CN105063201A - Molecular marker of corn chromosome 9 ear row number major QTL and application thereof - Google Patents
Molecular marker of corn chromosome 9 ear row number major QTL and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker of corn chromosome 9 ear row number major QTL and application thereof. The invention also provides a primer pair for identification or assistant identification of corn ear row number traits of maize ear rows, and the primer pair is obtained by amplification. The corn genome DNA is adopted as the template, and the primer pair shown as sequence 1 and sequence 2 is employed to perform PCR amplification, thus obtaining the primer pair of the DNA fragment, i.e. the primer pair composed of two single-stranded DNA shown as the sequence 1 and sequence 2. The corn genome is adopted as the template, the primers are employed to perform PCR amplification, and the obtained product is the molecular marker closely linked to the corn chromosome 9 ear row number major QTL. Experiments show that the additive effect of the corn ear row number major QTL is 0.9-1.3 rows, and the explicable phenotypic variation is 3.72%-10.78%, the QTL is closely linked with the SSR marker jsr21051, which can be effectively used for selection of the seedling stage ear row number, also can be used for molecular marker breeding of corn ear row number, thus providing the marking basis for fine localization of the corn ear row number QTL, and accelerating the high yield breeding process of corn.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of molecule marker and application thereof of corn Chromosome 9 tassel row number main effect QTL.
Background technology
Corn is important grain, feed and cash crop, according to the numeral that national grain and oil information center announces, within 2014, Chinese maize sown area reaches 3,620 ten thousand hectares, becomes the first food crop of China, occupies more and more consequence in China's agriculture production and the national economic development.But along with the continuous increase of population and the continuous minimizing of cultivated land resource, improve corn unit surface grain yield and be still task more and more urgent in China's Maize Production.
Under specific planting density, the output of unit surface is made up of corn single ear grain yield and spike number, and single tassel seed output is made up of tassel row number, row grain number and 100-grain weight.Visible, output is a very complicated proterties, and directly carrying out genetic analysis to yield traits is very complicated, a difficult research topic, and yield traits is resolved into each the factors of yield study respectively beyond doubt one select preferably.Tassel row number is an important the factors of yield, positive correlation remarkable in output, and the Genetic Mechanisms verifying tassel row number formation contributes to the Genetic Mechanisms of illustrating Yield Traits In Corn formation.The excavation of tassel row number relative new gene is its Forming Mechanism of research, realizes efficient conventional and Molecular Selection, improves the key of breeding efficiency and effect.
Classical quantitative genetics research shows, mealie line number is quantitative character, and there is complicated interaction between other the factors of yields; And proterties is by controlled by multiple genes, there is complicated interaction between gene, and the performance of proterties is easily subject to the impact of environment.Anederon just emphasized as far back as nineteen forty-four: " tassel row number is convenient to accurate-metering, is the good model of a research quantitative character ".The feature that tassel row number is easy to add up make early stage many scholars all with it for model research genetic development.19th century the forties, Emerson chooses the corn inbred line that a set of tassel row number is all 12, carries out plantation for many years, and carries out single cross, the double cross of storeroom, finds that tassel row number proterties is not only stable but also change.After this, investigator adopts the methods such as different generations, diallel cross to carry out Quantity Genetic Analysis to mealie line number proterties.
Summary of the invention
First object of the present invention be to provide a kind of for the identification of or the primer pair of assistant identification mealie line number proterties.
Provided by the present invention for the identification of or the primer pair of assistant identification mealie line number proterties, for the primer pair obtaining following DNA fragmentation that can increase: described DNA fragmentation take corn gene group DNA as template, primer pair shown in sequence 1 and sequence 2 in sequence table is adopted to carry out the DNA fragmentation of pcr amplification gained.
In the present invention, described primer pair is specifically made up of two single stranded DNAs shown in sequence in sequence table 1 and sequence 2.
The preparation method of described primer pair also belongs to protection scope of the present invention.
The preparation method of described primer pair, comprises the step of individually being packed by described two single stranded DNAs.
Second object of the present invention be to provide a kind of for the identification of or the test kit of assistant identification mealie line number proterties.
Provided by the present invention for the identification of or the test kit of assistant identification mealie line number proterties, containing described primer pair and following at least one: dNTP, archaeal dna polymerase and pcr amplification damping fluid.
The preparation method of described test kit also belongs to protection scope of the present invention.
The preparation method of described test kit, comprise by primer pair and following at least one step of individually packing: dNTP, archaeal dna polymerase and pcr amplification damping fluid.
3rd object of the present invention is to provide the method for the tassel row number proterties of a kind of qualification or assistant identification corn hybridization offspring.
The method of the tassel row number proterties of qualification provided by the present invention or assistant identification corn hybridization offspring, specifically can comprise the steps:
(a1) respectively with the genomic dna of each individuality in corn A, corn B and the corn hybridization progeny population of being hybridized by described corn A and described corn B and coming for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification respectively, obtain the PCR primer of each individuality in the PCR primer of described corn A, the PCR primer of described corn B and described corn hybridization progeny population;
The tassel row number of described corn A is more than the tassel row number of described corn B;
(a2) PCR primer of each individuality in the PCR primer of the PCR primer of described corn A, described corn B and described corn hybridization progeny population is carried out electrophoresis, respectively according to electrophoresis result according to the tassel row number proterties determining described corn hybridization offspring as follows: the tassel row number of the described corn hybridization offspring identical with the banding pattern of the electrophoretic band of the PCR primer of described corn A more than or candidate more than the identical described corn hybridization offspring of the banding pattern of the electrophoretic band of the PCR primer with described corn B.
4th object of the present invention is to provide a kind of method obtaining the relatively high individuality of tassel row number from corn hybridization progeny population.
The method obtaining the relatively high individuality of tassel row number from corn hybridization progeny population provided by the present invention, specifically can comprise the steps:
(b1) respectively with the genomic dna of each individuality in corn A, corn B and the corn hybridization progeny population of being hybridized by described corn A and described corn B and coming for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification respectively, obtain the PCR primer of each individuality in the PCR primer of described corn A, the PCR primer of described corn B and described corn hybridization progeny population;
The tassel row number of described corn A is more than the tassel row number of described corn B;
(b2) PCR primer of each individuality in the PCR primer of the PCR primer of described corn A, described corn B and described corn hybridization progeny population is carried out electrophoresis respectively, choose individuality identical with the banding pattern of the electrophoretic band of the PCR primer of described corn A in described corn hybridization progeny population, to be or candidate is the relatively high individuality of tassel row number described in described corn hybridization progeny population.
For above-mentioned two kinds of methods, in the present invention, described corn A is specially corn inbred line B73; Described corn B is specially corn inbred line west 5 211.Accordingly, described corn hybridization progeny population is specially the corn hybridization progeny population come by corn inbred line B73 and corn inbred line west 5 211 hybridization.
Certainly, described corn A also can be the corn of arbitrary kind of eligible A except corn inbred line B73; Described condition A is: take genomic dna as template, adopts described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, gained PCR primer is carried out electrophoresis, and the banding pattern of electrophoretic band is identical with reference to banding pattern A; Described with reference to banding pattern A be with the genomic dna of corn inbred line B73 for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, gained PCR primer carried out the banding pattern of electrophoresis gained.RIL system as identical with corn inbred line B73 in target section (be template with genomic dna, adopt described primer pair (sequence 1 and sequence 2) to carry out the amplified production of pcr amplification).
Accordingly, described corn B also can be the corn of arbitrary kind of eligible B except corn inbred line west 5 211; Described condition B is: take genomic dna as template, adopts described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, gained PCR primer is carried out electrophoresis, and the banding pattern of electrophoretic band is identical with reference to banding pattern B; Described with reference to banding pattern B be with the genomic dna in corn inbred line west 5 211 for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, gained PCR primer carried out the banding pattern of electrophoresis gained.RIL system as identical with corn inbred line west 5 211 in target section (be template with genomic dna, adopt described primer pair (sequence 1 and sequence 2) to carry out the amplified production of pcr amplification).
RIL is RIL (RecombinantInbredLines) RIL, and for a types of populations of genetic analysis and mapping in biology, produced through inbreeding of more generation by the material after hybridizing, in colony, each strain is isozygotied.
In described corn A and described corn B, arbitrary is maternal, and another is male parent.
In the above described two methods, described corn hybridization offspring specifically can be the corn hybridization offspring of isozygotying, as RIL system, DH system, or isozygoty F2 generation, isozygoty BC generation etc.
5th object of the present invention is to provide a kind of method of cultivating the corn variety that tassel row number increases.
The method of the corn variety that cultivation tassel row number provided by the present invention increases, choose the corn meeting following condition to carry out breeding as parent: take genomic dna as template, described primer pair (sequence 1 and sequence 2) is adopted to carry out pcr amplification, gained PCR primer is carried out electrophoresis, and the banding pattern of electrophoretic band is identical with reference to banding pattern A;
Described with reference to banding pattern A be with the genomic dna of corn inbred line B73 for template, adopt described primer pair (sequence 1 and sequence 2) to carry out pcr amplification, gained PCR primer carried out the banding pattern of electrophoresis gained.
In aforementioned three kinds of methods, the annealing temperature adopted during described pcr amplification specifically can be 58 DEG C.
Further, the reaction conditions of described pcr amplification is specially: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C of 10min; 12 DEG C of preservations.
In aforementioned three kinds of methods, described electrophoresis specifically can be polyacrylamide gel electrophoresis, and in described polyacrylamide gel electrophoresis, the concentration of described polyacrylamide gel is 8% (mass percentage).Described polyacrylamide gel electrophoresis is native polyacrylamide gel electrophoresis.
6th object of the present invention is to provide a kind of molecule marker relevant to mealie line number proterties.
The molecule marker relevant to mealie line number proterties provided by the present invention, being specially with corn gene group DNA is template, adopts described primer pair (sequence 1 and sequence 2) to carry out the DNA fragmentation of pcr amplification gained.
Wherein, the annealing temperature adopted during described pcr amplification specifically can be 58 DEG C.Further, the reaction conditions of described pcr amplification is specially: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C of 10min; 12 DEG C of preservations.
The application in arbitrary as follows of described primer pair (sequence 1 and sequence 2) or described test kit or described molecule marker also belongs to protection scope of the present invention:
(1) qualification or assistant identification mealie line number proterties;
(2) corn breeding.
First the present invention is just located by QTL, corn Chromosome 9 detects a tassel row number QTLqKRN9, its additive effect is that 0.9-1.3 is capable, explainable phenotypic variation is 3.72%-10.78%, this QTL and SSR marker jsr21051 close linkage, jsr21051 can be effective to the selection of tassel row number in seedling stage, can be used for the molecular marker breeding of mealie line number simultaneously, Fine Mapping for mealie line number QTL provides mark basis, accelerates the process of corn with high yield breeding.
Accompanying drawing explanation
Fig. 1 is the genetic linkage maps on the genetic linkage maps of corn Chromosome 9 and target interval.Wherein, A is the genetic linkage maps of Chromosome 9; B is the genetic linkage maps on target interval.
Fig. 2 is for utilizing jsr21051 to the F in B73 (male parent) × west 5 211 (female parent)
6the genotype call results of part individual plant in the segregating population produced after the individual plant selfing of the upper heterozygosis of target section (umc1191-umc2343) selected in generation.Wherein, A represents identical with male parent B73 genotype (electrophoresis banding pattern is identical); B represents and female parent west 5 211 genotype identical (electrophoresis banding pattern is identical); H represents heterozygosis banding pattern;-represent disappearance.
Fig. 3 is by the F in B73 (male parent) × west 5 211 (female parent)
6the tassel row number distribution plan of the segregating population produced after the individual plant selfing of the upper heterozygosis of target section (umc1191-umc2343) selected in generation.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Corn inbred line B73: be recorded in " Zhao Yongping. corn inbred line B73 protonitrate response miRNA and target gene qualification thereof. the Chinese Academy of Agricultural Sciences, Master's thesis in 2013 " literary composition, the public can obtain for repeating the present invention's experiment from applicant.
Corn inbred line west 5 211: be recorded in " Li Yan, Wang Jiangmin. the breeding and application of corn inbred line " west 5 211 ". Crop Diseases in Yunnan science and technology, 01 phase in 1998 " literary composition, the public can obtain for repeating the present invention's experiment from applicant.
The location of embodiment 1, mealie line number main effect QTL qKRN9
One, experiment material
1, the young leaflet tablet in good inbred lines B73 and west 5 211 extracts the screening for differential primer after DNA.
The F in 2, B73 (male parent) × west 5 211 (female parent)
2colony, totally 148 individual plants, for the first location of genetic map construction and tassel row number QTL.
3, the F in B73 (male parent) × west 5 211 (female parent) is chosen continuously
2, F
3, F
4, F
5in on target section the strain of heterozygosis carry out selfing produce segregating population, carry out the detection of tassel row number main effect QTL.
Two, the location of mealie line number main effect QTL qKRN9
1, corn gene group DNA extracts (modified CTAB method) in a large number
(1) get milpa young leaflet tablet 10-20mg, be placed in 1.5mlEppendorf pipe, add liquid nitrogen, be ground into fine powder.
(2) in centrifuge tube, 1 × CTAB Extraction buffer 600 μ l of 65 DEG C of preheatings is added, mixing of vibrating gently.
(3) temperature bath 30min in 65 DEG C of water-baths, and every 10min carefully shakes centrifuge tube once.
(4) take out centrifuge tube after 30min, in stink cupboard, add equal-volume chloroform: primary isoamyl alcohol (volume ratio 24:1), and careful fully shake centrifuge tube 2-3min, then leave standstill to organic phase by colourless → green → deep green.
(5), under room temperature, the centrifugal 10min of 10000rpm, then sucts in the centrifuge tube that 500 μ l are extremely new clearly.
(6) in supernatant, add the Virahol (-20 DEG C) of equal-volume precooling, after careful mixing, place 15min in-20 DEG C.
(7), under room temperature, the centrifugal 5min of 10000rpm, adds 70% ethanol 600 μ l and carries out rinsing, gentle agitation centrifuge tube, DNA is suspended after carefully outwelling supernatant.
(8), under room temperature, the centrifugal 3min of 10000rpm, then carefully outwells supernatant and places Air drying.
(9) after DNA dries, appropriate sterilizing ddH is added
2o, abundant dissolving DNA.
(10) by concentration and the purity of UV spectrophotometer measuring DNA, exist in-20 DEG C of refrigerators for subsequent use.
2, the amplification of target fragment
PCR reaction system (10 μ l system) is as follows: 40ng μ L
-1template DNA 2.0 μ L; 10 × PCRbuffer1.0 μ L; DNTPs0.2 μ L; Primer 1.0 μ L; RTaqDNA polysaccharase 0.1 μ L; ddH
2o5.7 μ L.
PCR response procedures (10 μ l system) is as follows: 94 DEG C of 5min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 36 circulations; 72 DEG C of 10min; 12 DEG C of insulations.
3, polyacrylamide gel electrophoresis detects
A. glue
(1) clean two pieces of sheet glass and pad clip are stepped up, the glue (ml) of 8%: 20%APS (μ l): TEMED (μ l) prepares mixing in the ratio of 1:10:1, quick back cover;
(2) prepare clean comb, treat that the gelling of back cover is solid, according to every plate 35ml 8% glue join glue in same ratio, encapsulating immediately; If any bubble, then beat sheet glass gently and drive bubble out of, the comb that parallel insertion is clean;
(3) for preventing cull, after gelling is solid, carefully extracts comb in time, offset plate is fixed on electrophoresis chamber, adds 1 × tbe buffer liquid.
40%PAGE solution (5L): deionized water 2000ml; N, N '-methylene diacrylamide 50g; Acrylamide 1950g; Agitator stirs, and fully dissolves, is settled to 5L.
8%PAGE solution: namely 40%PAGE solution dilution 5 doubly presses 40%PAGE:5 × tbe buffer liquid: the dilution proportion of deionized water=1:1:3.
5 × tbe buffer liquid (5L): Tris270.00g; Boric acid 137.50g; EDTA-Na
218.61g; Deionized water is settled to 1L.
B. electrophoresis
(1) in the PCR primer of amplification, add the 6 × LoadingBuffer of 2.0 μ l, after centrifugal, every hole point sample 4 μ l and every plate have the 100bpDNALadder of 1 hole point 4 μ l;
(2) under normal temperature,
200V prerunning 2min, then electrophoresis about 5h under 120V constant voltage in GT nucleic acid electrophoresis system (Bio-Rad, USA).
6 × LoadingBuffer (100ml): 0.5MEDTA (pH8.0) 2ml; Deionized formamide 98ml; Bromjophenol blue 0.05g; Dimethylbenzene cyanogen 0.05g.
1 × TBE electrophoretic buffer: 5 × tbe buffer liquid dilutes 5 times namely by 5 × tbe buffer liquid: the dilution proportion of deionized water=1:4.
C. silver dye
(1) 0.1% staining fluid: treat that electrophoresis terminates, gets clean silver dye basin, takes the AgNO of 0.50g
3, 500ml deionized water, mixing;
(2) dye: unloaded by offset plate, 0.1% staining fluid put into by stripping glue, and every basin silver dye 4 plate glue, then shake gently, dyeing 15min on shaking table;
(3) nitrite ion: NaOH10.00g, anhydrous Na
2cO
30.20g-0.30g, deionized water 500ml, formaldehyde 750 μ l;
(4) develop the color: after 15min to be dyed, carefully outwell nitrite ion and use deionized water short rinse 30s, every basin adds nitrite ion 500ml, continues to be placed on shaking table, shakes about 10min gently;
(5) according to glue: treat that gel becomes pale yellow, DNA band manifests completely, outwells nitrite ion, clear water rinses, by the band situation of the every plate glue of camera record, to read banding pattern.
4, the screening of corn Chromosome 9 SSR marker and the structure of genetic map
The SSR primer sequence that PCR reaction uses, from maize database (www.maizegdb.org), is synthesized by Tian Yihuiyuan bio tech ltd, Beijing.Through between parent pcr amplification and 8% native polyacrylamide gel electrophoresis detect and microcommunity checking, final obtain polymorphism obviously and banding pattern SSR marker clearly.
SSR marker is codominant marker, F
2what in individual plant, the banding pattern of marker site was identical with male parent B73 banding pattern is designated as A, and identical with female parent west 5 211 is designated as B, and heterozygosis banding pattern is designated as H, and disappearance is designated as "-", and application JoinMap4.0 software carries out genetic linkage maps structure.
Utilize from the F of polymorphism SSR marker in B73 (male parent) × west 5 211 (female parent) the Chromosome 9 of maize database (www.maizegdb.org) screening acquisition
2genotype in colony, construct the corn molecular marker linkage maps (in Fig. 1 A) that comprises 7 codominance SSR marker, this collection of illustrative plates is 36.6cM altogether, between mark, mean distance is 5.23cM, the chromosomal foci of most primer identical with the genetic linkage map of IBM (www.maizegdb.org).
5, tassel row number QTL locates
According to each individual plant phenotype in the genotype of each SSR marker and colony, by the genetic linkage maps on composite interval mapping method (CIM) (Zeng, 1994) the establishing target section of JoinMap4.0.Wherein, Kosambi function is selected recombination value to be converted to genetic distance unit (cM).Then WinQTLcartgrapher2.0 software is run, by composite interval mapping (CompositeIntervalMapping, CIM) method carries out QTL initial analysis location (ZengZB.Precisionmappingofquantitativetraitloci.Genetics, 1994,136:1457-1468).
Result shows: a mealie line number main effect QTL detected at molecule marker umc1191 and umc2343 interval, called after qKRN9, its LOD value is 3.0488, and additive effect is 0.644 row, explainable tassel row number heritable variation is 3.72%, and synergy gene is from male parent B73.
Primer pair for amplifier molecule mark umc1191:
Upstream primer: 5 '-AAGTCATTGCCCAAAGTGTTGC-3 ' (sequence 3);
Downstream primer: 5 '-ACTCATCACCCCTCCAGAGTGTC-3 ' (sequence 4).
Primer pair for amplifier molecule mark umc2343:
Upstream primer: 5 '-TCATCTTCCCCACAAATTTTCATT-3 ' (sequence 5);
Downstream primer: 5 '-GACTGACAACTCAGATTTCACCCA-3 ' (sequence 6).
The acquisition of embodiment 2, mealie line number main effect QTL qKRN9 close linkage SSR marker jsr21051
One, the exploitation of target interval SSR marker
For corn Chromosome 9 main effect QTL qKRN9 target interval, according to the Genomic sequence information announcing corn inbred line B73, from maize database MaizeGDB (http://www.maizegdb.org), transfer the sequence in target interval on different section, design corresponding primer in conjunction with primer3.0.Then, through pcr amplification and 8% the detection of native polyacrylamide gel electrophoresis and microcommunity checking, be chosen at B73,5 211 polymorphisms in west obviously and banding pattern clearly SSR primer for labeled analysis, in final acquisition QTL interval, polymorphism obviously and banding pattern SSR marker clearly, totally 9 is right, is designated as polymorphic molecular marker jsr16931, jsr20512, jsr21671, jsr19851, jsr21311, jsr21051, jsr18135, jsr22023 and jsr22381.
Two, the structure of target section genetic map
For target interval umc1191-umc2343, with reference to the genome sequence of corn inbred line B73, through design screening, develop B73,5 211 obvious differences in west and banding pattern as above 9 pairs of polymorphic molecular markers clearly altogether.Utilize known 3 SSR marker (the good umc2343 of umc1191, umc1078) on 9 pairs of SSR marker newly developed and target interval, for the F in B73 (male parent) × west 5 211 (female parent)
2offspring's segregating population that colony obtains in this section genotype heterozygosis individual plant selfing, identifies and adds up the genotype of each individual plant.By JoinMap4.0 software, wherein " A " is designated as 2, and " B " is designated as 0, and " H " is designated as 1, and "-" is designated as-1 (identical with male parent B73 banding pattern is designated as A, and identical with female parent west 5 211 is designated as B, and heterozygosis banding pattern is designated as H, and disappearance is designated as '-').Utilize Kosambi function, obtain the genetic linkage maps (in Fig. 1 B) in new genetic distance between the optimal ordering of 12 molecule markers on target section and mark and target section.
Three, the acquisition of close linkage mark jsr21051
Build the genotype data of encryption genetic map according to each generation, in conjunction with corresponding tassel row number data from generation to generation, utilize composite interval mapping legal position tassel row number main effect QTL qKRN9.Result shows, at the F in B73 (male parent) × west 5 211 (female parent)
2, F
3, F
4, F
5in on target section the strain of heterozygosis carry out selfing and produce in segregating population, the equal QTL that control tassel row number can be detected near mark jsr21051, its additive effect is respectively 1.3 row, 2.2 row, 0.94 row, 0.9 row, explainable phenotypic variation is 3.72%-10.78%, and its effect-increasing loci is all from male parent B73 (table 1).
The each QTLqKRN9 detected result from generation to generation of table 1 corn Chromosome 9
Note: " F in table
2, F
3, F
4, F
5" represent respectively by F
2, F
3, F
4, F
5the segregating population produced after the individual plant selfing of the upper heterozygosis of target section (umc1191-umc2343) selected in generation.
Primer pair for the SSR marker jsr21051 that increases:
Upstream primer: 5 '-tgaagagaagcttttgagacg-3 ' (sequence 1);
Downstream primer: 5 '-ggctgcagcagagagataat-3 ' (sequence 2).
Embodiment 3, the close linkage SSR marker jsr21051 application on mealie line number is selected
One, experiment material
Utilize the F in B73 (male parent) × west 5 211 (female parent)
6the segregating population produced after the individual plant selfing of the upper heterozygosis of target section (umc1191-umc2343) selected in generation, the application of qualification close linkage SSR marker jsr21051 on tassel row number is selected.
Two, experimental technique
For the F in B73 (male parent) × west 5 211 (female parent)
6the segregating population produced after the individual plant selfing of the upper heterozygosis of target section (umc1191-umc2343) selected in generation, seedling stage individual plant list sampling after, extract its genomic dna and with it for template, carry out pcr amplification with the amplimer (sequence 1 and sequence 2) of the close linkage mark jsr21051 of the tassel row number main effect QTL qKRN9 obtained, detect and add up the genotype of each individual plant.Meanwhile, after corn maturation, examine or check and add up F
6the main fringe tassel row number of each individual plant in colony.
PCR reaction system (10 μ L): Mix5.0 μ L; ddH
2o2.5 μ L; Upstream and downstream primer 1.5 μ L (50ng); Concentration is the DNA profiling 1.0 μ L of 20ng/ μ L.Wherein, Mix is Kang Run Cheng Ye bio tech ltd, Beijing product.
PCR reaction is reacted on instrument at GeneAmpPCRSystem9700PCR and is carried out, and PCR response procedures is as follows: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 circulations; 72 DEG C of 10min; 12 DEG C of preservations.
After reaction terminates, carry out 8% native polyacrylamide gel electrophoresis according to the method for embodiment 1.
Fig. 2 utilizes SSR marker jsr21051 to the genotype call results of part individual plant in segregating population.Meanwhile, after corn maturation, examine or check and add up the main fringe tassel row number of each individual plant in segregating population.
Carry out statistical study (table 2, Fig. 3) to the genotype of individual plant each in segregating population and tassel row number to find, utilize SSR marker jsr21051, the 27 pnca gene types that filter out altogether in 119 individual plants of segregating population are identical with the genotype of parent B73, and (namely the banding pattern of electrophoretic band is identical, for A), its tassel row number mean value is 11.55; 45 pnca gene types are identical with the genotype in parent west 5 211, and (namely the banding pattern of electrophoretic band is identical, for B), its tassel row number mean value is 10.53, compared with the plant tassel row number mean value identical with B73 with 27 pnca gene types, reduce by 1.02 row, there is significant difference (P<0.05) statistically.This shows, utilize SSR marker jsr21051 newly developed, the many corns of tassel row number can be gone out at Effective selection in seedling stage, save experimental cost, improve efficiency of selection, thus rapid screening goes out the many strains of tassel row number, for the SOYBEAN IN HIGH-YIELD BREEDING of corn.
The F of table 2ZC16 × C7-2
2the tassel row number statistics of colony
Based on the above results, the present invention utilizes material construction F based on Elite inbred B73 and west 5 211
2colony, a tassel row number main effect QTL qKRN9 detected in SSR marker mark umc1191 and the umc2343 interval of Chromosome 9, its LOD value is 3.0488, and additive effect is 0.644, and explainable tassel row number heritable variation is 3.72%.On this basis, adopt the construction of strategy segregating population of continuous selfing, target section is carried out to the exploitation of molecule marker simultaneously.Utilize institute develop mark gene type is carried out and the genetic linkage maps of establishing target section to the segregating population that each generation derives.To F
2, F
3, F
4and F
5segregating population derivative from generation to generation carries out QTL and detects discovery, the equal QTL that control tassel row number can be detected near mark jsr21051, its additive effect is respectively 1.3 row, 2.2 row, 0.94 row, 0.9 row, explainable phenotypic variation is 3.72%-10.78%, its effect-increasing loci is all from male parent B73, show thus, the tassel row number QTLqKRN9 stable existence on Chromosome 9, and with SSR marker jsr21051 close linkage.In addition, this close linkage is utilized to mark by F
6segregating population derivative from generation to generation carries out detection and finds, the genotype plant tassel row number mean value identical with B73 1.02 row more than the mean value of genotype and parent west 5 211 identical plant, show that jsr21051 can be effective to the selection of tassel row number in seedling stage, can be used for the molecular marker breeding of mealie line number simultaneously, Fine Mapping for mealie line number QTL provides mark basis, accelerates the process of corn with high yield breeding.
Claims (10)
1. for the identification of or the primer pair of assistant identification mealie line number proterties, for the primer pair obtaining following DNA fragmentation that can increase: described DNA fragmentation take corn gene group DNA as template, primer pair shown in sequence 1 and sequence 2 in sequence table is adopted to carry out the DNA fragmentation of pcr amplification gained.
2. primer pair according to claim 1, is characterized in that: the primer pair of described primer pair for being made up of two single stranded DNAs shown in sequence in sequence table 1 and sequence 2.
3. the method for the primer pair of preparation described in claim 1 or 2, comprises the step of individually being packed by two single stranded DNAs described in claim 1 or 2.
4. for the identification of or the test kit of assistant identification mealie line number proterties, containing the primer pair described in claim 1 or 2 and following at least one: dNTP, archaeal dna polymerase and pcr amplification damping fluid.
5. a method for the tassel row number proterties of qualification or assistant identification corn hybridization offspring, comprises the steps:
(a1) respectively with the genomic dna of each individuality in corn A, corn B and the corn hybridization progeny population of being hybridized by described corn A and described corn B and coming for template, adopt primer pair described in claim 1 or 2 to carry out pcr amplification respectively, obtain the PCR primer of each individuality in the PCR primer of described corn A, the PCR primer of described corn B and described corn hybridization progeny population;
The tassel row number of described corn A is more than the tassel row number of described corn B;
(a2) PCR primer of each individuality in the PCR primer of the PCR primer of described corn A, described corn B and described corn hybridization progeny population is carried out electrophoresis, respectively according to electrophoresis result according to the tassel row number proterties determining described corn hybridization offspring as follows: the tassel row number of the described corn hybridization offspring identical with the banding pattern of the electrophoretic band of the PCR primer of described corn A more than or candidate more than the identical described corn hybridization offspring of the banding pattern of the electrophoretic band of the PCR primer with described corn B.
6. from corn hybridization progeny population, obtain a method for the relatively high individuality of tassel row number, comprise the steps:
(b1) respectively with the genomic dna of each individuality in corn A, corn B and the corn hybridization progeny population of being hybridized by described corn A and described corn B and coming for template, adopt primer pair described in claim 1 or 2 to carry out pcr amplification respectively, obtain the PCR primer of each individuality in the PCR primer of described corn A, the PCR primer of described corn B and described corn hybridization progeny population;
The tassel row number of described corn A is more than the tassel row number of described corn B;
(b2) PCR primer of each individuality in the PCR primer of the PCR primer of described corn A, described corn B and described corn hybridization progeny population is carried out electrophoresis respectively, choose individuality identical with the banding pattern of the electrophoretic band of the PCR primer of described corn A in described corn hybridization progeny population, to be or candidate is the relatively high individuality of tassel row number described in described corn hybridization progeny population.
7. cultivate the method for the corn variety that tassel row number increases for one kind, choose the corn meeting following condition to carry out breeding as parent: take genomic dna as template, the primer pair described in claim 1 or 2 is adopted to carry out pcr amplification, gained PCR primer is carried out electrophoresis, and the banding pattern of electrophoretic band is identical with reference to banding pattern A;
Described is with the genomic dna of corn inbred line B73 for template with reference to banding pattern A, adopts the primer pair described in claim 1 or 2 to carry out pcr amplification, gained PCR primer is carried out the banding pattern of electrophoresis gained.
8., according to described method arbitrary in claim 5-7, it is characterized in that: the annealing temperature adopted during described pcr amplification is 58 DEG C.
9. the molecule marker relevant to mealie line number proterties, for taking corn gene group DNA as template, adopts primer pair described in claim 1 to carry out the DNA fragmentation of pcr amplification gained.
10. the application in arbitrary as follows of the primer pair described in claim 1 or 2 or test kit according to claim 4 or molecule marker according to claim 9:
(1) qualification or assistant identification mealie line number proterties;
(2) corn breeding.
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