CN107400703A - Chain molecular labeling and application with corn seed keeping quality main effect QTL qFSW-2 and qFSW-5 - Google Patents
Chain molecular labeling and application with corn seed keeping quality main effect QTL qFSW-2 and qFSW-5 Download PDFInfo
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Abstract
The present invention relates to the molecular labeling chain with corn seed keeping quality main effect QTL qFSW 2 and qFSW 5 and application.The corn seed keeping quality main effect QTL is the qFSW 2 in No. 2 region of chromosome Bin 2.06 of the corn and qFSW 5 in No. 5 chromosome Bin5.03 region of corn.Wherein, mark umc1079 and umc1028 is included with the molecular labeling of the close linkages of qFSW 2；Include mark umc2063 and umc2400 with the molecular labeling of the close linkages of qFSW 5.2 main effect QTL site qFSW 2 and qFSW 5 that the present invention passes through mapping maize seed keeping quality, it was found that 4 SSR molecular markers with 2 main effect QTL compact linkages, a feasible technological approaches is provided for corn seed storage endurance molecular breeding.
Description
Technical field
The present invention relates to biology field, specifically, is related to the molecular labeling chain with corn seed keeping quality main effect QTL qFSW-2 and qFSW-5 and application.
Background technology
Corn seed keeping quality has important value for the safe storage of seed, China's corn seed annual requirement is more than 1,570,000 tons, but annual actual production is much larger than consumption figure, but also must store a certain amount of seed of preparing against natural disasters, and substantial amounts of seed needs short-term or long-term storage.Generally use low temperature storage method at present, even if cryopreservation, its seed vitality also declines year by year, finally lose Seed practical value, research shows, keeping quality differs greatly between corn variety, some kinds through storing the germination percentage for still keeping higher for many years, but some seeds substantially reduce by short term storage germination percentage, the character dominant inheritance, therefore the strong corn variety of seed selection seed keeping quality is the most effective means for solving this problem, and the strong breed breeding of keeping quality depends on keeping quality resource, efficient breeding technique and the grasp to Genetic Mechanisms.
The assay method of seed keeping quality is mainly natural aging method and artificial accelerated aging method.It is general that the leading indicators such as potentiality of seed, germination percentage, vitality index and life index after aging are determined using artificial accelerated aging test to be evaluated and detected seed keeping quality due to the method detection keeping quality phenotype index cycle length of natural aging.The method is to be used for predicting the life-span of seed by Delouche propositions earliest.Conventional artificial accelerated aging method mainly includes hot and humid method, hot bath method and chemical-agent technique etc. at present.It is mainly hot and humid method, hot bath method wherein for corn keeping quality assay method, these methods respectively have advantage and disadvantage, and standard differs.Because corn seed keeping quality character inheritance basis is complicated, influenceed seriously by factors such as temperature, humidity, harvest times, the result obtained using different aging method and material is also not quite similar, and not yet establishes the corn seed keeping quality authentication method of stability and high efficiency.
With the development of marker assisted selection technology so that the genetic research and the assignment of genes gene mapping of seed keeping quality are possibly realized, and are also made great progress in recent years.Research for seed keeping quality and seed vitality so far is concentrated mainly in rice, wheat, arabidopsis isotype plant, but perfect with technology, also increasing in the research of corn, tomato, Chinese cabbage etc. crops kind.
Then Miura etc. carries out QTL positioning after carrying out potassium bichromate immersion artificial ageing using 98 long-grained nonglutinous rices and the backcross population of japonica rice, 3 gene locis related to keeping quality are have found, respectively on 2,4, No. 9 chromosomes.The contribution rate of gene loci wherein on No. 9 chromosomes is up to 59.5%, qLG-2 and qLG-4 contribution rate then than relatively low respectively 13.4%, 11.6%.Emile is material using the RIL of arabidopsis, method by determining the germination percentage of seed, ABA and environment stress after seed sugared content, aging, a series of QTLs of control seed keeping qualities is detected, and finds that all characters there are one or more common QTL sites.After the RIL of long-grained nonglutinous rice japonica rice is stored 1 year, 2 years, 3 years by Sasaki etc. respectively, carry out qtl analysis, it is found that the QTL site 12 of control germination percentage and seedling growth, respectively on No. 7 chromosomes and on No. 9 chromosomes, contribution rate is between 6.7%~17.3%, wherein the site RC9-2 contribution rate highests of the seedling growth of storage 2 years.
S.Landjeva etc. carries out qtl analysis to the germination percentage of the RIL of the wheat containing D genomes, seed vitality, seed longeivity and seedling growth, detect that 20 QTLs results show to control the gene of seed longeivity to be located on 1D or 5D altogether, for contribution rate between 15.3%~31.3%, the related locus wherein on 1D chromosomes is relatively abundanter.Fujino etc. carries out qtl analysis using 122 sufficient self-mating systems and finds that 3 QTLss (qLTG-3-1, qLTG-3-2 and qLTG-4) and qLTG-3-1 related to low temperature germination have been cloned.
Research is carried out primarily directed to the related morphological indexes of germination above, and the QTLs com-parison and analysis on the physiologic index of seed keeping quality is few.Cui etc. detects the QTL of the seedling vigor indexs such as control total amylase activity, soluble sugar content, alpha-amylase activity using long-grained nonglutinous rice and the RIL of japonica rice, 31 QTL are detected altogether, 5 different indexs are controlled respectively, it is predominantly located on 3,5, No. 6 chromosomes, and it was found that control the QTL of these characters to be located at similar region.It is basically identical on the index measured by seed keeping quality and seed vitality, main difference is that seed keeping quality is the indices of seed after measurement aging, so seed vitality is more similar to the QTL site of seed keeping quality.
The content of the invention
It is an object of the invention to provide the molecular labeling chain with corn seed keeping quality main effect QTL qFSW-2 and qFSW-5 and application.
In order to realize the object of the invention, the present invention and the molecular labeling of corn seed keeping quality main effect QTL compact linkage, the corn seed keeping quality main effect QTL are the qFSW-2 in No. 2 region of chromosome Bin 2.06 of the corn and qFSW-5 in No. 5 chromosome Bin5.03 region of corn.
Wherein, the molecular labeling with qFSW-2 close linkages includes 2 SSR markers umc1079 and umc1028；Include 2 SSR markers umc2063 and umc2400 with the molecular labeling of qFSW-5 close linkages.The primer for expanding each molecular labeling is as follows：
Umc1079 forward primer and reverse primer sequences is respectively SEQ ID NO.1 and 2；
Umc1028 forward primer and reverse primer sequences is respectively SEQ ID NO.3 and 4；
Umc2063 forward primer and reverse primer sequences is respectively SEQ ID NO.5 and 6；
Umc2400 forward primer and reverse primer sequences is respectively SEQ ID NO.7 and 8.
Using SEQ ID NO.1 and 2 in the strong corn inbred line east 156 of seed keeping quality it is amplifiable go out size be 121bp characteristic bands；
Using SEQ ID NO.3 and 4 in the strong corn inbred line east 156 of seed keeping quality it is amplifiable go out size be 138bp characteristic bands；
Using SEQ ID NO.5 and 6 in the strong corn inbred line east 156 of seed keeping quality it is amplifiable go out size be 91bp characteristic bands；
Using SEQ ID NO.7 and 8 in the strong corn inbred line east 156 of seed keeping quality it is amplifiable go out size be 114bp characteristic bands.
The present invention also provides application of the molecular labeling in identification corn seed keeping quality main effect QTL site qFSW-2 and qFSW-5.
The present invention also provides application of the molecular labeling in screening or identifying the strong corn variety of keeping quality.
Aforementioned applications comprise the following steps：
1) genomic DNA of plant to be measured is extracted；
2) using the genomic DNA of plant to be measured as template, using the primer for expanding above-mentioned molecular labeling, pcr amplification reaction is carried out；
3) pcr amplification product is detected.
The present invention also provides application of the molecular labeling in corn molecular mark.
The present invention also provides the PCR detection kit of the strong corn variety of identification keeping quality, and the kit includes the primer for expanding above-mentioned molecular labeling.
The present invention further provides the molecular labeling with corn seed keeping quality main effect QTL compact linkage according to above-mentioned molecular markers development.
2 main effect QTL site qFSW-2 and qFSW-5 that the present invention passes through mapping maize seed keeping quality, it was found that 4 SSR molecular markers with 2 main effect QTL compact linkages, a feasible technological approaches is provided for corn seed storage endurance molecular breeding.
Brief description of the drawings
Fig. 1 is the polymorphism result that agarose electrophoresis detects corn parent in the embodiment of the present invention 1.
Fig. 2 is the polymorphism result that polyacrylamide gel electrophoresis detects corn parent in the embodiment of the present invention 1.
Fig. 3 is primer bnlg1265 in the embodiment of the present invention 1 to F2 ： 3The agarose gel electrophoretogram of segregating population genotype detection.
Fig. 4 is primer phi027 in the embodiment of the present invention 1 to F2 ： 3The polyacrylamide gel electrophoresis collection of illustrative plates of segregating population genotype detection.
Fig. 5 is (east 156 × east 237) F in the embodiment of the present invention 22 ： 3Percentage of seedgermination distribution map after colony's artificial accelerated aging processing.
Fig. 6 is (east 156 × east 237) F in the embodiment of the present invention 22 ： 3Potentiality of seed distribution map after colony's artificial accelerated aging processing.
Fig. 7 is (east 156 × east 237) F in the embodiment of the present invention 22 ： 3Seedling fresh weight distribution map after colony's artificial accelerated aging processing.
Fig. 8 is (east 156 × east 237) F in the embodiment of the present invention 22 ： 3Seed germination index distribution map after colony's artificial accelerated aging processing.
Fig. 9 is (east 156 × east 237) F in the embodiment of the present invention 22 ： 3Seed Integrated vigor Index distribution map after colony's artificial accelerated aging processing.
Figure 10 is to be based on F in the embodiment of the present invention 32The maize genetic linkage map spectrum of informative population；Wherein, the QTL position ★ positioned with seedling fresh weight；The QTL position positioned with vitality index ▲；The QTL position positioned with germinating energy ◆.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment is according to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.
The foundation and genotyping of the corn seed keeping quality correlated traits genetic research colony of embodiment 1
1. test material
According to the corn inbred line (table 1) for selecting 2 parts of seed keeping quality significant differences early stage, its seed storage endurance of Middle East 156,8 years germination percentages remain at more than 90% under natural holding conditions；1 year germination percentage is stored intolerant to storage, under natural conditions and is down to 80% or so in east 237.Test material seed is provided by corn research institute of Northeast Agricultural University.
The relevant information of the corn inbred line of 1 two keeping quality significant differences of table
2 test methods
2.1F2The plantation of informative population and material
Spring in 2011 prepares east 156 × eastern autumn in 237,2011 in Harbin and plants F in Hainan1, it is selfed and obtains F2Seed.Spring in 2012 plants east 156 and east 237, F simultaneously on Heilungkiang Harbin Xiangfang farm1、F2.Single-strain planting, line-spacing 0.70m, spacing in the rows 0.3m, row are long 6.0 meters.Parent's respectively 3 row strains of plantation, F1Plant 3 rows, F2Plant 25 rows.
2.2 genotyping
2.2.1 maize leaf DNA extraction
2012 in growth of maize to 3 leaf, 1 heart stage maize leaf, selection parent, F1And F2Single-strain blade carries out DNA extraction, F2Individual plant numbers in order.Extraction DNA uses CTAB methods.Take young tender leaf agreement that contracts a film or TV play to an actor or actress 1.0g liquid feeding nitrogen to be ground into powdery rapidly, be transferred in 10mL centrifuge tubes.Add the 1mol/L Tris-HCl 7.5mL containing pH8.0 in the CTAB extraction buffer 100mL buffer solutions of 65 DEG C of preheatings of 4mL；PH8.0 0.5mol/L EDTA3.0m；NaCl 6.2g；CTAB 2.0g；0.2% mercaptoethanol (used time now adds) fully vibration mixes.50~60 minutes being incubated in 65 DEG C of water-baths (take out shake several times therebetween), being taken out, 4mL chloroforms are added after being cooled to room temperature：Isoamyl alcohol (24：1), put and slowly shaken 10 minutes on shaking table.To be centrifuged 10 minutes under the conditions of 4 DEG C of 12000r/min centrifuges, turn supernatant into another clean centrifuge tube.Again plus 4mL chloroforms：Isoamyl alcohol (24：1) repeat the above to shake, take supernatant to add isometric isopropanol into another clean centrifuge tube, gently shake up, a period of time is stood in 4 DEG C of refrigerators after centrifugally operated, go out DNA precipitations with crochet hook hook.DNA precipitations are washed three times with 70% ethanol, drying, add appropriate ddH2O and RNAase solution (10mg/ml) is fully mixed in the centrifuge tube of drying, warm bath 1h at 37 DEG C, and the DNA of extraction is carried out to the measure of concentration using ultraviolet specrophotometer, is diluted to after 25ng/ul -20 DEG C and is preserved for SSR marker analysis.
2.2.2SSR the screening of primer
From 949 pairs of SSR marker sites being distributed on 10 chromosomes of corn, primer sequence derives from MaizeGDB genome databases (http://www.maizegdb.org/).To the east of 156 with east 237 genomic DNA for template enter performing PCR amplification, filter out east 156 and east 237 between have polymorphism primer be used for F2The genotype detection of individual plant.PCR primer 3% agarose or 8.0% polyacrylamide gel electrophoresis, silver staining program are carried out with reference to Bassam et al. method.Its specific operation process is as follows：After all reactants are mixed by following system, the covering of 18uL mineral oil is added, in being expanded in PCR instrument.
Table 2SSR reaction systems
The touchdown PCR amplified reaction program of table 3
2.2.3 the detection of amplified production
According to pcr amplification product clip size, detected with 3% agarose gel electrophoresis or denaturing polyacrylamide gel electrophoresis, the primer of polymorphism between Selection parent be present.
1. prepared by agarose gel electrophoresis
The types of electrophoresis apparatus BIO RAD Power pac 300, electrophoresis tank DYCP-34A types.
Buffer solution：0.5 × TBE (45mmol/L Tris- borates, 1mmol/L EDTA), specific glue process and points for attention referring to《Molecular biology experiment instructs》.
2. polyacrylamide gel electrophoresis
Using the polyacrylamide native gel of gel 8%, instrument BIO RAD Power pac3000 type electrophoresis apparatuses.
3. electrophoretic procedures：
The assembling of glass plate：Between glass plate and offset plate plus parting bead, bottom alignment, is clamped and to load onto encapsulating base stand-by.Running gel is recorded：Glue produces polyacrylamide gel solution after fully shaking up, with inhaling between the glue prepared is gently circulated into two glass plates by bottle, plug comb in encapsulating mouth, pays attention to preventing comb bottom generation bubble.Put room temperature and it is more than half an hour to allow its polymerization.
Deposition condition：Applied sample amount is 1.0uL pcr amplification products, and using PBR322 as molecular weight Marker, electrophoretic buffer is 1 × TBE, firm power 85W prerunnings about 60 minutes.
4. silver staining program
It is fixed：It is placed under film is drawn with syringe needle in about 70cm × 50cm × 15cm plastic casing, adds 1.8L glacial acetic acid solution (10%), gently shake 10 minutes.
Rinsing：Rinsed 3 times with 2L ultra-pure waters, each 15s.
Silver staining：Add the dyeing liquor (0.1%AgNO that 1.5L newly matches somebody with somebody3), gently rock 10 minutes.
Rinsing：Rinsed with 2L ultra-pure waters, the time is no more than 10 seconds.
Development：Gel slab is put into the 1.5L developer solutions (22.5gNaOH, 1mL formaldehyde) newly matched somebody with somebody, gently rocked, until with line than more visible.
2.2.4 genotype counts
F2There are three kinds of banding patterns on each site of population sample：Banding pattern from east 156 is designated as 2, and the banding pattern from east 237 is designated as 0, and heterozygous is designated as 1, and missing is designated as -1.
3 results and analysis
3.1 parent's polymorphisms are examined
949 pairs of SSR primers are uniformly chosen on MaizeGDB using polymorphism between 3% agarose gel electrophoresis and eastern 156 (the resistance to storages) of polyacrylamide gel electrophoresis detection, east 237 (intolerant to storage) parent, 223 pairs of polymorphism primers are obtained, polymorphic sex ratio is 23.49%.To F2Colony carries out genotyping, wherein 192 pairs of SSR primers amplification banding patterns are clear, reproducible, available for the structure of genetic linkage mapses, wherein 1-10 chromosomes have 21,23,21,24,20,18,16,13,17,19 marks (table 4) respectively.Part primer pair parent's polymorphism primer the selection result such as Fig. 1 and Fig. 2, while to F2Colony's family carries out polymorphism inspection, and part primer detection result is shown in Fig. 3 and Fig. 4.
Polymorphism SSR primers between the east of table 4 156 and east 237
3.2 colony's genotype compositions and marker site separation situation
267 F are analyzed using 192 pairs of SSR markers2The genotype composition of individual, its gene of Middle East 156 account for the 47.71% of whole colony's genotype composition, and eastern 237 genes account for 52.28%.There are 58 site deviation theory segregation ratios 1: 2: 1 in 192 sites, account for 30.2% (table 5) in total site.The segregation ratio of genotype is further examined, there are 29 theoretical segregation ratio for deviating significantly from 1: 2: 1 in 58 sites, accounts for 50.00%.These deviate sites be concentrated mainly on the 1st, 2,7, on 9 chromosomes, the particularity of these chromosome segment structures may cause site to be deviateed, and surely belong to normal condition, the still structure for linkage map.
The marker site frequency distribution of table 5 and Chi-square statistic
The phenotype test of the resistance to storage correlated traits of the corn seed of embodiment 2
1 test material
The east 156 of preparation × eastern 237F2Colony's totally 267 individual plants.
2 test methods
2.1 Artificial ageing
Each individual plant takes out 150 seeds at random, 3 repetitions, repeats 50 every time, after sterilizing 30min with 1% javelle water, is put into small mesh bag, and after water-bath regulation is stablized into 10 minutes to 58 DEG C, small mesh bag is put into aging 60min in water-bath.Small mesh bag is taken out out of water-bath after aging, 2 times is rinsed with running water and uses distilled water flushing again 2 times.The seed rinsed is put in room temperature after drying 2-3 days equilibrium water contens and measures seed vitality index of correlation.
2.2 standard germinations are tested
Standard germination tests reference《National Seed Inspection code》The germinating method of middle GB/T3543.4-1995 technical stipulations carries out germination test.By the sand of sterilization, mixed thoroughly with distilled water, make its moisture consistent, be placed in germination box, thickness is about 3cm, is then uniformly put into seed, then covers 2cm wet sands.Germination box is put into 25 DEG C of illumination boxs, per 50 seeds of box, every two box once repeats, and each handles 4 repetitions.In germination process, normal chitting piece number, 4d statistics germinating energies are counted day by day, and 7d counts germination percentage, takes out seedling after 7d, cleaned with clear water, measures seedling fresh weight.Calculate germination index and vitality index.
Germination index Gi=∑s (Gt/Dt) (Gt:Germinative number within t days, Dt:Germinate number of days)
Vitality index Vi=Gi*St (Gi:Germination index, St:Seedling fresh weight)
The normal distribution-test of 2.3 characters
5 results such as germination percentage, germinating energy, germination index, vitality index, seedling fresh weight are analyzed using SPSS softwares, its degree of bias, kurtosis, average etc. is analyzed, detects whether normal distribution.
3 results and analysis
Early-stage Study shows, 58 ± 1 DEG C of hot bath aging processes handle the laggard quasi- germination test of rower, identification of indicator is evaluated using germinating energy, germination percentage, germination index, vitality index and relative germination rate, relative germination rate, relative germination index, relative activity index etc. as keeping quality, is the proper method for evaluating corn seed keeping quality.5 indexs such as germination percentage, germinating energy, germination index, vitality index and seedling fresh weight of seed after burin-in process can only be detected because test material is limited.
Using SPSS16.0 softwares to F2 ： 3The seed type of resistance to storage index of correlation after colony's aging carries out normal distribution detection, it the results are shown in Table 6, as seen from Table 6, indices difference is extremely notable between parent after hot bath aging, germinating energy is respectively 84.00% and 7.00%, and germination percentage is respectively 90.00% and 8.00%, and germination index is respectively 32.80 and 2.68, vitality index is respectively 13.54% and 1.23%, and relatively small seedling fresh weight difference is respectively 0.41g and 0.45g.The normal distribution of five characters is shown in Fig. 5-Fig. 9, and from Fig. 5 and table 6, percentage of seedgermination luffing is 0~98.66%, average is 62.39%, and skewness and kurtosis has two obvious peaks between-1 and 1, significant difference between family, super close phenomenon be present, substantially conform to just be distributed very much.Understand that germinating energy luffing is 0~98% by Fig. 6 and table 6, average 55.66%, skewness and kurtosis is between -1 and 1, without obvious peak, significant difference between family, super close phenomenon be present, substantially conforms to just be distributed very much.Understand that seedling fresh weight luffing is 0~1.84g by Fig. 7 and table 6, average 0.78g, the degree of bias has an apparent peak between -1 and 1, and difference is small between parent, but significant difference between family, super close phenomenon be present, substantially conforms to just be distributed very much.Understand that germination index luffing is 0~69.07 by Fig. 8 and table 6, average 23, skewness and kurtosis has an obvious peak between -1 and 1, significant difference between family, super close phenomenon be present, substantially conforms to just be distributed very much.Understand that vitality index luffing is that 0~67.98 average is 19.07 by Fig. 9 and table 6, skewness and kurtosis has an obvious peak between -1 and 1, significant difference between family, in the absence of super close phenomenon, substantially conforms to just be distributed very much.
The parent of table 6 and F2：3 colony's seed keeping quality index of correlation statistical parameters
The exploitation of the corn seed keeping quality qtl analysis of embodiment 3 and molecular labeling
1 test material
On Microsoft excel, the SSR marker genotypic database, F of 267 individual plants of mapping population established respectively2 ： 3Seed keeping quality index of correlation phenotypic data storehouse, including germination percentage, germinating energy, seedling fresh weight, germination index, vitality index (each index takes 3 repetition average values).
2 methods
2.1 structure SSR collection of illustrative plates
The structure of genetic linkage map is carried out to the SSR marker site of F2 colonies using the softwares of Icimapping 4.0 (2014), first part mark is grouped with " group " order, then each linkage group tag arrangement order (LOD=3.0) is determined with " order " order.Recombuination value is converted into map distance (cM) from " Kosambi " function.With reference to corn SSR Bin map, structure genetic linkage mapses are instructed with " map ".
2.2QTL is positioned and effect analysis
Use χ2Test method detects whether each marker genetype segregation ratio meets 1:2:1, and normal distribution-test is carried out to colony's gene frequency and field Assortment of characters.The softwares of Icimapping 4.0 are run, qtl analysis is carried out to resistance to storage correlated traits using composite interval mapping method.Corresponding operational factor is Window size=5.00cM；Model：ICIMADD；LOD=3.0.By 1000 repetition displacement tests, the LOD threshold values in the level of α=0.05 in genome range are estimated.With LOD>3.0 be threshold value existing for detectable QTL site, while analyzes QTL interpretable phenotypic variation rate, additive effect (A) and dominant effect (D).
3 results and analysis
The structure of 3.1 linkage maps
Information is expanded according to 192 pairs of primers, genetic map (LOD=3.0) is built using Icimapping4.0.Recombuination value is converted into map distance (cM) from " Kosambi " function, build SSR marker genetic linkage mapses (Figure 10), 192 SSR sites are fitted on Figure 10 altogether, cover the 2204.3cM of Maize genome, average distance is 11.48cM between mark.10 chromosomes of corn are covered, number of labels is 21,23,21,24,20,18,16,13,17,19 respectively.
The QTL initial analyses of the resistance to storage correlated traits of 3.2 corn seeds
QTL initial analyses and gene effect analysis are carried out to it using composite interval mapping method according to the genetic linkage mapses of structure and colony resistance to storage correlated traits, wherein not detecting QTL as index using germination percentage and germination index.
3.2.1 the keeping quality qtl analysis using vitality index as index
Qtl analysis is carried out by index of vitality index, 4 QTL (table 7) of control seed vitality are detected altogether, respectively on 5,7 and 10 chromosomes, the qSVI-5 on 5.02 sites on the 5th chromosome is between umc2167 and umc1597, contribution rate 6.51%；7th article chromosome detects two QTL sites, and contribution rate is respectively 9.16% and 20.87%；10th article chromosome detects 1 QTL, and contribution rate is up to 24.35%, between umc2043 and umc1367.All QTL additive effects are on the occasion of it is enhancing shelf-stable to show effect of the parent east 156 on these sites.
The application composite interval mapping method of table 7 carries out the qtl analysis of corn vitality index
3.2.2 the keeping quality qtl analysis using seedling fresh weight as index
Qtl analysis is carried out by index of seedling fresh weight, detects the QTL of control seedling fresh weight 5 QTL (table 8) altogether, the qFSW-1 on the site of the 1st chromosome 1.08 is between mark bnlg1671 and bnlg1643, contribution rate 9.75%；QFSW-2 on 2nd chromosome on 2.06 sites is between umc1079 and umc1028, contribution rate 17.09%；QFSW-5 is located on umc2063 and umc2400 on 5.03 sites on 5th article of chromosome, contribution rate point 22.68%；QFSW-7 on the site of the 7th chromosome 7.05 is between mark umc1545 and umc2333, contribution rate 4.37%；QFSW-10 on 10th chromosome on 10.03 sites is between umc2043 and umc1367, contribution rate 10.83；All QTL additive effects are on the occasion of it is enhancing shelf-stable to show effect of the parent east 156 on these sites.
The application composite interval mapping method of table 8 carries out the qtl analysis of corn seedling fresh weight
3.2.3 the keeping quality qtl analysis using germinating energy as index
Qtl analysis is carried out by index of germinating energy, 1 QTL (table 9) of control seedling fresh weight is detected altogether, positioned at the qGE-8 sites of the position of 8 chromosome 8.02, between bnlg2235 and primer bnlg1194, contribution rate 5.58%, additive effect be on the occasion of, show parent to the keeping quality on the site rise humidification.
The application composite interval mapping method of table 9 carries out the qtl analysis of corn germination gesture
4 corn seed keeping quality main effect QTLs of common location of the present invention, wherein qSVI-7-2 and qSVI-10 is the corn seed keeping quality main effect QTL obtained using vitality index as index, and qFSW-2 and qFSW-5 are the corn seed keeping quality main effect QTLs obtained using seedling fresh weight as index.
Although above the present invention is described in detail with a general description of the specific embodiments, on the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Bibliography
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Claims (8)
1. the molecular labeling with corn seed keeping quality main effect QTL compact linkage, its feature exist
In the corn seed keeping quality main effect QTL is to be located at No. 2 area of chromosome Bin 2.06 of corn
QFSW-2 in the domain and qFSW-5 in No. 5 chromosome Bin5.03 region of corn；
Wherein, the molecular labeling with qFSW-2 close linkages includes 2 SSR marker umc1079
And umc1028；Include 2 SSR marker umc2063 with the molecular labeling of qFSW-5 close linkages
And umc2400；The primer for expanding each molecular labeling is as follows：
Umc1079 forward primer and reverse primer sequences is respectively SEQ ID NO.1 and 2；
Umc1028 forward primer and reverse primer sequences is respectively SEQ ID NO.3 and 4；
Umc2063 forward primer and reverse primer sequences is respectively SEQ ID NO.5 and 6；
Umc2400 forward primer and reverse primer sequences is respectively SEQ ID NO.7 and 8.
2. molecular labeling according to claim 1, it is characterised in that utilize SEQ ID
NO.1 and 2 in the strong corn inbred line east 156 of seed keeping quality it is amplifiable go out size be 121
Bp characteristic bands；
It can be expanded in the strong corn inbred line east 156 of seed keeping quality using SEQ ID NO.3 and 4
Increase the characteristic bands for that size is 138bp；
It can be expanded in the strong corn inbred line east 156 of seed keeping quality using SEQ ID NO.5 and 6
Increase the characteristic bands for that size is 91bp；
It can be expanded in the strong corn inbred line east 156 of seed keeping quality using SEQ ID NO.7 and 8
Increase the characteristic bands for that size is 114bp.
3. the molecular labeling of claim 1 or 2 is in identification corn seed keeping quality main effect QTL
Application in site qFSW-2 and qFSW-5.
4. the molecular labeling of claim 1 or 2 is screening or identified the strong corn of keeping quality
Application in kind.
5. application according to claim 4, it is characterised in that comprise the following steps：
1) genomic DNA of plant to be measured is extracted；
2) using the genomic DNA of plant to be measured as template, using expanding claim 1 or 2
The primer of the molecular labeling, carry out pcr amplification reaction；
3) pcr amplification product is detected.
6. the molecular labeling of claim 1 or 2 answering in corn molecular mark
With.
7. identify the PCR detection kit of the strong corn variety of keeping quality, it is characterised in that
The kit includes the primer of the amplification molecular labeling of claim 1 or 2.
8. molecular markers development according to claim 1 or claim 2 with corn seed keeping quality master
The molecular labeling of effect QTL compact linkage.
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| CN110423838A (en) * | 2019-07-11 | 2019-11-08 | 东北农业大学 | The molecular labeling of main effect QTL section close linkage related to corn seed keeping quality is located at and its application |
| CN110777214A (en) * | 2019-07-11 | 2020-02-11 | 东北农业大学 | SSR (simple sequence repeat) marker closely linked with corn seed storage resistance and application thereof in molecular marker-assisted breeding |
| CN111073996A (en) * | 2020-02-12 | 2020-04-28 | 中国农业科学院作物科学研究所 | Molecular marker closely linked with corn rough dwarf resistant main effect QtL and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110423838A (en) * | 2019-07-11 | 2019-11-08 | 东北农业大学 | The molecular labeling of main effect QTL section close linkage related to corn seed keeping quality is located at and its application |
| CN110777214A (en) * | 2019-07-11 | 2020-02-11 | 东北农业大学 | SSR (simple sequence repeat) marker closely linked with corn seed storage resistance and application thereof in molecular marker-assisted breeding |
| CN110423838B (en) * | 2019-07-11 | 2022-05-31 | 东北农业大学 | Molecular marker closely linked with major QTL (quantitative trait locus) segment related to corn seed storage tolerance and application thereof |
| CN111073996A (en) * | 2020-02-12 | 2020-04-28 | 中国农业科学院作物科学研究所 | Molecular marker closely linked with corn rough dwarf resistant main effect QtL and application thereof |
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