CN106591435A - Molecular marker method for site of low-amylose-content gene of dark endosperm mutant w54 of japonica rice variety Koshihikari - Google Patents
Molecular marker method for site of low-amylose-content gene of dark endosperm mutant w54 of japonica rice variety Koshihikari Download PDFInfo
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Abstract
The invention relates to a molecular marker method for the site of a low-amylose-content gene of a dark endosperm mutant w54 of a japonica rice variety Koshihikari. The site of the low-amylose-content gene of the w54 is located beside a terminal marker W57 of the long arm of a chromosome No. 6, and the site and Wx are non-allelic. If a primer pair of an Indel marker developed in the invention, i.e., the W57 marker, can amplify a fragment with a length of 183 bp, it is proved that the site of the low-amylose-content gene of the w54 exists. As the molecular marker linked with the gene site is used for detecting whether a derived variety (line) of the dark endosperm mutant w54 of Koshihikari contains the site, selection efficiency of paddy rice with low amylose content can be improved, the process of breeding can be accelerated, and breeding cost is reduced; and the molecular marker method is of important value to cultivation of novel varieties (lines) of paddy rice with low amylose content.
Description
Technical field
The invention provides the more molecule labelling method of brightness endosperm mutant w54 gene locus with low-content of amylose,
Belong to molecular genetics field, be exclusively used in the seed selection of low amylose content paddy rice and the utilization of germ plasm resource.
Background technology
With people's quality of the life raising and rice food become more diverse, the rice quality related to food flavor and processing
Proterties is more and more widely paid close attention to.Rice quality includes milled quality, exterior quality, Cooking and eating quality and nutriment
Four main aspects such as matter, wherein particularly important with exterior quality and Cooking and eating quality, and cooking quality is mainly by sinking
Starch quality of the product in endosperm is determined.Starch generally accounts for more than the 90% of rice paddy seed endosperm dry weight, mainly by straight chain
Starch and amylopectin are constituted, and wherein amylose content is the decisive factor of rice boiling processing and edible quality.
The rice of low amylose content is the ideal material for improveing rice grain amylose content and Cooking Quality.It is to be situated between
Intermediate form between general glutinous rice and glutinous rice, its rice hardness is little, viscosity is big, rice grain is sparkling and crystal-clear, mouthfeel it is fragrant it is soft it is sweet it is glutinous, cold after
It is not hardened and does not bring back to life, with higher commodity.The paddy rice mutator of 15 low amylose contents, root are had reported at present
It is different from the equipotential sexual intercourse of cured matter gene (Wx) according to them, can be divided into and Wx equipotentials and the big class of non-equipotential two.Wherein Wx-mq is
Japanese Rice kind is processed by chemical mutagen N-methyl-N-nitrosourea (N-methyl-N-nitrosourea, MNU) to get over
Light, and the low amylose content mutant gene for obtaining.Carry the rice varieties of the genetic mutation site its Endosperm appaearances to present
Cloud, milky, the characteristic of the dark endosperm of the slightly poor grade of transparency, therefore referred to as dark endosperm ultro-microstructure.Wx-mq is widely used in training
Educate the rice varieties of low amylose content.The dark endosperm mutant w54 of the present invention gets over also with chemical mutagen MNU process
What light was obtained.W54 seeds show as dark endosperm, and amylose content is substantially less than more light, and only 6.4% or so.
The content of the invention
It is an object of the invention to provide the more molecular labeling of brightness endosperm mutant w54 gene locus with low-content of amylose
Method, the molecular labeling W57 obtained by the present invention can be used for the assisted selection of rice grain amylose content.
The purpose of the present invention can be achieved through the following technical solutions：
The molecular labeling W57 of one gene loci for getting over brightness endosperm mutant w54 low amylose contents is in low straight chain
Application in content of starch Rice molecular breeding, the primer of the molecular labeling is：SEQ ID in upstream primer P1 such as sequence table
NO：Shown in 1, SEQ ID NO in downstream primer P2 such as sequence table：Shown in 2.
A kind of primer pair for detecting described molecular labeling W57, SEQ ID NO in upstream primer P1 such as sequence table：1
It is shown, SEQ ID NO in downstream primer P2 such as sequence table：Shown in 2.
Application of the described primer pair in low amylose content Rice molecular breeding.
More the molecule labelling method of the gene locus with low-content of amylose of brightness endosperm mutant w54, uses molecular labeling
W57 primer SEQ ID NO.1/SEQ ID NO.2, for expanding dark endosperm mutant w54 derived material DNA, if can expand
Go out the fragment of 183bp, then indicate the presence of gene locus with low-content of amylose.
Application of the molecular labeling of the present invention in low amylose content rice breeding.
A kind of method for detecting molecular labeling of the present invention, using primer pair of the present invention, PCR amplifying rice bases
One section of sequence in because of group, PAGE glue is separated, and silver staining colour developing judges whether 183bp specific bands occur.
Method of the present invention, preferably includes following steps：
1) oryza sativa genomic dna is extracted with SDS methods；
2) expanded using primer pair PCR of the present invention, reaction total system is preferably 10 μ L：10mM Tris-HCl pH
8.3,50mM KCl,1.5mM MgCl2, 50 μM of dNTPs, 0.2 μM of primer, 0.5U Taq polymerase (TaKaRa, greatly
Connect) and 20ng DNA profilings.Amplified reaction is carried out in A200 (the bright base in Hangzhou) PCR instrument：94℃4min；94℃1min,55℃
1min, 72 DEG C of 1.5min, 35 circulations；72℃7min.Amplified production is separated with 8% Native PAGE glue, aobvious by silver staining
Color, silver staining program is formulated according to the method for Sanguinetti et al. (1994) and formed.The DNA bands of amplification are entered using lamp box
Row observation.
If 3) fragment of 183bp can be amplified, dark endosperm mutant w54 low amylose content genes position is indicated
The presence of point.
Beneficial effect
Early stage, applicant screens dark endosperm mutant w54 in the more light mutant library that MNU is processed, and its total starch contains
Amount is only declined slightly relative to wild type, but amylose content is significantly reduced, only the 45.1% of wild type.The present invention is utilized
The dark endosperm mutant w54 of low amylose content hybridizes the F for obtaining with high amylose content rice variety Nanjing 112Colony
Carry out Genes location analysis, it was demonstrated that w54 mutators are located near No. 6 chromosome long arm end mark W57, non-with Wx etc.
Position.Being detected by the molecular labeling chain with said gene site in more brightness endosperm mutant w54 derived varieties (being) is
It is no containing the site, the efficiency of selection of low amylose content paddy rice can be improved.More brightness endosperm mutation provided by the present invention
The molecule labelling method of body w54 gene locus with low-content of amylose, with advantages below：
(1) amylose content is the main factor for determining that rice quality is inferior.The present invention adopts molecular biology side
With the more brightness endosperm mutant w54 of low amylose content as material, development reduces the molecule of rice grain amylose content to method
Mark, for the assisted selection of fine quality rice, the improvement to China's rice quality has generality.
(2) by the paddy rice gene locus with low-content of amylose locality specific of Marker-assisted selection of the present invention, identification side
Just.By detection and the chain molecular labeling of gene locus with low-content of amylose, can be used for the base of w54 derived varieties (being)
Because of type detection.
(3) assistant breeding selection target is clearly, cost-effective.In traditional breeding way, first have to collect with low straight
The parent of chain content of starch carries out a series of hybridization with cultivar, and to carry out individual plant measure to amylose content.Cause
This low amylose content breeding is extremely time-consuming.The molecular labeling of the present invention can be used for w54 gene locus with low-content of amylose
Marker-assisted selection breeding.By detecting the gene loci, the individual plant of low amylose content can be just identified in seedling stage,
Eliminate other plant, overcome the cycle the time required to conventional breeding methods it is long the shortcomings of, not only save production cost but also significantly
Improve the efficiency of selection of low amylose paddy rice.Meanwhile, can be targetedly by the low amylose content gene in laboratory
The interior polymerization for selecting to obtain and purposefully carry out multiple Fineness genes, so as to cultivate the paddy rice new product being possessed of good qualities
Kind.
Description of the drawings
The seed phenotype of Fig. 1 wild types more light and mutant w54.A. wild type and mutant mature seed；B. wild type
With mutant brown rice；The translucency of C, D. wild type and mutant brown rice.
Fig. 2 wild types more light and mutant w54 seeds total starch and amylose content.A. total starch content；B. straight chain
Content of starch.
Finely positioning of Fig. 3 w54 mutators on No. 6 chromosome.
The electrophoresis pattern of Fig. 4 w54 gene locus with low-content of amylose compact linkage molecule mark.M:Marker；1:
w54；2:Nanjing 11；3:F2The individuality of middle heterozygosis banding pattern；4-13:F2In have w54 banding patterns individuality；14-23:F2In have south
The individuality of the banding pattern of capital 11.
Specific embodiment
The molecular labeling of embodiment 1, more brightness endosperm mutant w54 low amylose content genes
Materials and methods：
(1) genetic analysis of w54
F will be obtained after wild type more light and mutant w54 hybridization1For cenospecies, F1F is obtained for a cenospecies selfing generation2Group
Body.Mature seed sloughed and observed under lamp box after clever shell, and the number seeds for counting dark endosperm and transparent endosperm are respectively 183 Hes
595。χ2=0.829<χ2 0.05,1=3.84, card side's result shows, actual observation ratio meets 3:1 theoretical ratio, therefore be mutated
The dark endosperm phenotype of body w54 is controlled by a pair of Recessive genes.
(2) w54/ Nanjing 11F2Informative population and phenotypic evaluation
(1) to get over brightness endosperm mutant w54 as female parent, rice variety Nanjing 11 is male parent, and hybridization is constructed
W54/ Nanjing 11F2Colony, harvests F2Colony's seed.
(2)F2After colony's mature seed drying, slough clever shell and observe under lamp box, the dark endosperm of control mutation parent w54
Phenotype, it is individual to select the dark endosperm consistent with w54 phenotypes from 1000 particle swarm body seeds, 83 altogether.
(3) measure of amylose content is carried out by agriculture ministerial standard NY147-88.Half granule seed makes 100 mesh fineness
Ground rice, weighs respectively half granule seed powder sample and standard sample 0.0100g, in inserting the test tube of 20ml with closures.Add
The ethanol of 0.10ml 95% and 0.90ml NaOH solutions, boiling water bath gelatinization 10min, plus distilled water are settled to 10ml, shake up.Draw
Sample solution 5ml is in the 100ml volumetric flasks for filling half bottle of distilled water, plus 1.0ml 1mol/L acetic acid solutions, then adds 1.5ml iodine
Liquid, distilled water constant volume, shakes up, and determines the absorbance (OD) under 620nm.The amylose content and OD values of according to standard sample
Calibration curve is set up, according to calibration curve the amylose content of sample is calculated.
(3) w54/ Nanjing 11F2The molecular marker analysis of colony
(1) parent and F are extracted with SDS methods2The individual DNA of the dark endosperm of colony.
(2) program of the ssr analysis with reference to Chen et al. (1997).10 μ L reaction systems include：10mM Tris-HCl
pH 8.3,50mM KCl,1.5mM MgCl2, 50 μM of dNTPs, 0.2 μM of primer, 0.5U Taq polymerase (TaKaRa,
Dalian) and 20ng DNA profilings.Amplified reaction is carried out in A200 (the bright base in Hangzhou) PCR instrument：94℃4min；94℃1min,55
DEG C 1min, 72 DEG C of 1.5min, 35 circulations；72℃7min.Amplified production is separated with 8% Native PAGE glue, by silver staining
Colour developing, silver staining program is formulated according to the method for Sanguinetti et al. (1994) and formed.The DNA bands of amplification utilize lamp box
Observed.
(3) using the existing molecular labeling in laboratory, screen more between light and Nanjing 11 polymorphism preferably and be completely covered 12
The SSR molecular marker of bar chromosome；At a distance of the SSR molecule marks of 10cM or so between choosing from these SSR molecular markers
Remember row linkage analysis into；Artificial regulation：Mutant w54 banding patterns are designated as " 1 ", and the banding pattern of Nanjing 11 is designated as " 2 ", and heterozygous is designated as " 3 ".
Screening in SSR marker is entirely " 1 " type or the molecular labeling for having chain trend.Using MapMaker softwares, target base is built
The genetic linkage mapses of cause.
(4) according to the result of just positioning, in target gene near zone continual exploitation molecular labeling, while screening is more
Exchanging individual plant is used for finely positioning；And the Japanese fine database sequence information announced according to bioinformatics website, find corresponding
The BAC clones that mark is located, build the physical map of coverage goal gene.
(4) result and analysis
Using the existing 703 pairs of SSR and InDel primers being uniformly distributed on 12 chromosomes of paddy rice in laboratory, to w54
Primer polymorphism screening is carried out with Nanjing 11, the primer 160 that polymorphism is preferable and genetic distance is appropriate is therefrom chosen, base is carried out
Because of Primary Location.Genotype data shows occur being divided at No. 6 chromosome long arm end mark RM5814 and RM1370
From, illustrate target gene and the two marks it is chain.Further with the positioning group that the mark of its both sides is constituted to 83 individual plants
Body carries out linkage analysis, recycles MAPMAKER3.0 softwares to carry out linkage analysis, and w54 low amylose content genes is preliminary
Near positioning RM5463 marks；From w54/ Nanjing 11F2Dark endosperm is selected in about 5000 parts of colony's seed individual 612 parts, using
It is published in Gramene database (http://www.gramene.org/Oryza_sativa) and NCBI (http://
Www.ncbi.nlm.nih.gov) No. 6 fine chromosome sequence of long-grained nonglutinous rice 9311 and Jing rice Japan, independent development on database
The InDel molecular labelings such as W12, W39, W57, and most at last w54 low amylose content genes are positioned at No. 6 chromosome long arm
End mark W57 is nearby (Fig. 3).
Molecular labeling W57 primers：
Upstream primer P1：ACATACCAAGGCAGGAAGTC(SEQ ID NO.1)
Downstream primer P2：CACATCATTTTGCTCCCTTT(SEQ ID NO.2)
If the amplified fragments of 183bp can be amplified with molecular labeling W57 primers, dark endosperm mutant is indicated
The presence of w54 gene locus with low-content of amylose.
It is southern to w54/ further to verify Detection results of the molecular labeling W57 to w54 gene locus with low-content of amylose
Capital 11F2Colony's difference seed, records respectively phenotype, and cuts half extraction DNA.Using above-mentioned 10 μ L reaction systems, W57 is used
Primer expands seed DNA, and its electrophoresis product is presented the band of 3 types, i.e. low amylose content parent w54 banding patterns
(183bp), the banding pattern of high amylose content parent Nanjing 11 (177bp) and while the heterozygosis banding pattern (177bp with parents' banding pattern
And 183bp) (Fig. 4).With remaining half measure amylose content, as a result show, the grain endosperm outward appearance with w54 banding patterns
For dark endosperm, amylose content is similar to w54, is low amylose content；Outside grain endosperm with the banding pattern of Nanjing 11
Sight is essentially transparent, and amylose content is similar to Nanjing 11, is high amylose content；Seed embryo with heterozygosis banding pattern
Newborn outward appearance and amylose content (table 1) similar to Nanjing 11.Therefore, w54 can effectively be distinguished using molecular labeling W57 low straight
Chain content of starch gene loci, improves the efficiency of selection to its gene, and the seed selection for accelerating low amylose content rice varieties is entered
Journey.
The rice grain amylose content of table 1 and its corresponding genotype
Note：D is w54 genotype, and H is heterozygosis banding pattern, and N is the genotype of Nanjing 11.
<110>Agricultural University Of Nanjing
<120>Jing rice varieties get over the molecule labelling method of brightness endosperm mutant w54 gene locus with low-content of amylose
<160> 2
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer P1
<400> 1
acataccaag gcaggaagtc 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>Primer P2
<400> 2
cacatcattt tgctcccttt 20
Claims (5)
1. the molecular labeling W57 of a more gene loci for brightness endosperm mutant w54 low amylose contents forms sediment in low straight chain
Application in powder content Rice molecular breeding, it is characterised in that the primer of the molecular labeling is：In upstream primer P1 such as sequence table
SEQ ID NO：Shown in 1, SEQ ID NO in downstream primer P2 such as sequence table：Shown in 2.
2. it is a kind of for test right require 1 described in molecular labeling W57 primer pair, it is characterised in that upstream primer P1 is such as
SEQ ID NO in sequence table：Shown in 1, SEQ ID NO in downstream primer P2 such as sequence table：Shown in 2.
3. application of the primer pair described in claim 2 in low amylose content Rice molecular breeding.
4. a kind of method that the gene loci of brightness endosperm mutant w54 low amylose contents is got in detection, it is characterised in that profit
With primer pair described in right 2, PCR amplifying rice genomic DNAs, there is length 183bp fragment, then indicate dark endosperm mutant
The presence of w54 gene locus with low-content of amylose.
5. method according to claim 4, it is characterised in that the reaction total system of the PCR amplifications is 10 μ L, including：
10mM Tris-HCl pH 8.3,50mM KCl,1.5mM MgCl2, 50 μM of dNTPs, 0.2 μM of primer pair, 0.5U Taq
Polymerase and 20ng DNA profilings；Amplification program：94℃4min；94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1.5min, 35
Circulation；72℃7min.
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| CN111118201A (en) * | 2020-02-23 | 2020-05-08 | 云南省农业科学院农业环境资源研究所 | Molecular marker closely linked with gene du13(t) for regulating and controlling low amylose content of rice and application thereof |
| CN111118201B (en) * | 2020-02-23 | 2020-11-03 | 云南省农业科学院农业环境资源研究所 | Molecular marker closely linked with gene du13(t) for regulating and controlling low amylose content of rice and application thereof |
| CN115125318A (en) * | 2022-04-26 | 2022-09-30 | 浙江大学 | SSCP molecular marker related to dark endosperm character of rice grain, gene and application thereof |
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