CN103882145A - PCR (Polymerase Chain Reaction) molecular marking method for identifying allele mutation of rice long-grain gene qGL3 - Google Patents
PCR (Polymerase Chain Reaction) molecular marking method for identifying allele mutation of rice long-grain gene qGL3 Download PDFInfo
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Abstract
The invention discloses a PCR (Polymerase Chain Reaction) molecular marking method for identifying allele mutation of rice long-grain gene qGL3, and belongs to the technical field of agro-biological engineering. According to the PCR molecular marking method, four specific molecular marking primers are designed and synthesized for PCR amplification of DNA of different rice based on the difference of long-grain and large-grain variety TD70 and short-grain and small-grain variety Kasalath on the nucleotide base sequence of the long-grain gene qGL3, the amplified product is subjected to electrophoresis detection with 2.0% of agarose gel, and DNA brands of different dimension represent different allele types of qGL3 after DuRed nuclear acid developing. With the adoption of the molecular marking method, the difference of different alleles of the long-grain gene qGL3 in rice germplasm resources or breeding population can be quickly and accurately identified; in addition, the selection efficiency for the long-grain homozygous genotype qGL3-TqGL3-T is obviously improved; and therefore, the coordinative improvement of the appearance quality and output traits of rice can be realized.
Description
One, technical field
The invention discloses a kind of PCR molecule marking method of identifying paddy rice grain length gene qGL3 allelic variation, belong to agro-biological engineering technical field, be exclusively used in containing qGL3-
tqGL3-
tscreening, qualification and the breed breeding of the long grain of genotype high-quality, high-yield rice resource.
Two, background technology
Paddy rice is the important food crop of China, approximately 3,000 ten thousand hectares of cultivated areas throughout the year, account for 30% of national cereal cultivated area, paddy ultimate production exceedes 20,000 ten thousand tons, accounts for the more than 40% of national total grain output, therefore, Rice Production is to ensure China grain security, promote national economic development to have very important status (Lu Jingbo, Chinese rice, 2002,6:17-20).
In recent years, along with the raising of population growth and living standards of the people, domestic market constantly increases the demand of fine quality rice.Rice breeding, in paying close attention to variety yield raising, is focused on the lifting (Luo Yukun etc., rice in China science, 2004,18 (2): 135-139) of rice quality more.Grain type is to weigh the important indicator of paddy morphological specificity, it by grain length, grain wide and grain thick three fundamentals form, wherein the relation of grain length proterties and rice yield and exterior quality is the closest, thereby the extremely concern of breeding man always in yield and quality of rice improvement.Ensureing, under the wide constant prerequisite of grain, suitably to increase grain length, thousand seed weight, the output of rice increases, the white grain of chalk rate reduces, exterior quality becomes excellent.Genetic research shows, grain length is a complicated quantitative character, shows as different modes of inheritance under different genetic group or envrionment conditions, and it both may be subject to the effect of 1 or several major genes, also may be subject to multiple polygenic control, show the hereditary feature of normal distribution.Due to the additivity of grain length gene complexity, dominant with epistasis effect, be difficult to it to carry out character improvement by the method for traditional breeding method, therefore only have utilize with grain length gene close linkage or be divided into from molecule marker carry out assistant breeding and could carry out accurate and effective selection to grain length proterties (it is rich etc. that poplar is loved and respect one's elder brother, Molecular Plant Breeding, 2010,8 (1): 59-66).
It is the prerequisite of carrying out marker assisted selection that the heredity of grain length gene is resolved, in recent years along with the structure of paddy rice dense genetic map spectrum and completing of gene order-checking, (the Quantitative Trait Loci of grain length quantitative trait locus, QTL) location work has obtained impressive progress (Wang Zhonghua etc., life science, 2009,21 (3): 444-451).According to the statistics of Gramene website, individual by the end of the QTL76 that altogether navigate to control grain length proterties by more than 20 dissimilar genetic groups in March, 2014, they are distributed widely on 12 karyomit(e)s of paddy rice, the QTL number that wherein the 3rd karyomit(e) detects is maximum and effect is relatively stable, thereby (high will is strong etc. to be considered to the hot spot region of paddy rice grain length gene distribution, heredity, 2011,33 (4): 314-321).
At present, the grain length gene of having cloned in paddy rice has GS3 and qGL3, and their positions are respectively at the 3rd chromosomal different zones.Wherein, being positioned near the grain length major gene GS3 in kinetochore not only affects the length of seed, also affects the thousand seed weight of paddy simultaneously.Compared with short, granule paddy rice; on exon 2, there is a single nucleotide variations in long, large grain kind GS3 gene; cause the TGC codon mutation of original encoding aminothiopropionic acid to become terminator codon TGA; make protein translation premature termination; cause the sequence of 4 conservative regions such as class PEBP structural domain to lose; and then forfeiture gene function; seed length and weight are increased; the albumen of this explanation GS3 genes encoding has effect (the Fan C C of negative regulation; et al.; Theoretical and Applied Genetics, 2006,112 (6): 1164-1171; Mao H L, et al., Proc Natl Acad Sci USA, 2010,107 (45): 19579-19584).Different from GS3, grain length gene qGL3 is positioned at paddy rice the 3rd long-armed middle part of karyomit(e), and its coding is containing the serine/threonine Phosphoric acid esterase of Kelch structural domain.Compared with 9311, long, large grain strain N411 exists a C to A mononucleotide nucleotide variation in exon10, causes the 364th amino acid to replace with L-glutamic acid by aspartic acid, causes clever hülle cell longitudinal splitting to accelerate, and then causes that seed is elongated, it is large to become.By the not homoallelic hereditary effect analysis of qGL3 is found; qGL3 is 0.33～1.17mm to the additive effect of grain length; 19.20%～70.70% of soluble phenotypic variation; be 2.49～5.43g to the additive effect of thousand seed weight; 23.09%～58.50%(Zhang X J of soluble phenotypic variation, et al., Proc Natl Acad Sci USA; 2012,109 (52): 21534-21539; Hu Z et al., Journal of Integrative Plant Biology, 2012,54 (12): 979-990).As can be seen here, as the larger new gene of grain length effect in paddy rice, qGL3 is with a wide range of applications in yield of brown rice and quality breeding.
The order-checking research of gene shows; the distribution of the grain length synergy genotype of qGL3 in common rice is very rare; only exist in some special long grain germ plasm resources as N411, DT108 and CW23 etc.; lacking allelic variation is principal element (the Zhang X J that qGL3 is not yet utilized effectively in breeding; et al.; Proc Natl Acad Sci USA, 2012,109 (52): 21534-21539; Hu Z et al., Journal of Integrative Plant Biology, 2012,54 (12): 979-990).Meanwhile, due to the allelic hereditary effect of qGL3 grain length synergy and GS3 gene very similar, be difficult to by grain measurement of length effectively being distinguished to this two kinds of different genes.Therefore obtain and can identify that the molecule marker of qGL3 different genotype utilizes significant to qualification and the breeding of long grain Rice Germplasm Resources.But, the linked marker that can be used for qGL3 genotype screening of having reported all comes from Xian, the Jing Jiao target group that hereditary difference is larger, in breeding selection, not only lack polymorphism, and difficulty has ensured accuracy (the Zhang X J to offspring's individual plant genotype identification, et al., Proc Natl Acad Sci USA, 2012,109 (52): 21534-21539).Therefore, set up a kind of molecule marking method of PCR-based technology and distinguish fast and accurately the allelotrope that qGL3 is different, will contribute to length, the screening of Large grain rice germplasm new resources, qualification and breeding utilization.
Three, summary of the invention
Technical problem: the present invention is directed to that existing linked marker is difficult to fast, the situation of the different allelotypes of precise Identification grain length gene qGL3, in qGL3 exon10, there is the mononucleotide base difference of C to A according to length, large grain kind and short, granule kind, design gene specific PCR primer pair paddy DNA increases, develop the color and identify fast and accurately the not isoallele of qGL3 by agarose gel electrophoresis and DuRed nucleic acid, to improve long grain homozygous genotype qGL3-
tqGL3-
tefficiency of selection, the screening, qualification and the breed breeding that accelerate a long grain high-quality, high-yield rice resource.
Technical scheme:
The present invention's " a kind of PCR molecule marking method of identifying paddy rice grain length gene qGL3 allelic variation ", is characterized in that: utilize qGL3 gene-specific primer
Forward outer primer qGL3-O-F sequence: 5'-GGCCACTCATGCACCATAACTACACTTG-3'
Oppositely outer primer qGL3-O-R sequence: 5'-CAGGTTTTCTTACCTCCTCGTAGACCTCCA-3'
Forward inner primer qGL3-I-F sequence: 5'-TGCACGATTCTATCTGGTTCAGTGCTAAAC-3'
Oppositely inner primer qGL3-I-R sequence: 5'-TGTCGCACCAGACTCCAGCAGCTATT-3'
The PCR molecule marking method of fast, accurately distinguishing the different allelic variations of paddy rice grain length gene qGL3 is:
(1) extraction of rice plant genomic dna (SDS method), with reference to Dellaporta S L, et al., Plant Mol Biol Rep, 1983,1 (1): 19221;
(2) above-mentioned specific molecular marker primer qGL3-O-F, qGL3-O-R, qGL3-I-F and qGL3-I-R are added to same PCR reaction system, and the DNA of Rice Germplasm Resources or breeding population plant is increased; 20 μ L PCR reaction systems comprise: the DNA2.0 μ L of 20ng/ μ L, and the primer 2 .0 μ L of 4nmol/L, the wherein each 0.5 μ L of primer qGL3-O-F, qGL3-O-R, qGL3-I-F and qGL3-I-R, containing 25mmol/L MgCl
210 × PCR Buffer2.0 μ L, the dNTP0.4 μ L of 2.5mmol/L, Taq0.5 μ L and the ddH of 2U/ μ L
2o13.1 μ L;
PCR response procedures comprises: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 1min, circulate 33 times; Then 72 DEG C are extended after 10min, and amplified production is added to sample-loading buffer termination reaction.
(3) 100V electrophoresis 50min on the sepharose that reaction product is 2.0% in quality than concentration, observes through DuRed nucleic acid staining and under gel imaging system, records.If contain two feature bands of 448bp and 288bp simultaneously, for containing grain length genotype qGL3-
tqGL3-
thomozygote; If contain two feature bands of 448bp and 216bp simultaneously, for containing grain length genotype qGL3-
kqGL3-
khomozygote, if there are three feature bands of 448bp, 288bp and 216bp simultaneously, for containing grain length genotype qGL3-
tqGL3-
kheterozygote.
Beneficial effect
" a kind of PCR molecule marking method of identifying paddy rice grain length gene qGL3 allelic variation " provided by the invention, has the following advantages:
(1) molecule marker provided by the invention is according to the functional mark of the allelic difference design of qGL3, its nucleotide variation can directly reflect the phenotype of plant, do not exist the heredity exchange between mark and gene to cause genotypic wrong qualification, efficiently solve the drawback that in Molecular Detection process, mark polymorphism is poor, accuracy is not high;
(2) molecule marking method provided by the invention can be realized the Rapid identification to qGL3 different genotype rice germplasm by simple pcr amplification.Because paddy rice grain length proterties is the quantitative character by controlled by multiple genes, and hereditary effect and the GS3 of qGL3 gene are closely similar, only be difficult to determine in paddy rice whether there is its target gene type according to its grain length phenotype, can overcome this defect by molecule marking method of the present invention;
(3) molecule marking method provided by the invention can be effective to long grain high-quality, high-yield rice varietal labeling assistant breeding.Because traditional breeding method mainly relies on visual inspection and linear measure to the selection of grain length proterties, such selection is not only subject to the impact in vine growth and development stage, and consuming time, consumption power.Utilize molecule marking method of the present invention, just can be by the specific band of primer amplification in seedling stage, judge the allelotrope difference that qGL3 is different, predict the phenotype of Grain Length in Rice with this, this has improved the efficiency of selection of rice varieties to a great extent, shorten breeding cycle and reduced breeding cost, having accelerated the improvement process of paddy rice exterior quality and output.
Four, brief description of the drawings
Fig. 1 is long, grain strain TD70 and comparison short, granule kind Kasalath seed length greatly
(A: containing qGL3-
tqGL3-
thomozygous genotype is long, Large grain rice strain-TD70; B: containing qGL3-
kqGL--
khomozygous genotype is short, granule rice varieties-Kasalath)
Fig. 2 utilizes TD70/Kasalath recombinant inbred lines location grain length QTL-qGL3
Fig. 3 is for detection of the PCR molecule marker layout strategy of grain length gene qGL3 allelic variation
(note: in sequence comparison diagram, square frame represents the base difference site of qGL3 gene growing, in large grain strain TD70 and short, granule kind Kasalath; Arrow represents primer location and length, and qGL3-O-F represents forward outer primer, and qGL3-O-R represents reverse outer primer, and qGL3-I-F represents forward inner primer, and qGL3-I-R represents reverse inner primer)
Fig. 4 specific PCR molecule marker is to the allelic detection of different rice varieties qGL3
(M:DNA molecular weight standard, 100-2,000bp; 1:TD70; 2:Kasalath; 3～17: military round-grained rice 13, fine, the Suyunuo of Japan, osmanthus are extensive 527 towards No. 2, Long Tefu, another name for Sichuan Province towards glutinous, southern round-grained rice 44, military fortune round-grained rice 23,9311, bright extensive 63, precious Shan 97, osmanthus, another name for Sichuan Province is extensive 158, China accounts for and Nanjing 11)
Fig. 5 specific PCR molecule marker is to the allelic detection of the different strain qGL3 of TD70/Kasalath recombinant inbred lines (M:DNA molecular weight standard, 100-2,000bp; 1:TD70; 2:Kasalath; 3:TD70/KasalathF
1; 4～7: part RIL plant)
Fig. 6 molecule marking method of the present invention is for the improved, process of rice varieties Kasalath grain length proterties
Fig. 7 Kasalath and Kasalath-qGL3
-Timprovement is the comparison of grain length proterties
(A: containing qGL3
-KqGL3
-Khomozygous genotype is short, granule paddy rice Kasalath; B: containing qGL3
-TqGL3
-Thomozygous genotype is long, Large grain rice Kasalath-qGL3
-T)
Five, embodiment
By more than 3000 parts of Rice Germplasm Resources of agricultural germ plasm resource storehouse in mid-term, Jiangsu Province cryopreservation are carried out to a qualification for type proterties, analysis, therefrom screen 1 part of length, large japonica rice that derives from Swan Valley ///9520//(72-496/ is imperial glutinous) filial generation and stablize strain TD70.Utilize TD70 and short, granule India rice variety Kasalath hybridization, build the recombinant inbred lines containing 240 strains through single seed descent, by drafting and the 2010-2011 QTL positioning analysis of continuous 2 years of Molecular Markers Linkage Map, on rice chromosome, navigate to multiple QTLs relevant to grain type, wherein on the 3rd karyomit(e), navigate to 1 grain length QTL-qGL3-2.By the sequencing analysis to TD70 and Kasalath DNA, determine that qGL3-2 is exactly the qGL3 gene of having reported, one is positioned at the new main effect QTL (Fig. 2) of the long-armed control grain length of paddy rice the 3rd karyomit(e) proterties.Compared with short, granule rice varieties Kasalath, there is the variation of a single base C-A at 1092 Nucleotide places of this gene exon10 in long, large grain strain TD70, cause the 364th amino acid to replace with L-glutamic acid by aspartic acid, cause clever hülle cell longitudinal splitting to accelerate, and then cause that seed is elongated, become large phenotype.Design specific PCR molecule marker according to this mononucleotide difference, utilized the method can effectively identify the not isoallele of grain length gene qGL3 in Rice Germplasm Resources or breeding population, improved grain length synergy genotype qGL3
-TqGL3
-Tefficiency of selection, shorten breeding cycle, reduce breeding cost, accelerate screening, qualification and the breed breeding of long grain high-quality, high-yield rice resource.
For fully openly the present invention's " a kind of PCR molecule marking method of identifying paddy rice grain length gene qGL3 allelic variation ", be illustrated below in conjunction with method validation and embodiment.Its concrete implementation step is as follows:
(1) test materials
Derive from Swan Valley ///9520//(72-496/ is imperial glutinous) filial generation, long, large grain japonica rice strain TD70(grain length 13.70 ± 0.10mm, thousand seed weight 80.75 ± 2.35g); Short, granule India rice variety Kasalath, grain length 8.09 ± 0.06mm, thousand seed weight 20.25 ± 0.12g) and derive from TD70/Kasalath240 strain (F
8the recombinant inbred lines forming from generation to generation).
6 conventional japonica rice kinds: Wujin, military round-grained rice 13(changzhou rice wheat breeding station), Japan fine (agriculture examination hall, Aichi, Japan), Suyunuo (TAI HU AREA, Jiangsu farm variety), osmanthus is towards glutinous (Agricultural College Affiliated to Yangzhou Univ.), Nan Jing 44(Cereal Crops Research Inst., Jiangsu Agricultural Science Academy) and military Wujin, the round-grained rice 23(changzhou rice research institute that transports);
9 indica conventional rice materials: raise rice No. 6 (Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.), bright extensive 63(Sanming, Fujian Province Institute of agricultural sciences), precious Shan 97(Wenzhou District of Zhejiang Province Institute of agricultural sciences), osmanthus is towards No. 2 (Guangxi Academy of Agricultural Sciences), Long Tefu (Zhangzhou, Fujian Institute of agricultural sciences), Shu Hui 527(Inst. of Paddy Rice, Sichuan Agriculture Univ.), Shu Hui 158(Inst. of Paddy Rice, Sichuan Agriculture Univ.), China accounts for (rice in China institute) and Nanjing 11(Cereal Crops Research Inst., Jiangsu Agricultural Science Academy).
Above material is public material, and agricultural germ plasm resource storehouse in mid-term, Jiangsu Province can provide free.Concrete reference is: Zhang Yadong etc., rice in China science, 2013,27 (2): 122-128; Money Jin Ao etc., Jiangsu agricultural sciences, 2005,4:305; Jiang Yunhong etc., Shandong agricultural sciences, 1981,2:28-29; Wu Jinglun etc., Jiangsu agricultural sciences, 1992,3:6-8; Wang Feihua, Yangzhou University's Master's thesis, 2009,23; Wang Cailin etc., Chinese rice, 2007,13 (2): 33-34; Wang Zhiming etc., China seed industry, 2011,5:75; Wear positive element etc., Jiangsu agricultural sciences, 1997,4:13-14; Wu Fangxi etc., Fujian Journal of Agricultural Sciench, 2011,26 (6): 1101-1112; Chen Jianming etc., seed, 2002,3:50-51; Lin Shicheng, Min Shaokai, rice in China kind and pedigree thereof. Shanghai: the .1991:312 of Shanghai science tech publishing house; Height is bright etc., hybrid rice, 2001,16 (2): 5-6; Wang Yu equality, hybrid rice, 2004,19 (4): 12-14; Wu Xianjun etc., China seed industry, 2007,10:70; Yu Shengmiao etc., hybrid rice, 2009,24 (6): 42-44; Lin Shicheng, Min Shaokai, rice in China kind and pedigree thereof. Shanghai: the .1991:318 of Shanghai science tech publishing house.
(2) exploitation of grain length gene qGL3 molecule marker
(1) screening of length, Large grain rice germ plasm resource
By more than 3000 parts of Rice Germplasm Resources of agricultural germ plasm resource storehouse in mid-term, Jiangsu Province cryopreservation are carried out to a qualification for type proterties, analysis, therefrom screen 1 part and derive from Swan Valley ///9520//(72-496/ is imperial glutinous) filial generation, long, large grain japonica rice is stablized strain TD70.By grain type phenotype test for years, TD70 grain length 13.70 ± 0.10mm, thousand seed weight 80.75 ± 2.35g, short, granule India rice variety Kasalath grain length 8.09 ± 0.06mm, thousand seed weight 20.25 ± 0.12g, both difference highly significants (Fig. 1).
(2) the QTL Position Research of TD70/Kasalath RIL grain length gene
Utilize long, Large grain rice strain TD70 and short, granule rice variety Kasalath hybridization, build the recombinant inbred lines containing 240 strains through single seed descent, utilize 141 Molecular Markers Linkage Maps that exist the microsatellite marker of polymorphism difference to draw Liao Gai colony between parent, taking grain length measured value as former data, utilize QTL IciMapping3.1 software, using LOD value 2.5 as threshold value, adopt complete Interval Mapping to scan in full genome range, detect grain length proterties QTL site, on paddy rice the 3rd karyomit(e) is long-armed, navigate to 1 main effect grain length QTL-qGL3-2.Analyze the qGL3-2 gene order of TD70 and Kasalath, find to exist between them many places base difference.Wherein TD70 exists C to A mononucleotide nucleotide variation at 1092 Nucleotide places of this gene exon10, cause the 364th amino acids to replace with L-glutamic acid by aspartic acid, cause the speed of clever hülle cell longitudinal splitting to be accelerated, and then cause that seed is elongated, become large phenotype, therefore determine that qGL3-2 is exactly the grain length gene qGL3 having reported.
(3) design of primers of mark
There is the single nucleotide variation of C to A according to TD70 and Kasalath at 1092 Nucleotide places of qGL3 site exon10, download the qGL3 genome BAC cloned sequence (sequence number is: AK288069) of having announced from NCBI website, designed specific PCR molecule marker (Fig. 3), its primer sequence is:
Forward outer primer qGL3-O-F sequence: 5'-GGCCACTCATGCACCATAACTACACTTG-3'
Oppositely outer primer qGL3-O-R sequence: 5'-CAGGTTTTCTTACCTCCTCGTAGACCTCCA-3'
Forward inner primer qGL3-I-F sequence: 5'-TGCACGATTCTATCTGGTTCAGTGCTAAAC-3'
Oppositely inner primer qGL3-I-R sequence: 5'-TGTCGCACCAGACTCCAGCAGCTATT-3'
(3) molecular marker analysis
(1) extraction of rice varieties genomic dna
The extraction (SDS method) of rice varieties genomic dna, with reference to Dellaporta S L, et al., Plant Mol Biol Rep, 1983,1 (1): 19221.Concrete steps are: get rice seedling blade, in the mortar of-20 DEG C of precoolings by liquid nitrogen grinding and pack 1.5mL centrifuge tube into; Add 600uL extracting solution (20%SDS, 1M Tris-HCl, 0.5M EDTA, 5M NaCl, 65 DEG C of preheatings), shake up, 65 DEG C of temperature are bathed 30min, middle vibration 3～4 times; Add 1/4 volume 5MKAC, shake up postposition 30min on ice; Add chloroform-primary isoamyl alcohol (24:1) 300～400uL, fully vibration on shaking table, 120rpm, 30min; Centrifugal 15 minutes of 8000～10000rpm, liquid level layering, lower floor's color is darker, and the micro-band yellow-green colour in upper strata is got supernatant (400uL left and right) to another centrifuge tube; Add equal-volume chloroform-primary isoamyl alcohol (24:1), fully vibration on shaking table, 80～90rpm, 30min; Centrifugal 15 minutes of 8000rpm, shifts supernatant (400uL left and right) to new centrifuge tube; Add the dehydrated alcohol of 2 times of volume-20 DEG C precoolings, shake up gently until have floss generation, the centrifugal 6min of 12000rpm; Abandon dehydrated alcohol, add 4 DEG C of 70% ethanol, place 10min, abandon supernatant, air-dry 1h on Bechtop; Add 100～200uLTE ,-20 DEG C of preservations.
(2) amplification of molecule marker and electrophoresis detection
QGL3 gene specific molecular marker primer sequence is:
Forward outer primer qGL3-O-F sequence: 5'-GGCCACTCATGCACCATAACTACACTTG-3'
Oppositely outer primer qGL3-O-R sequence: 5'-CAGGTTTTCTTACCTCCTCGTAGACCTCCA-3'
Forward inner primer qGL3-I-F sequence: 5'-TGCACGATTCTATCTGGTTCAGTGCTAAAC-3'
Oppositely inner primer qGL3-I-R sequence: 5'-TGTCGCACCAGACTCCAGCAGCTATT-3'
Described molecule marker primer qGL3-O-F, qGL3-O-R, qGL3-I-F and qGL3-I-R are added to same PCR reaction system, the DNA of different rice varieties and the each strain of TD70/Kasalath recombinant inbred lines is carried out to pcr amplification;
20 μ L PCR reaction systems comprise: the DNA2.0 μ L of 20ng/ μ L, and the primer 2 .0 μ L of 4nmol/L, the wherein each 0.5 μ L of primer qGL3-O-F, qGL3-O-R, qGL3-I-F and qGL3-I-R, containing 25mmol/L MgCl
210 × PCRBuffer2.0 μ L, the dNTP0.4 μ L of 2.5mmol/L, Taq0.5 μ L and the ddH of 2U/ μ L
2o13.1 μ L;
PCR response procedures comprises: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 1min, circulate 33 times; Then 72 DEG C are extended after 10min, and amplified production is added to sample-loading buffer termination reaction; 100V electrophoresis 50min on the sepharose that reaction product is 2.0% in quality than concentration, observes through DuRed nucleic acid staining and under gel imaging system, records.
(4) specific PCR molecule marker of the present invention is to the allelic detection of different rice varieties qGL3
Taking length, large grain strain TD70 and short, granule kind Kasalath as contrast,, Suyunuo fine to the military round-grained rice 13 of 6 japonica rice varieties, Japan, osmanthus are extensive 527 towards No. 2, Long Tefu, another name for Sichuan Province towards glutinous, southern round-grained rice 44,23 and 9 rice varieties 9311 of military fortune round-grained rice, bright extensive 63, precious Shan 97, osmanthus, another name for Sichuan Province is extensive 158, China accounts for, the DNA in Nanjing 11 carries out the genotypic detection of qGL3.Pcr amplification product can show the band of two types through 2.0% agarose gel electrophoresis.Wherein all occurred in each DNA sample by the band of the 448bp of forward and reverse outer primer qGL3-O-F and qGL3-O-R amplification, it not only plays positive control (can detect DNA effectively be increased), also can effectively reduce the amplification of non-specific PCR product and the formation of primer dimer simultaneously.Except the total band of 448bp, only there is long, large grain strain TD70 can amplify the feature band of a treaty 288bp, this band is by forward outer primer qGL3-O-F and oppositely inner primer qGL3-I-R amplification generation, that its increases is loci (i.e. long, large grain strain TD70 genotype, the qGL3 that exon10 1092 Nucleotide in qGL3 site are A
-TqGL3
-T); And other all Xian, japonica rice variety all can only amplify the feature band of the treaty 216bp except 448bp, this band is by forward inner primer qGL3-I-F and oppositely outer primer qGL3-O-R amplification generation, that its increases is loci (i.e. short, granule kind Kasalath genotype, the qGL3 that exon10 1092 Nucleotide in qGL3 site are C
-KqGL3
-K) (Fig. 4).This has also confirmed the grain length synergy gene qGL3 of qGL3
-TqGL3
-Tdistribution in common rice is really very rare, exists only in the special long grain germ plasm resource of only a few.By to thering is qGL3
-KqGL3
-K15 Xian of homologous genes type, japonica rice variety carry out the analysis of grain length proterties, and result shows that the reason that causes these kinds to have grain length difference is only that they have different GS3 allelotypes (table 1).
Table 1 Xian, japonica rice variety GS3 and qGL3 different genotype and grain length phenotypic number
(5) specific PCR molecule marker of the present invention is to the different detections of allelotype of TD70/Kasalath recombinant inbred lines qGL3 and the comparison of grain length phenotypic number
240 strains that derive from TD70 and Kasalath recombinant inbred lines are carried out to allelotype detection with the exceptional function mark of qGL3 gene.Result shows only have in 131 strains of grain length gene qGL3 allelomorphism difference the strain (qGL3 identical with TD70 banding pattern
-TqGL3
-T) there are 17, the strain (qGL3 identical with Kasalath banding pattern
-KqGL3
-K) there are 114 (Fig. 5).By the analysis of two kinds of Genotype Strains grain length variation situations is found, containing qGL3
-TqGL3
-Tgenotype Strains grain length mean value is 10.19mm, containing qGL3
-KqGL3
-Kgenotype Strains grain length mean value is 8.72mm, and both exist utmost point significant difference (table 2).In this explanation qGL3 gene pairs TD70/Kasalath recombinant inbred lines, the grain length of different strains has considerable influence, is the important main effect site of grain length proterties.
Table 2 has the comparison of two kinds of different genotype strain grain length mean values of grain length gene qGL3
(6) analysis of GS3 and qGL3 gene genetic effect in TD70/Kasalath recombinant inbred lines
240 strains of RIL can be divided into four kinds of genotype combination: GS3 according to the banding pattern of qGL3 and GS3 gene specific PCR molecule marker
-TqGL3
-T, qGL3
-KgS3
-T, qGL3
-TgS3
-Kand qGL3
-KgS3
-K.By finding the comparative analysis of different genotype combination strain grain length mean value, containing qGL3
-TgS3
-Tthe grain length mean value of Genotype Strains is 11.10mm, and the utmost point is significantly higher than other three kinds of genotype combinations; Containing qGL3
-Kand GS3
-Tthe grain length mean value of Genotype Strains is 9.98mm, a little less than containing qGL3
-TgS3
-Kthe grain length mean value of Genotype Strains is 10.19mm, but there is no significant difference between the two; And containing qGL3
-TgS3
-Tthe grain length mean value of Genotype Strains is minimum is 8.72mm, and their the large I of the hereditary effect to grain length is expressed as qGL3
-TgS3
-T>qGL3
-KgS3
-T≌ qGL3
-TgS3
-K>qGL3
-KgS3
-K(table 3).As can be seen from the above analysis, single grain length synergy gene qGL3
-Tand GS3
-Twhile independently existence, their effect does not have significant difference, and the in the situation that of a synergy genetically deficient, another synergy gene just shows the hereditary effect of self; GS3
-Tand qGL3
-Twhen two synergy genes exist simultaneously, although the phenotypic number maximum of grain length does not show the additive effect of gene completely; And in the time that two synergy genes do not exist, grain length phenotypic number minimum.As can be seen here, the mechanism of action of qGL3 and GS3 gene pairs grain length is also incomplete same, and qGL3 gene has the value of equal importance with GS3 in breeding.Moreover, by the polymerization of qGL3 and GS3 synergy gene, can also further improve length and the thousand seed weight of rice grain, and then promote the quality and yield of rice varieties.
The comparison of tetra-kinds of genotype combination strain grain length mean values of table 3qGL3 and GS3
(7) molecule marking method of the present invention is for the improvement of rice varieties Kasalath grain length proterties
Selecting long, large grain strain TD70 is grain length gene qGL3
-Tdonor, short, granule kind Kasalath is acceptor, adopts the strategy of back cross breeding, the grain length proterties of orderly improvement Kasalath.In the process of continuous backcross and selfing, utilize qGL3 of the present invention
-Tthe specific PCR molecule marker of gene is followed the tracks of detection, and selects in conjunction with the system of economical character, to BC
3f
3it is qGL3 that generation obtains grain length genotype
-TqGL3
-T, grain length improvement strain-Kasalath-qGL3 that other economical character and original Kasalath are basically identical
-T, its concrete Breeding Process refers to Fig. 6.By to Kasalath-qGL3
-Tfind with the grain length of original kind Kasalath and the comparative analysis of thousand seed weight, containing qGL3
-TqGL3
-Tthe improvement of homozygous genotype is that grain length is 10.07 ± 0.08mm, and thousand seed weight is 25.5 ± 0.09g, containing qGL3
-KqGL3
-Kthe Kasalath grain length of homozygous genotype is 8.09 ± 0.06mm, and thousand seed weight 20.25 ± 0.12g exists utmost point significant difference (Fig. 7) between the two.This shows that molecule marking method of the present invention can select the genotype of grain length gene qGL3, and then realizes grain length proterties is improved fast and accurately.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
Mono-kind of <120> identifies the PCR molecule marking method of paddy rice grain length gene qGL3 allelic variation
<130> 0
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 28
<212> DNA
<213> 1
<220>
<221> qGL3-O-F
<222> (1)..(28)
<223>
<400> 1
ggccactcat gcaccataac tacacttg 28
<210> 2
<211> 30
<212> DNA
<213> is artificial
<220>
<221> qGL3-O-R
<222> (1)..(30)
<223>
<400> 2
caggttttct tacctcctcg tagacctcca 30
<210> 3
<211> 30
<212> DNA
<213> is artificial
<220>
<221> qGL3- I -F
<222> (1)..(30)
<223>
<400> 3
tgcacgattc tatctggttc agtgctaaac 30
<210> 4
<211> 26
<212> DNA
<213>
<220>
<221> qGL3- I -R
<222> (1)..(26)
<223>
<400> 4
tgtcgcacca gactccagca gctatt 26
Claims (2)
1. a qualification paddy rice grain length gene
qGL3the PCR molecule marking method of allelic variation, is characterized in that:
With
qGL3gene-specific primer
Forward outer primer
qGL3-o-F sequence: 5'-GGCCACTCATGCACCATAACTACACTTG-3'
Oppositely outer primer
qGL3-o-R sequence: 5'-CAGGTTTTCTTACCTCCTCGTAGACCTCCA-3'
Forward inner primer
qGL3-i-F sequence: 5'-TGCACGATTCTATCTGGTTCAGTGCTAAAC-3'
Oppositely inner primer
qGL3-i-R sequence: 5'-TGTCGCACCAGACTCCAGCAGCTATT-3'
The genomic dna of amplifying rice kind, if contain 448 bp and two feature bands of 288 bp simultaneously, for containing grain length genotype
qGL3- t qGL3- t homozygote; If contain 448 bp and two feature bands of 216 bp simultaneously, for containing grain length genotype
qGL3- k qGL3- k homozygote, if there are three feature bands of 448 bp, 288 bp and 216 bp simultaneously, for containing grain length genotype
qGL3- t qGL3- k heterozygote.
2. method according to claim 1, is characterized in that:
(1) extraction of rice plant genomic dna;
(2) by molecule marker primer claimed in claim 1
qGL3-O-F,
qGL3-O-R,
qGL3-I-F and
qGL3-I-R adds same PCR reaction system, and the DNA of Rice Germplasm Resources or breeding population plant is increased; 20 μ L PCR reaction systems comprise: the DNA 2.0 μ L of 20 ng/ μ L, the primer 2 .0 μ L of 4 nmol/ L, wherein primer
qGL3-O-F,
qGL3-O-R,
qGL3-I-F and
qGL3the each 0.5 μ L of-I-R, containing 25 mmol/L MgCl
210 × PCR Buffer, 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 2 U/ μ L's
taq0.5 μ L and ddH
2o 13.1 μ L;
PCR response procedures comprises: 94 DEG C of denaturation 5 min; Then 94 DEG C of sex change 30s, 55 DEG C of renaturation 30s, 72 DEG C of extension 1min, circulate 33 times; Then 72 DEG C are extended after 10 min, and amplified production is added to sample-loading buffer termination reaction;
(3) 100V electrophoresis 50 min on the sepharose that reaction product is 2.0% in quality than concentration, observe through DuRed nucleic acid staining and under gel imaging system, record.
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| CN106755434A (en) * | 2016-12-28 | 2017-05-31 | 海南波莲水稻基因科技有限公司 | A kind of molecular labeling of paddy rice grain length gene qGL3 and its application |
| CN108239673A (en) * | 2016-12-26 | 2018-07-03 | 中国科学院植物研究所 | A kind of method for cultivating high-yield crop and its special specific gene pack section and special primer pair |
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