 1295; (b): umc 1671; (c) the method comprises the following steps phi 328175; (d) the method comprises the following steps phi 082; (e) the method comprises the following steps umc 1367; (f) the method comprises the following steps phi 050; (g): phi 054; (h) the method comprises the following steps umc1648 (i): umc2043. numerals (1 to 4) respectively represent: (1): east 237; (2): east 156; (3): resisting the pond; (4): and (6) sensing the pond.

Detailed Description

The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. It is to be understood that the embodiments are illustrative only and are not to be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 localization of maize seed storage tolerance-related QTL

1 test Material

The corn tolerance inbred line east 156 and the maize intolerance inbred line east 237 are used as parents, and are inbred for 7 generations through a single seed transmission method to obtain an RIL group containing 288 families, which is used for QTL analysis and marker development of seed tolerance. Wherein the east 156 seeds have strong storability, the seeds are hard, and the germination rate is still kept above 90% in 8 years under the natural storage condition; east 237 has poor storability, seeds are half-horse-tooth-shaped, and the germination rate is reduced to about 80 percent after 1-year storage under natural conditions. The two inbred lines are bred and stored by the corn research institute of northeast agriculture university.

2 test method

2.1 genotype testing

2.1.1 extraction of Total DNA from maize leaves

Total DNA of parent strains of 156, 237 and 288 RIL group families of 3 leaves at the 1 heart stage of seedlings leaves is extracted by a CTAB small amount method, and the individual plants are numbered in sequence. DNA concentration and quality were determined by UV spectrophotometer and diluted to 25 ng/. mu.L before storage at-20 ℃ for SSR marker analysis.

2.1.2 screening of SSR primers

Selecting 1349 SSR markers distributed on 10 chromosomes of maize, wherein the primer sequences are from a MaizeGDB database (C)http://www.maizegdb.org/) And carrying out the polymorphism detection among parents of the RIL group, and using the screened polymorphism markers for the genotype detection of the F7 individual strain.

The PCR amplification system is as follows: the total amount of the system was 10. mu.L, which contained ddH₂O6. mu.L, 10 XTaq Buffer 1. mu. L, dNTP (2.5mM/mL), 0.6. mu. L, Primer-F (20U) 0.1. mu. L, Primer-R (20U) 0.1. mu. L, Taq (5U/. mu.L) 0.2. mu. L, DNA (20 ng/. mu.L-50 ng/. mu.L) 2. mu.L, after all the reactants are mixed according to the system, 15. mu.L of mineral oil is added for covering, and amplification is carried out on a PCR instrument;

the amplification procedure was: setting the temperature of the stage 1 at 94 ℃, 5min, and circulating for 1 time; stage 2 is set at 94 ℃, 45s and 35 times of circulation; stage 3 is set at 62 ℃, 2min and 35 times of circulation; stage 4, setting 72 ℃, 60s, and circulating for 35 times; stage 5, setting the temperature at 72 ℃, 10min, and 1 circulation; stage 6 sets 8 ℃ for preservation.

Detection of the amplification product: the amplification products were detected by 8% native polyacrylamide gel electrophoresis. The loading was 2.0uL 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 nitrate silver staining and developing:

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, and gently shaken for 10 min.

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

Silver staining: 1.5L of fresh staining solution (0.1% AgNO3) was added and gently shaken for 10 min.

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

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.

Each SSR locus of the sample has three band types, the band type consistent with east 156 is 2, the band type consistent with east 237 is 0, the heterozygote type is 1, the deletion is-1, and a genotype database is established.

2.1.3 seed storage-related phenotype assays

2.1.3.1 Artificial aging method

Sterilizing uniformly-sized and undamaged corn seeds for 30min by using 1% sodium hypochlorite disinfectant, putting the corn seeds into a small mesh bag, and aging the corn seeds in a water bath kettle for 45min at the temperature of 58 +/-1 ℃; washing with tap water for 3 times and distilled water for 3 times; drying at room temperature for 2-3 days, balancing water content, and performing standard germination test.

2.1.3.2 Standard Germination experiments

A standard germination test is carried out according to a method specified in GB/T3543.4-1995 technology in crop seed inspection regulations, 50 seeds are placed in each box, an aging treatment group and a control group are set in the test, each group is repeated for 3 times, 50 seeds are repeated, the germination number is recorded every day, the germination potential of the seeds is calculated at the 4 th day, the germination rate and the germination index are calculated at the 7 th day, then the length of the seedlings is measured, and the vitality index is calculated.

2.1.3.3 determination of seed storage tolerance related index

The measurement indexes include germination percentage (GE), germination vigor (GP), Germination Index (GI), Vitality Index (VI), Seedling Length (SL) and Simple Vitality Index (SVI), and the specific detection method is based on the principle and method of seed vitality measurement of the color inspiration and the like^[67]The calculation formula of the measurement method in (1) is as follows:

germination vigor (GE) ═ number of germinated seeds sown in the germination test at the 4 th day/number of seeds tested × 100%;

the Germination Percentage (GP) ═ number of normal seedlings grown at the 7 th sowing time/number of seeds to be tested x 100%;

germination Index (GI) ═ Sigma Gt/Dt, where Dt is the number of germination days and Gt is the number of germination at different times corresponding to Dt;

the Vitality Index (VI) is GI multiplied by S, wherein S is the length (cm) of the 7 th seedling sowed in the germination test, and GI is the germination index;

The Simple Vitality Index (SVI) is GP multiplied by S, wherein S is the length (cm) of the 7 th seedling sowed in the germination test, and GP is the germination rate;

seedling length (cm) SL: sowing seeds 7d in a germination test between the root tip and the tip, and randomly taking 10 seedlings of each family for determination;

in order to avoid the influence of the original activity of the seeds among families on the storage tolerance evaluation, the relative value of each phenotypic character is used as the storage tolerance evaluation index in the research. The relative value of each phenotypic trait is the trait value of the aged germination test/the trait value of the standard germination test, and all indexes are the average of 3 replicates.

And establishing a storage phenotype database of the population, and analyzing the data by using software SPSS19.0 and Microsoft Excel 2010 for variance, average value, standard deviation, variation amplitude, variation coefficient and the like.

2.1.4 maize seed storability-related QTL analysis

2.1.4.1 construction of genetic linkage map

The genetic linkage map is constructed on an SSR genotype database by utilizing Isimiping 4.1 software, partial markers are grouped by using a group command, the sequence of the markers of each linkage group is determined by using an order command (LOD is 3.0), and a Kosambi function is selected to convert a recombination value into a map distance (cM). And (3) constructing a genetic linkage map by referring to a corn SSR Bin map and using a map instruction.

2.1.4.2QTL analysis

3 results and analysis

3.1 construction of genetic linkage map

In the MaizeGDB genome database (http://www.maizegdb.org/) 1349 SSR markers uniformly distributed on 10 chromosomes of corn are selected, polymorphism detection is carried out between the east 156 parents and the east 237 parents, 226 primers with polymorphism and clear bands among the parents are screened out for constructing a genetic linkage map, and the names, the sites and the sequences of the screened primers are shown in table 1.

TABLE 1 polymorphic SSR primers

A genetic map was constructed with the aid of the icimapping4.1 software, based on a genotype database of 226 SSR markers detected on the RIL population (LOD 3.0). 226 SSR marker loci are fitted in the map, 10 chromosomes of corn are covered, the total length is 3467.4cM, the average marker interval is 15.34cM, the marker numbers of chromosomes 1-10 are 28, 30, 28, 20, 16, 20 and 16 respectively, and the constructed genetic linkage map is shown in the map 1.

3.2RIL population F_7:8Pedigree seed storability-associated phenotype detection

288 family seeds of RIL group treated by hot water bath aging method are subjected to standard germination test, and storage-related phenotypic data are determined. As can be seen from Table 2, the test lines showed large variation range among the 6 storage tolerance-related indexes, and the difference was very significant (P < 0.01). The coefficient of variation of other storage related indexes except relative seedling length is more than 40%, wherein the coefficient of variation of relative germination potential is the largest and is as high as 57.07%.

The results of the frequency distribution analysis of the 6 traits of the RIL population are shown in figure 2, and the 6 storage-tolerance related trait values such as relative germination percentage, relative germination vigor, relative vitality index and the like are normally distributed, so that the method is suitable for phenotypic evaluation of seed storage tolerance.

TABLE 2 statistical analysis of the familial storability-related indices of the RIL population

3.3 QTL analysis related to maize seed storability

And (3) combining the genotype detection result of the RIL group and the phenotype detection result of the seed storability, and performing QTL analysis on the corn seed storability by adopting a composite interval mapping method. As shown in fig. 1 and table 3, the 17 QTLs that affect the 6 maize seed storability related indicators, such as relative germination percentage, relative germination vigor, and relative germination index, were co-detected and distributed on

chromosomes

1, 2, 7, 9, and 10 of maize, and the single QTL explained the phenotypic variation from the lowest 2.33% to the highest 20.11%. The additive effect values of 9 QTLs are positive values, and the allele of east 156 has synergistic effect on the loci, accounting for 52.94% of the number of the QTLs; while the east 237 allele has a synergistic effect on the remaining 8 QTL sites, accounting for 47.06% of the total number of QTLs.

3 main QTLs related to storability, namely qRGP-7, qRGE-7 and qRGI-7, exist between the marks umc1295 and phi082 on chromosome 7 and take relative germination rate, relative germination potential and relative germination index as indexes, and the contribution rates of the phenotypes are respectively 10.52%, 12.23% and 14.34%; there are 5 storability-related major QTLs qRGP-10, qRGE-10, qRGI-10, qRVI-10 and qRSVI-10 between markers phi050 and umc2043 on chromosome 10 as indexes of relative germination rate, relative germination potential, relative germination index, relative vitality index and relative simple vitality index, and the phenotype contribution rates are 12.43%, 20.11%, 14.33%, 10.08% and 10.85%, respectively; the synergistic effect of both storability-related major QTL segments is from east 156.

3.4 maize seed storability-related major QTL relocation

QTL mapping results using RIL populations with previous F_2:3The results of population mapping were compared, and 2 consensus major QTL segments were found, which were located on cQTL-7 on chromosome 7.05 and cQTL-10 on chromosome 10.03, respectively, and the QTL mapped in the two segments are shown in Table 4.

TABLE 4 QTL of concordance detected by two different populations

Note: LOD value (LOD): judging the existence of the QTL by adopting the threshold LOD value which is more than or equal to 3.0; additive effect (a): an additive effect value of "+" indicates that the east 156 allele is synergistic at this site; contribution ratio (PVE): interpretation of the percentage of phenotypic variation

The consensus QTL sections cQTL-7 and cQTL-10 are located between chromosome 7, umc1295 and umc2333, respectively, at a size of about 9.73Mb, and between chromosome 10, phi054 and umc2043, at a size of about 93.13 Mb.

The encryption of SSR markers is carried out near and inside the boundary of 2 consistency major QTL sections cQTL-7 and cQTL-10, wherein 9 markers are added to the cQTL-7 section of the chromosome 7, 22 markers are added to the cQTL-10 section of the chromosome 10, and the construction of genetic linkage maps and the QTL analysis are carried out again. The added tag names are shown in table 5.

TABLE 5 increased molecular markers

The total number of the encrypted SSR markers is increased to 257, the genotype detection result and the storability phenotype detection result are combined, the storage-tolerance related QTL is analyzed again by adopting a composite interval mapping method, and the positioning result is changed on the chromosomes 7 and 10, which is shown in figure 3.

4 QTLs were detected on chromosome 7, qRGP-7 affecting relative germination, qRGE-7 affecting relative germination potential, qRGI-7 affecting relative germination index, and qRSVI-7 affecting relative ease vitality index, all located between umc1671 and phi328175, with contributions of 11.72%, 14.63%, 10.04%, and 2.27%, respectively. The synergistic effect of 3 QTL sites other than qRSVI-7 was from east 156. 3 major QTL qRGP-7, qRGE-7 and qRGI-7 with contribution rate greater than 10% are located in the same marker segment (umc16171-phi328175 segment), and the segment is determined to be the storability related major QTL segment, and the synergistic effect is from east 156.

9 QTLs are detected on chromosome 10, namely qRGP-10 influencing relative germination rate, qRGE-10 influencing relative germination potential and qRGI-10 influencing relative germination index, which are all positioned between phi054 and umc2043, the contribution rates are 14.19%, 17.99% and 18.26%, and the additive effect values are 0.1466, 0.1786 and 0.1142 respectively; 3 qRVI-10-1, qRVI-10-2 and qRVI-10-3 which influence the relative vitality index are respectively positioned between markers phi050 and phi054, phi054 and umc2043 and umc1930 and umc1648, the contribution rates are respectively 22.39%, 12.80% and 19.71%, and the additive effect values are respectively 0.1418, 0.1425 and 0.1381; 3 qRSVI-10-1, qRSVI-10-2 and qRSVI-10-3 affecting the relative ease vitality index, respectively located between markers phi050 and phi054, phi054 and umc2043, and umc1930 and umc1648, with contributions of 19.59%, 19.78% and 14.36%, respectively, and additive effect values of 0.1567, 0.1571 and 0.1495, respectively. The 9 sites all received synergy from east 156.

3 major QTL qRGP-10, qRGE-10 and qRGI-10, all with contributions greater than 10% on chromosome 10, still localized to phi054-umc2043 segment; 2 major QTL qRVI-10 and qRSVI-10, each with a contribution rate greater than 15%, were mapped to the umc1648-phi050 segment, which was determined to be a storability-related major QTL segment, with synergism from east 156.

TABLE 6 relocating detected QTLs

3.5 Re-alignment analysis of major QTL segments for storage-related consistency

The QTL relocation result and F_2:3The result of population positioning is subjected to consistency analysis again, 2 consistency QTL sections relevant to corn seed storability are determined in total, and the sections are reduced compared with the previous sections

Chromosome 7 is located in the consensus QTL major segment cQTL-7 between markers umc1671 and phi328175, and comprises 3 QTL qRGP-7, qRGE-7 and qRGI-7, which respectively affect relative germination rate, relative germination potential and relative germination index, with phenotype contribution rates of 11.72%, 14.63% and 10.04%, and segment size of about 7.97 Mb.

Chromosome 10 is located in the consensus major QTL segment cQTL-10 between markers umc1648 and phi050, and comprises 2 QTL qRVI-10 and qRSVI-10, which respectively affect the relative viability index and the relative ease viability index, the phenotype contribution rate is 18.30% and 17.91%, respectively, and the segment size is about 39.15 Mb.

Test example 1 development and verification test of molecular marker

1. Test materials

141 American maize inbred lines were introduced from North Central Regional Plant Introduction Station (Table 7) by the northern northeast university of agriculture corn institute in 2012, and used for the development of new maize germplasm resources with good storability.

TABLE 7141 pedigree of the Extrinsic American inbred line

2. Test method

2.1 development of Linked markers

SSR markers near and inside the consistency main effect QTL section are selected to detect resistance pool and sensing pool genotypes, the genotype separation condition is subjected to X2 detection, and SSR markers which have polymorphism between parents and between resistance pool and are closely linked with the QTL are screened out. The amplification systems and methods and detection of the amplification products are as described above.

And (3) carrying out genotype detection on the screened molecular markers in 85 storability families of the RIL group, and screening out the storability related linkage markers according to the coincidence degree between the genotype detection result and the storability phenotype detection and the chi 2 test result.

2.2 application of molecular markers in breeding

2.2.1 molecular marker detection

141 parts of American corn inbred line genome DNA is extracted by a CTAN small quantity method, genotype detection is carried out by developed corn seed storability related SSR markers, and an inbred line containing the storability related major QTL is screened out. 2.2.2 Artificial aging seed storability phenotype detection and Material screening

141 American maize inbred lines were artificially aged by the high temperature High Humidity (HH) method. Modified according to the artificial accelerated aging seed method utilized by Zeng (2002)^[102]Selecting seeds with uniform seed size and no damage, sterilizing with 1% sodium hypochlorite disinfectant for 30min, and placing into a small mesh bag; placing on a screen mesh in a dryer, adding water under the screen mesh, and keeping the distance between the water surface and the screen mesh surface at 2 cm; balancing water content with vaseline sealed dryer, and aging in a constant temperature incubator at 45 deg.C for 72 hr; taking out the seeds, airing the seeds for 3d at room temperature, reducing the water content to the original state, and then carrying out a standard germination test.

An aging treatment group and a control group were set, each group was repeated 3 times, 50 grains each time, and the standard germination test method, measurement index and measurement method were as described above.

Performing descriptive analysis on data such as variance, mean value, standard deviation, variation amplitude, variation coefficient and the like by using software SPSS19.0 and Microsoft Excel 2010; carrying out cluster analysis on the seed storability of 141 American corn inbred lines by adopting a systematic classification method; determining a silage American corn inbred line, and verifying the availability of the developed linkage marker by combining with a genotype detection result; and (4) screening selfing lines containing the major QTL related to the storability, wherein the phenotype is identified as the storability, and the selfing lines are used as the storability materials.

3. Test results

3.1 development of linkage markers

Near and inside the boundaries of 2 storability-related consistency QTL sections cQTL-7 and cQTL-10, 9 markers such as umc1295, umc1671, phi328175, umc1367, phi054, umc1648 and phi050 are selected for detection of selection efficiency. Constructing an anti-infection pool formed by mixing equal amounts of DNA of extremely-resistant families in 30 RIL populations, mixing equal amounts of DNA of extremely-non-resistant families in 30 RIL populations, and performing genotype detection among the anti-infection pools by using the 9 SSR markers. The results are shown in FIG. 4, where the markers umc1295, phi082, umc1367, phi054 and umc2043 are polymorphic only between the two parents, and not in the resistant pool; and umc1671, phi328175, phi050 and umc1648 have polymorphism between parents and between influenza resistant pools, and the effective transmission rate of the polymorphic markers between the parents is 44.44%. And (3) carrying out individual plant genotype analysis on the screened differential markers by using individual plants in the resistant pool and the sensitive pool, and evaluating the relevance of the markers and the corn seed storability sites by using a Chi 2 fitness test.

The suitability detection analysis of chi 2 shows that the markers umc1671, phi328175, phi050 and umc1648 have obvious correlation with the main effect sites of the corn seed storage tolerance (see table 8), and chi 2 detection values are 0.862, 1.690, 0.133 and 1.286 which are respectively less than 3.84(p is 0.05 level, n is 1), so that the suitability detection method can be used for screening the main effect sites of the corn seed storage tolerance; and the other 5 markers χ 2 all detected values greater than 3.84(p ═ 0.05 levels).

TABLE 8 Chi 2 assay for storage-related markers in RIL population

And (4) combining the genotype detection results of the 4 markers in 85 storage-tolerant families and the storage-tolerant phenotype detection results to perform coincidence rate analysis. The genotype and storability phenotype concordance rates for the 4 markers were 83.12%, 80.82%, 88.89% and 82.93%, respectively, with an average of 83.94% (see table 9), with the highest concordance rate for marker umc 1648. Finally, 4 markers umc1671, phi328175, phi050 and umc1648 from the parent east 156 are determined to be related linkage markers of maize seed storability.

TABLE 9 degree of match between each marker genotype test and the genotype test of the RIL population pedigree

3.2 detection of Linked markers

141 American maize inbred lines were genotyped with 4 developed storability-associated linkage markers umc1671, phi328175, umc1648 and phi050, and the results are shown in Table 10. The inbred lines tested individually for tolerance to storage for 4 markers were 8, 13, 10 and 9, respectively.

TABLE 10 inbred lines of the United states containing storage-related major QTLs

3.3141 parts American inbred line artificial aging method seed storability phenotype detection

141 American corn inbred line seeds were subjected to artificial aging treatment and then tested for phenotypic data related to seed storability. As can be seen from table 11, the reference material showed a large variation range among 6 storage stability related indexes, with a very significant difference (P < 0.01); the coefficient of variation of other storage related indexes except relative seedling length is more than 35%, wherein the coefficient of variation of relative germination potential is the largest and is as high as 68.63%.

TABLE 11141 relative value statistical analysis of the storage-related traits of the American maize inbred lines

The seeds of 141 maize inbred lines were classified into 5 classes according to the storability by systematic clustering based on the relative values of the 6 storability-related trait indexes, and the results of the clustering analysis are shown in table 12. The selfing line with the strongest class I storability comprises 23 parts of N193, ND250, ND252 and the like; the II type selfing line with strong storability comprises 35 parts of 912 parts, W8555 parts, N209 parts and the like; class III self-bred line with moderate storability comprises 32 parts of N544, 3IIH6, N543 and the like; the IV self-line with weak storability comprises 33 parts of Mo48, RS710, MBWZ and the like; the selfing lines with the weakest storability in class V include 20 parts of OQ403, CQ702RC, MM402A, etc.

Inbred lines identified in the storability linked marker test to contain qRGE-7 and qRVI-102 major QTLs at the same time are ND248, ND252, ND246, SD65, N532, N209 and Tx 110; the ND248, ND252, ND246 and SD65 are identified as inbred lines with the strongest storability in the detection of the storability phenotype, and the N532, N209 and Tx110 are identified as stronger in the detection of the storability phenotype. The developed linked markers can be proved to be used and the inbred lines can be used for breeding new corn seed storage-resistant varieties.

TABLE 12141 clustering results of American maize inbred lines

SEQUENCE LISTING

<110> northeast university of agriculture

<120> SSR marker closely linked with corn seed storage tolerance and application thereof in molecular marker-assisted breeding

<130> HLJ-4006-190405A

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> DNA

<213> Artifical sequence

<400> 1

ctgcagtacg tgagcctgta cg 22

<210> 2

<211> 23

<212> DNA

<213> Artifical sequence

<400> 2

gcttgagctg tgaggaagtt ttg 23

<210> 3

<211> 25

<212> DNA

<213> Artifical sequence

<400> 3

taacatgcca gacacatacg gacag 25

<210> 4

<211> 22

<212> DNA

<213> Artifical sequence

<400> 4

atggctctag cgaagcgtag ag 22

Claims

1. The SSR molecular marker is applied to breeding of corn seed storage-resistant new varieties; the SSR molecular markers are phi050 and umc 1648; the nucleotide sequence of the primer for amplifying the SSR molecular marker umc1648 is shown as SEQ ID No. 1 and SEQ ID No. 2; the nucleotide sequences of the primers for amplifying the SSR molecular marker phi050 are shown as SEQ ID No. 3 and SEQ ID No. 4.

2, the application of SSR molecular markers in the detection of corn seed storability; the SSR molecular markers are phi050 and umc 1648; the nucleotide sequence of the primer for amplifying the SSR molecular marker umc1648 is shown as SEQ ID No. 1 and SEQ ID No. 2; the nucleotide sequences of the primers for amplifying the SSR molecular marker phi050 are shown as SEQ ID No. 3 and SEQ ID No. 4.

3. The application of the primer with the nucleotide sequence shown in SEQ ID No. 1-4 in the breeding of new corn seed storage-resistant varieties.

4. Application of primers with nucleotide sequences shown in SEQ ID No. 1-4 in detecting corn seed storability.

5. The application of the kit containing the primer with the nucleotide sequence shown in SEQ ID No. 1-4 in the breeding of new corn seed storage-resistant varieties.

6. The application of the kit containing the primer with the nucleotide sequence shown in SEQ ID No. 1-4 in detecting the corn seed storage stability.