CN106119376B - The molecule labelling method in rice chalkiness degree main effect QTL site and application - Google Patents

The molecule labelling method in rice chalkiness degree main effect QTL site and application Download PDF

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CN106119376B
CN106119376B CN201610526723.4A CN201610526723A CN106119376B CN 106119376 B CN106119376 B CN 106119376B CN 201610526723 A CN201610526723 A CN 201610526723A CN 106119376 B CN106119376 B CN 106119376B
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rice
main effect
effect qtl
chalk
chalkiness degree
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CN106119376A (en
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雷东阳
旷浩源
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Hunan Agricultural University
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Abstract

A kind of molecule labelling method in rice chalkiness degree main effect QTL site, this method is to utilize a pair or two pairs of primers in the primer pair of molecular labeling RM20660 or the primer pair of molecular labeling RM7412, PCR amplification is carried out to the DNA of rice material to be identified, amplified production carries out electrophoresis detection, the DNA fragmentation of corresponding size is such as amplified, then indicates that low chalkiness degree main effect QTL qCD6 exists.This method passes through 2 SSR markers with the rice whiteness measuring main effect QTL qCD6 close linkage navigated on low the 6th chromosome of chalk plain boiled water rice varieties Lemont that application screens, predict the chalkiness degree of rice material, the breeding for being conducive to the high-quality white new rice variety of low chalk accelerates the selection progress of high-quality low chalk plain boiled water rice varieties.

Description

The molecule labelling method in rice chalkiness degree main effect QTL site and application
Technical field
The invention belongs to field of biotechnology, and in particular to point in rice important quality character chalkiness degree main effect QTL site Sub- labeling method also relates to application of the molecule labelling method in the white high-grade rice kind of the low chalk of breeding.
Background technique
Under huge population and arable land pressure, China always pay attention to the improvement in rice yield traits, and in grain quality rice Research and application aspect more lag.As the improvement of people's living standards, to rice quality, more stringent requirements are proposed；This Outside, great change is just occurring for China's mode of agriculture, and large-scale planting is the inexorable trend of the following Rice Production development, The mainly commodity food of large-scale planting family kind, it is emphasised that high benefit, since the price of grain quality rice is higher by about than common rice 20-30%, under the premise of not sacrificing yield, every kind of one mu of grain quality rice can be about 100 yuan of synergy, therefore, scale Rice Production The demand to high-grade rice kind such as peasant household and farmer is more urgent.
It is the most key character for influencing rice quality that chalk is white, chalk rice seriously affect Appearance Quality of Paddy Rice, processing quality, Cooking Quality and nutritional quality, and it is directly related to the commodity and market value of rice.Research due to China to grain quality rice With pay attention to not enough, the rice varieties rice matter common manifestation of cultivation is not good enough, state examine rice variety in, chalkiness degree index it is high-quality Rate only has 40% or so, is the index that compliance rate is minimum in rice quality correlated traits.Although Chinese hybrid rice yield has reached To higher level, but rice quality it is particularly excellent kind it is seldom, therefore, how quickly, accurately the white top grade of the low chalk of breeding is excellent Matter rice varieties are the main targets that new period breeder pursues.
Summary of the invention
The technical problems to be solved by the present invention are: in view of the above shortcomings of the prior art, providing a kind of rice chalk The molecule labelling method in whiteness main effect QTL site, while this method answering in the white high-grade rice kind of the low chalk of breeding being also provided With.
In order to solve the above-mentioned technical problem, the technical scheme adopted by the invention is that: a kind of rice chalkiness degree main effect The molecule labelling method of QTL site, which is characterized in that this method is the primer pair and molecule mark using molecular labeling RM20660 Remember a pair or two pairs of primers in the primer pair of RM7412, PCR amplification, amplified production are carried out to the DNA of rice material to be identified Electrophoresis detection is carried out, the DNA fragmentation of corresponding size is such as amplified, then indicates that rice chalkiness main effect QTL qCD6 exists；
Wherein, the primer pair sequence of above-mentioned molecular labeling RM20660 are as follows: upstream primer 5 '- CGCTAGAGCTAGCTATGGAGATCG-3 ' (SEQ ID No.1), 5 '-CCCTCCTCTACAGTTTCCACTCC- of downstream primer 3 ' (SEQ ID No.1), after carrying out PCR amplification to DNA, electrophoresis can detect the item with rice varieties Lemont same position Band, amplified production size are 140-160bp；
The primer pair sequence of above-mentioned molecular labeling RM7412 are as follows: upstream primer 5 '-TCAGCTCAGCTCAGCATCAG-3 ' (SEQ ID No.3), downstream primer 5 '-ACTCATCAATCGTGTGCTGC-3 ' (SEQ ID No.4) carries out PCR expansion to DNA After increasing, electrophoresis can detect the band with rice varieties Lemont same position, and amplified production size is 135-145bp.
Reaction system when above-mentioned PCR amplification are as follows: 2 μm of 1.0 μ L of ol/L primer, 0.2 μ L of 2.5mmol/L dNTP, DNA mould 1.2 μ L of plate, 0.1 μ L of 5U/ μ L Taq archaeal dna polymerase, contain Mg2+10 × PCR buffer 1.0 μ L, ddH2O complements to 10 μ L. PCR reaction condition are as follows: 94 DEG C of denaturation 4min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s expand 35 circulations, and last 72 DEG C of ends prolong Stretch 5min.
Present invention simultaneously provides the molecule labelling methods in the rice chalkiness degree main effect QTL site in the low chalk of breeding Application in white high-grade rice kind.
The present invention uses the molecule labelling method of QTL site, be using screen in low chalk plain boiled water rice varieties 2 SSR markers of the rice whiteness measuring main effect QTL qCD6 close linkage navigated on the 6th chromosome of Lemont predict rice The chalkiness degree of material accelerates the selection progress of high-quality low chalk plain boiled water rice varieties.
Detailed description of the invention
Fig. 1 is positioning of rice varieties of the embodiment of the present invention Lemont rice chalkiness main effect QTL qCD6 in the 6th chromosome, Rice chalkiness main effect QTL qCD6 is positioned at the region 153kb between RM20660 and RM7412.
Fig. 2 is SSR primer RM20660 in the embodiment of the present invention in parent and Lemont/ Milyang 46 F6It expands and produces in group Raw electrophoretogram.
Wherein: M is DNA marker label, and swimming lane 1 is low chalk plain boiled water rice varieties Lemont, and swimming lane 2 is high chalk plain boiled water rice Kind Milyang 46, swimming lane 3-24 are Lemont/ Milyang 46 F6Group's difference strain.
Fig. 3 is SSR primer RM7412 in the embodiment of the present invention in parent's (Lemont and Milyang 46) and the close sun of Lemont/ 46F6The electrophoretogram of generation is expanded in group.
Wherein: M is DNA marker label, and swimming lane 1 is low chalk plain boiled water rice varieties Lemont, and swimming lane 2 is high chalk plain boiled water rice Kind Milyang 46, swimming lane 3-24 are Lemont/ Milyang 46 F6Group's difference strain.
Specific embodiment
With reference to embodiment, the embodiment of the present invention is furthur described in detail.Following embodiment is only used In illustrating the present invention, but it is not intended to limit the scope of the invention.
Embodiment 1
Material to be tested: it is female parent with high-quality low chalk peitunitze rice varieties Lemont, is with the white rice variety Milyang 46 of high chalk Paternal hybrid constructs the Milyang 46 RILs of the Lemont containing 162 strains × 4628 F with single-seed descent6Group.
Phenotype identification
Lemont, Milyang 46 and Lemont/ Milyang 46 RILs F are planted in Agricultural University Of Hunan proving ground within 20156Group 162 strains of body, May 5, the sowing of 2 phase of 15 bu in May, two phases were separated by 10 days, and transplanted respectively at May 30, June 4, single This plant, each material respectively plant 30 plants, and each strain plants 3 rows, 10 plants of every row, and seeding row spacing is 20cm × 20cm, and record begins respectively Fringe date and the heading stage of selection consistent 148 recombinant inbred lines and parent Lemont and Milyang 46 are analyzed for rice chalkiness. When mature, every strain randomly chooses 10 single fringe threshings, and the measurement of rice chalkiness is carried out after spontaneously drying 3 months at room temperature.
Referring to The Ministry of Agriculture of the People's Republic of China, MOA's standard meter matter measuring method (NY/T 593-2013), in the white scanner of chalk Upper scanning polished rice, 100 polished rice of grab sample are recorded in scanner scanning as a result, each material respectively analyzes 3 samples, with 3 times The average value of measurement is for statisticalling analyze.Chalkiness degree=chalky grain rate × Chalkiness size.
The building of genetic linkage maps
It chooses and is uniformly distributed on 12 chromosomes of rice, cover the low chalk plain boiled water rice product of 528 pairs of SSR primer pairs of full-length genome Two parent materials of kind Lemont and high chalk plain boiled water rice varieties Milyang 46 carry out polymorphism screenings.Wherein 188 pairs of primers are in amphiphilic This shows polymorphic, and the ratio of polymorphism primer is 35.61%, wherein the 4th, 11, on 12 chromosomes primer polymorphism compared with It is low.
Polymorphic apparent 188 pairs of codominance SSR markers between two parents are chosen, with the Lemont containing 148 strains × close Positive 46RIL F6Group constructs the Molecular Markers Linkage Map of 12 chromosome of full-length genome.The total length of the linkage map is 1700.68CM, average distance is 9.05CM between label, and label is relatively evenly distributed on 12 chromosomes.
The positioning of rice whiteness measuring QTL
Composite interval mapping, which is carried out, by 2.5 positioning software of Windows QTL Cartographer carries out rice chalkiness Qtl analysis is spent, detects the qCD6 of main effect, LOD value 8.24, phenotypic interpretation in the 6th section chromosome RM439~RM340 Rate is 16.02%, additive effect be -7.86, additive effect reduction allele effect from the white parent Lemont of low chalk etc. Position gene.The position view of the 6th article of chromosome rice chalkiness main effect QTL qCD6 of rice is shown in Fig. 1 in the genetic map of building, Part electrophoretogram is shown in Fig. 2-Fig. 3.
For the main effect QTL of this section of finely positioning, there is polymorphic SSR between this section screens 3 two parents again Label or InDel mark RM3307, RM2744 and InDel1, to encrypt the reference numerals of this section.With Lemont × close sun 46RIL F6The chalkiness degree index of group is that phenotype index further positions qCD6, is located in molecular labeling Between RM2744 and InDel1, the distance between the two labels is 435kb.The location area section further screen 5 There are polymorphic SSR marker or InDel label RM20648, InDel3, RM7412, RM20660 between two parent of Lemont and Milyang 46 And InDel2, by the distance of rice whiteness measuring major gene resistance qCD6 finely positioning 153kb between RM7412-RM20660.
With RM20660, RM7412 etc. molecular marker assisted selection can be carried out to the main effect QTL.Wherein, in the present embodiment PCR reaction system are as follows: 2 μm of 1.0 μ L of ol/L primer, 0.2 μ L of 2.5mmol/L dNTP, 1.2 μ L of DNA profiling, 5U/ μ L Taq 0.1 μ L of archaeal dna polymerase, contain Mg2+10 × PCR buffer 1.0 μ L, ddH2O complements to 10 μ L.PCR reaction condition are as follows: 94 DEG C of changes Property 4min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, expand 35 circulations, last 72 DEG C extend 5min eventually.
The molecular labeling in 1 rice whiteness measuring main effect QTL site of table
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.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Claims (4)

1. a kind of molecule labelling method in rice chalkiness degree main effect QTL site, which is characterized in that this method is to utilize molecule The primer pair of RM20660 and a pair or two pairs of primers in the primer pair of molecular labeling RM7412 are marked, to rice material to be identified The DNA of material carries out PCR amplification, and amplified production carries out electrophoresis detection, such as amplifies the DNA fragmentation of corresponding size, then indicate rice The white main effect QTL qCD6 of rice chalk exists；
Wherein, as shown in SEQ ID No.1 and SEQ ID No.2, amplification produces the primer pair sequence of above-mentioned molecular labeling RM20660 Object size is 140-160bp；
The primer pair sequence of above-mentioned molecular labeling RM7412 is as shown in SEQ ID No.3 and SEQ ID No.4, amplified production size For 135-145bp.
2. the molecule labelling method in rice chalkiness degree main effect QTL site as described in claim 1, which is characterized in that institute State the reaction system composition of PCR: 2 μm of 1.0 μ L of ol/L primer, 0.2 μ L of 2.5mmol/L dNTP, 1.2 μ L of DNA profiling, 5U/ μ L 0.1 μ L of Taq archaeal dna polymerase, contain Mg2+10 × PCR buffer 1.0 μ L, ddH2O complements to 10 μ L.
3. the molecule labelling method in rice chalkiness degree main effect QTL site as described in claim 1, which is characterized in that institute Stating the reaction condition of PCR are as follows: 94 DEG C of denaturation 4min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s expand 35 circulations, and last 72 DEG C Extend 5min eventually.
4. the molecule labelling method in rice chalkiness degree main effect QTL site as claimed in any one of claims 1-3 is selecting Educate the application in the low white high-grade rice kind of chalk.
CN201610526723.4A 2016-07-06 2016-07-06 The molecule labelling method in rice chalkiness degree main effect QTL site and application Expired - Fee Related CN106119376B (en)

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CN113122653B (en) * 2021-05-31 2023-03-24 浙江师范大学 Main effect QTL for regulating and controlling brown rice rate of rice, molecular marker and application
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CN104789559A (en) * 2015-03-23 2015-07-22 华南农业大学 Molecular marker for identifying genotype of rice chalkiness major effect gene and application of molecular marker

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CN104789559A (en) * 2015-03-23 2015-07-22 华南农业大学 Molecular marker for identifying genotype of rice chalkiness major effect gene and application of molecular marker

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利用高代回交导入系定位稻米外观品质性状QTL;雷东阳等;《湖南农业大学学报（自然科学版）》;20090228;摘要，第1.2节，表1，第4页左栏第2段 *
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