阅读说明：本技术 与玉米种子耐储性紧密连锁的ssr标记及其在分子标记辅助育种中的应用 (SSR (simple sequence repeat) marker closely linked with corn seed storage resistance and application thereof in molecular marker-assisted breeding ) 是由 邸宏 张�林 *** 封陈晨 周羽 曾兴 于 2019-07-11 设计创作，主要内容包括：本发明公开了与位于玉米种子耐储性相关主效QTL区段紧密连锁的分子标记及其应用；所述分子标记与位于玉米第10号染色体Bin10.03区域内玉米种子耐储性相关主效QTL区段cQTL-10紧密连锁,该分子标记为phi050和umc1648。本发明通过定位玉米种子耐储性的1个主效QTL区段,该区段包含包含2个QTL qRVI-10和qRSVI-10,分别影响相对活力指数和相对简易活力指数,发现了与玉米种子耐储性紧密连锁的2个SSR分子标记phi050和umc1648,本发明所提供的SSR分子标记可应用于提高玉米种子耐储能力的分子辅助育种。(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, 2SSR 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 2QTL 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.)

1. An SSR molecular marker for maize seed storage tolerance, wherein the SSR molecular marker is closely linked with a maize seed storage tolerance related major QTL segment cQTL-10 in a maize chromosome 10 Bin10.03 region.

2. A SSR molecular marker according to claim 1 wherein said molecular markers are phi050 and umc 1648.

3. The primer for amplifying the SSR molecular marker in claim 2, wherein 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.

4. A kit for detecting corn seed storage endurance, characterized in that, the kit comprises the primer of claim 3.

5. Use of the molecular marker of claim 1 or 2 in breeding new corn seed storage-tolerant varieties.

6. Use of the molecular marker of claim 1 or 2 for testing corn seed storability.

7. The use of the primer of claim 3 in breeding new corn seed storage-tolerant varieties.

8. Use of the primer of claim 3 for testing corn seed storability.

9. The use of the kit of claim 4 in breeding new corn seed storage-tolerant varieties.

10. Use of the kit of claim 4 for testing corn seed storability.

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 viability over a period of time, and the viability-maintaining times of different types of seeds vary, and deterioration and aging of seeds inevitably occur with prolonged 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 seeds.

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 stability of the seeds is improved by combining the molecular breeding technology, and a new variety with high self-activity and storage tolerance is bred.

Corn is the first large feed, grain and industrial raw material crop in China, the annual seed demand is about 9 hundred million kilograms, the actual annual seed production is about 10 to 11 hundred million kilograms, and in addition to barren seeds and germplasm resources for breeding, a large number of seeds for use need to be stored every year, so that the improvement of the seed storage capacity has important value for corn genetic breeding and agricultural production. The data of the national agricultural technology promotion service center (https:// www.natesc.org.cn) shows 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 need to be stored. The germination rate is reduced by the storage condition and the self reason of the seeds every year, the amount of the seeds losing the seed value reaches 3-5 jin, and the serious loss is caused to the seed 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 summer (2018), sweet corn varieties of 'Dongshan 88' and 'nonghan 99' are used as test materials, and SRAP marker detection shows that the genetic diversity of the aged seeds is reduced and the genetic diversity of the seeds with good storability is reduced. Li Chun Rei (2015) takes Zheng 958 and Xian Yu 335 seeds as test materials, and related researches on corn seed storability are carried out from the aspect of epigenetic DNA methylation by utilizing an MSAP molecular marking technology, and the research shows that the correlation between the seed storability and CG level DNA methylation is high.

Li et al (Li T, Zhang Y, Wang D, et al. Regulation of Seed Vigor bymanagement of Raffinose FamilyOligosaccharides in Maize and Arabidopsis thaliana [ J ]. Molecular Plant, 2017, 10(12):1540.) found that over-expressing both ZmGOLS2, ZmRS and AtSTS genes alone, both increased Arabidopsis thaliana Seed Vigor, whereas over-expressing ZmGOLS gene alone decreased Arabidopsis thaliana Seed Vigor.

Corn seedThe QTL positioning related to the child 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, GengG. QTL Analysis of gain Storage stability for size Under controlled degradation 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 mapping [ 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]Harbin: northeast agriculture university, 2015) detects 10 QTLs respectively located on chromosome 1, 2, 5, 7, 8 and 10, and the phenotypic contribution rate range is between 4.37% and 24.35%; hund et al (Hund A, Frachboud Y, Soldaiti A, et al. QTL controlling root and shootdraits of mail threads under Cold stress [ J]Theoretical 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, henna, 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 QTL of 4 related characters such as germination rate, germination vigor, germination index and vitality index; 172 QTLs related to seed vigor are detected by Hanzangping (2014), are distributed on 9 chromosomes except No. 6, and explain trait genetic variation is between 5.39% and 12.11%; zhao Rui Fang (Zhao Rui Fang, corn seed vigor phase under different conditions)QTL mapping analysis of relational traits [ D]The university of southern river agriculture, 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 storage stability, of which qGP5 were detected in both mapping 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) detected 4 germination potential-related QTLs 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 standard germination conditions; under the condition of low temperature stress, one qtlgp1 associated with the germination potential and located on chromosome 10 was detected, accounting for the phenotypic variation of 6.67%, and one qtlgr1 associated with the germination rate and located on chromosome 6, accounting for the phenotypic variation of 7.75%.

Disclosure of Invention

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

the second purpose of the invention is to apply the molecular marker obtained by screening to the molecular assisted breeding of a new corn seed storage-tolerant 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 resistance 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 resistance 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 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 consensus major QTL segment cQTL-10 between markers umc1648 and phi050, and comprises 2QTL 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 mixed in equal amount to form an anti-pool, selects the family DNA which is extremely non-storable in 30 RIL groups to be mixed in equal amount 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 storage-tolerant families of the RIL population, and screening out the storage-tolerant related linkage markers according to the coincidence degree between the genotype detection result and the storage-tolerant 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, but 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 a tolerant inbred line of 8, 13, 10 and 9 parts. Inbred lines identified as containing qRGE-7 major QTL in the storability linked marker assay were ND248, ND252, ND246, SD65, N532, N209, Tx 110; wherein ND248, ND252, ND246, SD65 are identified as the most storable inbred lines in the phenotypic test of storability, and N532, N209, Tx110 are identified as stronger storability in the phenotypic test of storability.

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 influencing relative germination rate, relative germination vigor and relative germination index, so that the method can be used for screening the molecular markers for the storage tolerance of the corn seeds to assist breeding.

Detailed description of the invention

Analysis and positioning of maize seed storage-resistant 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 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.

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 interregional 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 population positioning results to determine a consistency major 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 population, 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 the 9 QTLs are positive values, and the allele of east 156 plays a synergistic role in the loci and accounts for 52.94 percent 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 vigor, relative germination index, relative vigor index and relative simple vigor 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 contribution rates of the phenotypes 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. cQTL-7 and in 2 consensus major QTL segmentsAnd (3) encrypting SSR markers near and inside the boundary of cQTL-10, wherein 9 markers are added to the cQTL-7 section of chromosome 7, 22 markers are added to the cQTL-10 section of chromosome 10, and the construction of the genetic linkage map 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 storage tolerance 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 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 which affect 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 viability index, located between markers phi050 and phi054, phi054 and umc2043, umc1930 and umc1648, respectively, 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 within cQTL-10, the consensus major QTL segment between markers umc1648 and phi050, packageContains 2QTL qRVI-10 and qRSVI-10, which respectively affect the relative vitality index and the relative simple 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 and mixing the same amount to form an anti-pool, selecting the family DNA which is extremely non-storable in 30 RIL groups and mixing the same amount 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-pool formed by mixing equal amounts of DNA of extremely-resistant families in 30 RIL populations, and a sensitive pool formed by mixing equal amounts of DNA of extremely-non-resistant families in 30 RIL populations, and carrying out genotype detection among the anti-sensitive pools by utilizing 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 major effect sites of the corn seed storage resistance (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 major effect sites of the corn seed storage resistance; and the other 5 markers χ 2 all detected values greater than 3.84(p ═ 0.05 levels).

The results of genotype tests on 85 storage-tolerant families combined with the results of the storage-tolerant phenotype tests on 4 markers were used for the coincidence 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%, 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 corn seed storability related linked markers.

141 American maize inbred lines were genotyped with 4 storability-associated linkage markers developed umc1671, phi328175, umc1648 and phi 050. The 4 markers were individually tested as a self-cross line of 8, 13, 10 and 9 shares, respectively, of storage tolerance.

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 of qRVI-10 containing chromosome number 10 affecting relative viability index are ND248, ND252, ND246, SD65, N532, N209, Tx110, LH192, PHJ90, 3IIH6 and 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 maize inbred lines into 5 types according to the storability, and according to the clustering analysis result, the inbred line with the strongest I type storability comprises 23 parts of N193, ND250, ND252 and the like; the II type selfing line with strong storability includes 35 portions of 912, W8555 and N209, etc.; 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 seed and has the storage resistance can be used for breeding new corn seed storage-resistant varieties.

Drawings

FIG. 1 is based on F _7:8The relative germination rate is ★, the relative germination vigor is:

the relative germination index is ▲, the relative vitality index is ●, the relative seedling length is ■, and the relative simple vitality index is ◆;

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 shows the reconstructed genetic linkage maps of chromosome 7 and chromosome 10, the relative germination percentage: ★, the relative germination vigor:

relative germination index of ▲, relative vitality index of ●, relative seedling length of ■;

relative simple vitality index ◆

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 will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It should be understood that the illustrated embodiments are exemplary only, and are not intended to limit the scope of the present 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.