CN1130120C - Method for screening rice plant with low content of straght-chain starch from rice seeds - Google Patents

Abstract

The present invention provides a method for screening rice plants of which the seeds contain low or middle content of amylose. The method uses a DNA sequence analysis method to identify a basic group of the + 1 position of a donor in a first intron of a rice waxy gene, and then the rice plants of which the basic groups are T are selected. The DNA sequence analysis method comprises a PCR-enzyme cutting method, a southern hybridization method under a strict condition and a PCR-sequence measuring method. The method can select a single target plant for backcross in the seedling stage of the rice, so blindness is reduced. The phenotype of the amylose can be measured without needing to wait after a plant produces seeds, the influence of genetic segregation and the gene dosage effect on breeding seeds is avoided, work load is lightened, and breeding efficiency is improved greatly.

Description

The screening technique of the rice plant that the rice paddy seed amylose content is low

Present invention relates in general to the botany field, be specifically related to the crop breeding field.More particularly, the present invention relates to the screening technique of the low or medium rice plant of a kind of rice grain amylose content.

Paddy rice is one of most important cereal crops in China's agricultural production, and rice yield accounts for 43% in China's total output of grain.In the past few decades, China's genetic breeding scholar and agricultural experts have made very big contribution on the per unit area yield that improves paddy rice.Along with improving constantly of Increase of population and living standards of the people,, need to produce the grains that are more suitable for people's taste also in order to adapt to the needs of the commodity food export trade more.Therefore, cultivate the target that output is higher, the better new rice variety of edible quality remains the continuous pursuit of genetic breeding scholar.

China's agricultural was scored consecutive bumper harvests in recent years, and great change has taken place agricultural product supply and demand general layout, and grain-production has entered the buyer's market new stage, made the grain stock of most laboratories full cruelly, especially in paddy inferior overstock more.On the contrary, market to the demand of fine quality rice but in continuous increase.Along with the raising of living standards of the people, exterior quality, the food flavor quality of rice proposed new higher requirement, particularly part is taken in higher family, and is more and more higher to the requirement of daily rice quality.And the most rice varieties outward appearances of China are not good, mouthfeel is bad, and applied widely of less types of good quality and high output is especially for indica hybrid rice, all the more so.

The quality of rice edible quality to a great extent with the rice endosperm in the composition of starch, promptly amylose is relevant with the ratio of amylopectin, rice grain amylose content is too high, often makes that the meal qualitative change is hard, mouthfeel is poor, thereby influences the edible quality of rice.In conventional breeding, owing to usually be preferred object with output, therefore there is the yield level of quite a few rice varieties higher, but quality deviation often; In the conventional seed selection process of the new combination of hybrid rice, kinds such as some parent such as Long Tefu, special green grass or young crops, precious Shan 97 have high yield, disease-resistant characteristic, but because their amylose content is very high, so the edible taste of boiling of the hybridization rice of being produced by their preparations is relatively poor.Some indica hybrid rices particularly, the comparable conventional variety volume increase 15～20% of its output, but the hybridization rice quality is not ideal enough, causes stock, and on the low side.Tracing it to its cause, mainly is because the amylose content of hybridization rice is higher, be about 27% as the amylose content of the precious Shan 97 of the male sterile line parent who is applied to ternary hybrid rice at present (A), and Long Tefu (A) is more up to about 30%.

Starch in the rice partly is made up of amylose and amylopectin two, and amylose is the big molecule of being made up of the glucose residue that α-(1,4) key connects of line style long-chain polysaccharide, and amylopectin has the bifurcation that α-(1,6) key forms.Amylopectin generally accounts for more than 70%, and all the other then are amylose.The content of amylose all plays an important role on edible quality and the boiling processing quality at the decision yield of brown rice in the endosperm.Except cultivation condition to the amylose content in the rice influential, the content difference of amylose in different cultivars rice is heritable characteristic.

Khush equals 1984 at Genetics, reports among the 107:141-167, and the paddy rice waxy gene (waxy gene) on No. 3 chromosome of paddy rice (being called chromosome now No. 6) is being controlled the synthetic of amylose in the endosperm.This gene code is incorporated into amylosynthease (the Granule-bound starch synthase on the starch grain, GBSS) [EC.2.4.1.11], thereby synthetic (Okagaki and the Wessler of amylose in control paddy pollen, endosperm and the blastular, 1988, Genetics, 120:1137-1143), be to influence the key gene that starch is formed in the paddy endosperm.Because this gene only efficiently expresses in pollen, endosperm and the blastular of paddy rice, so it is space-time specificity expression's a gene.

The applicant has cloned waxy gene from paddy rice, and has finished sequence analysis.This gene is made up of 13 exons and 14 introns.The 1st intron of paddy rice waxy gene is present in 5 ' boot section, and length is 1124bp.And corn, monocotyledonous waxy gene such as barley is compared, and the paddy rice waxy gene has bigger the 1st intron order [Wang, ZY; Wu, ZL; Xing, YY; Zheng, FQ; Guo, XL; Zhang, WG andHong, MM, (1990) Nucleotide sequence of rice waxy gene.Nucleic Acids Research.18 (19): 5898.].

The gene that much plays an important role is not only regulated and control on transcriptional level, and is also regulated and control on post-transcriptional level.In the work of research paddy rice waxy gene expression regulation, the waxy gene that we observe japonica rice Han Feng has two kinds of transcription products of 2.3kb and 3.3kb to exist, wherein the 2.3kb transcription product is ripe mRNA, and prove that by analysis the 3.3kb transcription product is the not cut precursor RNA of the 1st intron.We have detected the relative quantity of two kinds of transcription products of waxy gene in the relative amount of content, Wx albumen of amylose in the seed of more than 30 rice varieties (long-grained nonglutinous rice, japonica rice and glutinous rice are wherein arranged) and the immature seed.The result shows between ripe mRNA (2.3kb) content of amylose, Wx albumen and waxy gene positive correlation is arranged; And negative correlation is arranged with the content of precursor RNA (3.3kb).The regulation and control of pointing out the expression of waxy gene in the different rice varieties to be subjected to the waxy gene post-transcriptional level thus, especially with transcribe elementary product in the 1st intron relevant by the degree of montage, i.e. the kind amylose content that montage efficient is high also high [Wang, ZY; Zheng, FQ; Shen, GZ Gao, JP; Snustad, D.P.; Li, MG; Zhang, JL and Hong, MM, (1995) The amylose content in rice endosperm is related to thepost-transcriptional regulation ofthe waxy gene.Plant Journal.7 (4): 613-622].

For understand fully in the rice amylose whether really therewith intron montage efficient relation is arranged, the applicant has made up two kinds of mosaic genes being made up of cold rich 6366 kind of low amylose content (in) Wx gene the 1st intron of long-grained nonglutinous rice 232 waxy gene promotors, long-grained nonglutinous rice 232 (kind of high amylose content) or japonica rice and gus reporter gene, the plasmid that will contain these two kinds of mosaic genes transforms respectively in the into different rice varieties, measures their resistant calli and the GUS enzymic activity in the transfer-gen plant immature seed endosperm.Measurement result shows that the mosaic gene GUS activity of the mosaic gene GUS specific activity japonica rice contain long-grained nonglutinous rice 232 (kind of high amylose content) Wx gene the 1st intron cold rich 6366 kind of low amylose content (in) Wx gene the 1st intron obviously exceeds tens times.

The applicant also measures by RT-PCR and binding sequence, has analyzed in the different rice varieties waxy gene the 1st intron by the situation of montage.The result is presented in the paddy rice of high amylose starches (＞20%) content, and the montage of waxy gene the 1st intron meets the GT-AG rule of intron montage fully; And in, in the paddy rice of low amylose (6～16%) content, the montage of waxy gene the 1st intron has the replacement phenomenon, they have utilized hidden confession, acceptor site [Cai, XL; Wang, ZY; Xing, YY; Zhang, JL and Hong, MM, (1998) Aberrantsplicing of intron 1 leads to the heterogeneous 5`UTR and decreased expression of waxygene in rice cultivars of intermediate amylose content Plant Journal.14 (4): 459-465.].About the specificity analysis of waxy gene also among further research.

What at present, conventional breeding method screening paddy rice low or medium amylose content relied on is the amylose content of directly measuring in the paddy endosperm.Yet this method exists some shortcomings.Paddy rice is a diplont, and its seed (being rice) mainly partly is made up of embryo and endosperm two.Endosperm is the storage tissue of an eggcase being made up of the triploid cell in the rice plant, has accumulated a large amount of carbohydrate and storage protein in the endosperm, and its dry matter weight can account for about 75% of seed dry weight.People are exactly endosperm part in the rice paddy seed as the rice of food.Because albuminous cell is triploid in rice plant, and than the late generation of diplontic contemporary somatic cell, therefore exist heredity in the genetic breeding of endosperm proterties (as amylose content) and separate and dosage effect of gene, hereditary different with other proterties.So with the conventional breeding method that detects amylose content it is improved not only very loaded down with trivial detailsly, and is difficult for obtaining the homozygous genotype of control rice grain amylose content.Though the rice amylose content of some hybrid rice combinations produce decreases at present, but because male sterile line is still the genotype of high amylose content, the hybridization rice that it was produced is an allometrosis, the amylose content of the different grain of rices has nothing in common with each other, still bad (the Mo Huidong of its boiling processing characteristics and palatability, Gu Minghong, Jiang Changjian etc. (1990) cereal quality trait genetic research progress, Jiangsu science tech publishing house).And because conventional breeding method need could be measured the amylose content of seed after plant is born seed, so the cycle of this method is longer.

In order to overcome existing these shortcomings of conventional breeding method, the objective of the invention is sequence and characteristic by further research paddy rice waxy gene the 1st intron, a kind of cycle weak point, result are provided paddy rice screening technique accurately.

The invention provides the screening technique of the low or medium rice plant of a kind of rice paddy seed amylose content, this method is to identify the base of donor+1 in paddy rice waxy gene first intron with the dna sequence analysis method, selects the rice plant that this bit base is T then.

By than higher and in, Wx gene the 1st intron of two class rice varieties of low amylose content, the applicant finds have 16 bases variant therebetween.Subsequently, the applicant has studied several mutational sites in paddy rice Wx gene the 1st intron to the influence of gene expression by the method for rite-directed mutagenesis and transformed plant.With the 1st intron part site in the Wx gene promoter of high amylose content kind according in, the base of low amylose rice varieties makes external rite-directed mutagenesis, transformed promotor and GUS code area are fused into chimeric plasmid, transform respectively and spend No. 11 in the japonica rice, measure the GUS activity in the transformed plant immature seed endosperm then.The result shows, the bases G of waxy gene the 1st intron donor+1, after being mutated into the T base, promptly 5 of the 1st intron ' end splice junction base is replaced the rapid decline that the GUS activity is caused in back (AGgt/AGtt), and other several detection site all do not have remarkable influence to expression of gene.Therefore, the applicant infers, the guanine (G) of the 1st intron 5 ' end is that efficient excision of Wx gene the 1st intron and the correct splicing of exon are necessary, this locates to be replaced by thymidine (T) and in the Wx gene of the rice varieties of middle low amylose content, thereby make excision efficient reduce greatly, and then influence the content of amylose.The applicant also further finds, in all long-grained nonglutinous rices measured and japonica rice plant, the base of waxy gene the 1st intron donor+1 is T or G.

Find based on these, the invention provides a kind of method of in breeding process, coming the lower rice plant of Screening of Rice seed amylose content by the base of differentiating waxy gene the 1st intron donor+1.In a preferable embodiment of the present invention, describedly be used for identifying that the method for waxy gene the 1st intron donor+1 bit base comprises the PCR-PCR sequencing PCR, Southern hybrid method under rigorous condition and PCR-enzyme blanking method.

The PCR-PCR sequencing PCR comprises the following steps: to design a pair of primer by the other adjacent DNA sequence of single base mutation point, with total DNA of sample plant as template, go out this dna fragmentation through pcr amplification, be cloned into it on order-checking plasmid vector or directly this dna fragmentation carried out sequencing, thereby judge which kind of base the mutational site is.Sequence measurement to dna fragmentation specifically comprises, but be not limited to, for example Sanger dideoxy chain termination and Maxam-Gilbert PCR sequencing PCR etc. are (referring to people such as Sambrook, " molecular cloning experiment guide ", the 2nd edition, Cold SpringHarbor Press (Cold Spring Harbor, NY, 1989)), wherein preferably the Sanger dideoxy-chain stop PCR sequencing PCR.

Southern cross method under the rigorous condition comprises the following steps: to stride by the other adjacent DNA sequence design of single base mutation point two dna probes of mutational site, this site base is with mutating alkali yl is not identical in the probe, and this site base is identical with mutating alkali yl in another probe.Two probes are after tagging, and the total DNA with the sample plant under the rigorous condition of control carries out the Southern blot hybridization.Because two probes are different with the annealing temperature of sample DNA, signal after the hybridization is strong and weak different, can judge that thus the mutational site is which kind of base is (equally referring to people such as Sambrook, " molecular cloning experiment guide ", the 2nd edition, Cold Spring Harbor Press (Cold SpringHarbor, NY, 1989)).

But, as described above as the present invention, the base that only need be tested and appraised paddy rice waxy gene intron donor+1 is that T or G are the amylose content level of bearing after the measurable paddy rice maturation in the seed, so the present invention here provides, and a kind of cost is low, the PCR-enzyme is cut detection method simply rapidly.

Therefore, in a preferable embodiment of the present invention, adopt PCR-enzyme blanking method to differentiate that described waxy gene the 1st intron donor+1 bit base is G or T, this method comprises the following steps:

1) designs and synthesizes the PCR primer according to paddy rice waxy gene the 1st exon and the 1st intron join domain two sides;

2) total DNA of extraction rice plant;

3) with the rice total dna be template, add synthetic primer, carry out pcr amplification;

4) with this amplified production of digestion with restriction enzyme;

5) make product electrophoresis on Ago-Gel;

6) EB dyeing, the DNA band position after observing electrophoresis under the UV lamp.

With waxy gene the 1st intron donor+1 is that the genotype of the rice varieties of G base is labeled as GG; The genotype that is the rice varieties of T base is labeled as TT.If the restriction enzyme that adopts can be discerned the genotypic sequence of GG, and digestion product is identical with DNA band swimming distance in the undigested product, shows that then this rice varieties is the TT genotype; If DNA swimming distance is more farther than the swimming distance of DNA in the digestion product in the undigested product, restriction enzyme can cut off pcr amplification product, shows that then this rice varieties is the GG genotype; If the DNA band of two kinds of swimming distances all exists in the digestion product, show the genotype that this paddy rice strain is the TG heterozygosis (seeing the electrophoresis schematic diagram of Fig. 2).Otherwise if employing is the restriction enzyme of specific recognition TT genotype sequence, what then DNA band swimming distance was identical with undigested product represents GG genotype (electrophoretogram does not show) through digestion product.Preferable, this restriction enzyme is the AccI enzyme of specific recognition GG genotype sequence.

Yet, should be appreciated that dna sequence analysis method of the present invention is not limited to above-mentioned this several method.In the prior art, the method for particular bases all is well-known in the known gene of mensuration/discriminating sequence.Those of ordinary skills obviously can be used for various modifications method of the present invention according to the instruction of this specification and change, and therefore, all these improvements and changes also all within the scope of the invention.

In another preferable embodiment of the present invention, described discriminating step can be carried out in any period of paddy growth.Better, described discriminating/determination step can carry out at paddy growth seedling stage.In another preferable embodiment, can choose individual plant and carry out described evaluation.

The rice paddy seed amylose content can be measured with conventional means well known in the art, for example including, but not limited to adopting method herein described in the embodiment to measure.In this article, if the amylose content that records in the paddy endosperm is higher than 20% (weight), then claim " amylose content is higher "; Or this amylose content is lower than 20% (weight), then claims " amylose content is low or medium ".

Adopt method of the present invention to have following significant advantage.At first, the present invention has found a molecular labeling directly related with the genotype of rice paddy seed amylose content.In this article, should " molecular labeling " be the base of waxy gene the 1st intron donor+1.This molecular labeling is different with breeding molecular labeling in general sense, it not only with amylose content complete linkage in heredity of rice paddy seed, and the base of paddy rice waxy gene the 1st intron donor+1 of being detected itself promptly is the important inherent cause of the different rice variety amylose contents of decision.The second, this law all can be measured the genotype of rice plant in any period of rice growing season, can select the genotype plant of isozygotying, the height of measurable rice grain amylose content.The 3rd, it can be measured by individual plant, thereby gets final product the preliminary election individual plant vegetative growth phase when the paddy rice backcross transformation as the parent of hybridizing or backcrossing.This method detects genotype and is simple and easy to usefulness, can select the individual plant backcrossed at rice seedling, has reduced blindness, also need not to wait for the phenotype of measuring amylose after plant is set seeds, avoid the influence of hereditary separation and dosage effect of gene, alleviated workload, increased the breeding probability greatly.The 4th, by constantly backcrossing, when selecting medium amylose content gene type, can keep other merit of modified kind.Thereby can be used for the seed selection of the medium amylose content gene type of hybrid rice male sterile line.

Further describe the present invention again below in conjunction with accompanying drawing.

Fig. 1 is the other adjacent partial dna sequence of middle waxy gene the 1st intron 5 ' donor of the rice varieties (GG genotype) of high amylose content, gtatac is the recognition sequence of AccI among the figure, and g becomes after being mutated into t in the rice varieties (TT genotype) of medium amylose content

Can not be discerned by AccI.Underscore has marked the position of upstream and downstream primer among the PCR.

Fig. 2 is the electrophoresis schematic diagram that the different genotype rice varieties detects after the PCR-enzyme is cut.The left side band is an initial point among the figure, and the right side is the DNA electrophoretic band.Swimming lane 1-5 is respectively: 1) the GG genotype PCR product of cutting through enzyme; 2) the TT genotype PCR product of cutting through enzyme: 3) the GT genotype PCR product of cutting through enzyme; 4) GG genotype PCR product; 5) TT genotype PCR product.

Fig. 3 is the flow chart of molecular mark improvement rice edible quality.

Further describe the present invention below in conjunction with embodiment.

The evaluation of embodiment 1 molecular labeling

1) extraction of the total DNA of blade

(20mmol/l EDTA pH8.0), adds 2cm to the extraction buffer solution of adding 400 μ l precoolings in little mortar for 1.4mol/l NaCl, 100mmol/l Tris.Cl ²Fresh rice leaf, homogenate.Homogenate is poured in the 1.5ml centrifuge tube, added 2 * CTAB buffer solution [2%CTAB (w/v), 1.4mol/l NaCl, 100mmol/l Tris.Cl, 20mmol/l EDTA, pH8.0] of 60 ℃ of preheatings of 500 μ l again, be incubated 30-60 minute at 60 ℃ behind the mixing.Add 450 μ l chloroforms: isoamyl alcohol (24: 1) mixed liquor shakes up under the room temperature with 13000rpm centrifugal 10 minutes.To go up the phase aqueous solution and move in the new centrifuge tube, add 600 μ l isopropyl alcohols, place 15 minutes for-20 ℃ behind the mixing.4 ℃ with 10000rpm centrifugal 5 minutes, abandon supernatant, precipitation with 500 μ l, 70% washing with alcohol once.The vacuum drying post precipitation dissolves with 30 μ l TE buffer solutions.

2) PCR reaction

In the 0.5ml centrifuge tube, add 5 μ l, 10 * PCR buffer solution (10mmol/l Tris.Cl, pH8.3,50mmol/l KCl, 0.001% gelatin) respectively, 3 μ l 25mmol/l MgCl ₂, 4 μ l 2.5mmol/l dNTPs, the 50pmol upstream primer (5 '-GCTTCACTTCTCTGCTTGTG-3 '), the 50pmol downstream primer (5 '-ATGATTTAACGAGAGTTGAA-3 '), the total DNA of 1 μ l blade, 1U Taq polymerase is used aseptic ddH ₂O complements to 50 μ l with cumulative volume.On the GeneAmp PCR System of PERKIN ELMER company 9600 type pcr amplification instrument by 30 circulations of following condition amplification: 94 ℃ of sex change 40s, 55 ℃ of annealing 40s, 72 ℃ are extended 40s, after last circulation again 72 ℃ of extensions 10 minutes.Amplified production is through agarose gel electrophoresis, and EB dyeing back can detect the DNA band of a 460bp under uviol lamp.

3) the AccI enzyme is cut evaluation

In the 0.5ml centrifuge tube, add 10 μ l pcr amplification products, 1.5 μ l, 10 * enzyme cutting buffering liquid (33mmol/lTris-acetate, 10mmol/l MgAc, 66mmol/l KAc, 0.5mmol/l DTT, pH7.9), 5U AccI enzyme is used aseptic ddH ₂O complements to 15 μ l with cumulative volume, and at 37 ℃ of insulation 1h, electrophoresis detection on 2% Ago-Gel compares with the pcr amplification product of cutting without enzyme behind the mixing.If have the AccI restriction enzyme site in the PCR product, then the dna fragmentation of 460bp is cut into 403bp and two bands of 57bp, on 2% Ago-Gel, can distinguish the DNA migration distance of 460bp and 403bp.

4) Sanger dideoxy chain termination order-checking

As step 1), 2) described, obtain pcr amplification product.As people such as Sambrook, " molecular cloning experiment guide ", the 2nd edition, described in the Cold Spring Harbor Press (Cold Spring Harbor, NY, 1989) like that, stop checking order with the Sanger dideoxy-chain this amplified production checked order.

5) the Southern blot hybridization under the rigorous condition

Design and synthesize two dna probes: 1) 5 '-GCAAGGTATACATATATGTT-3 ', 2) 5 '-GCAAGTTATACATATATGTT-3 '.With [γ- ³²P] P carries out tagging to two probes.Under the rigorous condition (6XSSC, 5X Denhardt ' s reagent, 0.5%SDS, 100 mcg/ml sex change fragmentation salmon sperm DNAs, 48 ℃ under hybridize) of control, make the total DNA of sample plant of probe and step 1) acquisition carry out the Southern blot hybridization.

With above-mentioned distinct methods the molecular labeling (being the waxy gene first intron donor+1 bit base) of 27 rice varieties is identified that the gained result is summarized in the following table 1.

The mensuration of embodiment 2 amylose contents

From the amylose of 68 ℃ of baking Sigma companies after 2 hours, amylopectin sample, take by weighing each 50mg of amylose and amylopectin, add in the volumetric flask of numbering respectively, respectively add the 1ml absolute ethyl alcohol then, starch is soaked into, respectively add 9ml 1mol/l NaOH again, boiling water bath 10 minutes, be settled to 100ml after the cooling, abundant mixing, draw in following ratio:

Volumetric flask numbers 012345678

Amylose (ml) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Amylopectin (ml) 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7

AC％ 0 4 8 12 16 20 24 28 32

Add 25ml ddH ₂O, 0.5ml 1mol/l HAG, 0.5ml I-KI reagent is settled to 50ml, and fully mixing was placed 10 minutes.Simultaneously, the configuration blank is inhaled 225 μ l 1mol/l NaOH, adds 25ml ddH ₂O, 0.5ml 1mol/lHAc, 0.5ml I-KI reagent is settled to 50ml, reads the above-mentioned optical density value of respectively managing at the 620nm place as control tube, sets up calibration curve.

Get that mature seed shells, threshing, clay into power, cross 100 mesh sieves, 68 ℃ of bakings are spent the night.Get 25mg sample dry powder, add 0.5ml absolute ethyl alcohol, 4.5ml 1mol/l NaOH, boiling water bath 10 minutes is settled to 50ml after the cooling, fully mixing.Get 2.5ml, add 25ml ddH ₂O, 0.5ml 1mol/l HAc, 0.5ml I-KI reagent is settled to 50ml, and fully mixing was placed 10 minutes, established blank, before method is shone, surveyed the 620nm optical density readings.On calibration curve, check in the amylose content of sample.

Listed qualification result in the following table 1 to amylose content and molecular labeling thereof in the seed of different rice varieties or hybrid rice parents.

The testing result of different rice quality amylose contents of table 1 and molecular labeling

	Amylose content	Molecular labeling
			232	20.0	GG
92103	27.3	GG
			IR 36	22.3	GG
Zhenshan 97B	31.2	GG
			Long Tefu B	31.9	GG
Special blue or green	29.2	GG
			The blue or green B early of association	30.6	GG
IP1529-68-3-2	31.7	GG
			Train short by 64 (S)	24.9	GG
Han Feng	16.0	TT
			Qiu Guang	9.2	TT
CG3	14.3	TT
			IR661	11.2	TT
Long grain rice	8.0	TT
			Salt extensive 559	12.5	TT
Raise rice 4	15	TT

Bright brightness 63	13.3	TT
			Extensive 136	17.4	TT
6078	13.5	TT
			9522	17.0	TT
SWR20	11	TT
			9516	17.5	TT
In spend 11	17.5	TT
			02428	16.9	TT
Land-reclaimable 57	16.9	TT
			Give glutinous 593	0.70	TT
Guanling perfume (or spice) is glutinous	0.54	TT

As can be seen from Table 1, in these 27 paddy rice, without exception have the genotypic rice varieties of GG or hybrid rice parents all has higher amylose content, and have the genotypic paddy rice of TT be in the rice varieties or the hybrid rice parents of low equal size.This proves that fully selected molecular labeling of the present invention and content of amylose in rice are closely linked.

Embodiment 3 molecular marker assisted selection breedings

According to the relation of the close linkage between selected molecular labeling of this paper and the rice paddy seed amylose content, design breeding method with the low or medium amylose content paddy rice of this molecular marker assisted selection.With a target variety to be improved or hybrid rice parents (the good and edible taste difference of cultivated character, the GG genotypic rice kind that contains high amylose starches, as Zhenshan 97B) and medium amylose content, has the genotypic rice varieties of TT (as 9511, salt extensive 559) hybridization, constantly using in the backcross process of GG genotypic rice kind and hybrid generation, detect the genotype of the first filial generation plant of backcrossing with above-mentioned PCR-enzyme blanking method, select the progeny plant of GT heterozygous to backcross as male parent, though with the method for setting up near-isogenic line to those output height but the relatively poor GG genotypic rice kind of edible quality is carried out genetic improvement, when keeping original rice-cultivating kind merit, reduce their amylose content, reach the purpose of improving food flavor.The flow chart of molecular mark improvement rice edible quality has been shown among Fig. 3.

For verifying our design, to after the hybridization of GG genotype and TT genotypic rice again through the F2 generation of selfing different individual plants molecular labeling and the amylose content of being tied in the seed measure, determination data shows that again the amylose content in this molecular labeling and the seed is close linkage between the two, separates jointly.Shown parent and hybridization F2 thereof testing result in the following table 2 for individual plant genotype, amylose content.

Table 2 parent and the testing result of hybridization F2 for individual plant genotype, amylose content

Amylose content (AC%)	Genotype and plant number
										The 9311X Zhenshan 97B			The 559X Zhenshan 97B			559	9311	Zhenshan 97B
	TT	GT	GG	TT	GT	GG	TT	TT	GG
										＜9.0	2			5
9.1-11.0	1			4
										11.1-13.0	1			2			3
13.1-15.0	5	1		3			1	2
										15.1-17.0	2	1			2	1		5
17.1-19.0	4	1			2			1
										19.1-21.0	1	1	1		5
21.1-23.0	1	4			8	1
										23.1-25.0		5	2		5
25.1-27.0		7	4		2	2			1
										27.1-29.0		3	4		1	1			6
29.1-31.0		1	4			2			5
										The plant sum	17	24	15	14	25	7	4	8	12

For example in Zhenshan 97B (GG genotype, amylose content be 31% wait to improve the target parent) with 9511 (TT genotype, amylose content is 15%) hybrid combination in, picked at random F1 is for 60 seeds being tied on the plant, obtain 56 strain F2 after the plantation for plant, it is the TT genotype that 17 strains are arranged after testing, and their seed bearing amylose contents of institute are between 7.1-23.0%, and wherein 15 strain amylose contents are lower than 19%; It is the genotype of GT heterozygosis that 24 strains are arranged, and their amylose content is between 13.1-31.0%, and wherein 19 strain amylose contents are at 21.1-29.0%; And have GG genotypic is 15 strains, and their amylose content is between 19.1-31.0%, and wherein the amylose content of 12 strains is higher than 25%.

And for example in Zhenshan 97B (GG genotype, amylose content be 31% wait to improve the target parent) with extensive 559 (the TT genotype of salt, amylose content is 13%) hybrid combination in, picked at random F1 is for the seed of being tied on the plant, obtain 46 strain F2 after the plantation for plant, it is the TT genotype that 14 strains are arranged after testing, and their seed bearing amylose contents of institute are between 7.1-15.0%, and the genotypic 14 strain amylose contents of all TT all are lower than 15%; It is the genotype of GT heterozygosis that 25 strains are arranged, and their amylose content is between 15.1-29.0%, and wherein the amylose content of 18 strains is at 19.1-25.0%; And have GG genotypic is 7 strains, and their amylose content is between 17.1-31.0%, and wherein the amylose content of 5 strains is higher than 25%.

Claims (7)

1. the screening technique of the low or medium rice plant of a seed amylose content is characterized in that, identifies the base of donor+1 in paddy rice waxy gene first intron with the dna sequence analysis method, selects the rice plant that this bit base is T then.

2. method according to claim 1 is characterized in that, described dna sequence analysis method comprises Southern cross method and PCR-Sanger dideoxy-chain termination PCR sequencing PCR under PCR-enzyme blanking method, the rigorous condition.

3. method according to claim 2 is characterized in that, described PCR-enzyme blanking method comprises the following steps:

1) according to the design of paddy rice waxy gene the 1st exon and the 1st intron join domain two sides, synthetic pcr primer thing;

2) total DNA of extraction rice plant;

3) with the rice total dna be template, add synthetic primer, carry out pcr amplification;

4) with this amplified production of digestion with restriction enzyme;

5) make product electrophoresis on Ago-Gel;

6) EB dyeing, the DNA band position after observing electrophoresis under the UV lamp.

4. method according to claim 3 is characterized in that described restriction enzyme is AccI.

5. method according to claim 1 is characterized in that, can carry out described mensuration in any period of rice growing season.

6. method according to claim 5 is characterized in that, carries out described mensuration in the seedling stage of rice growing season.

7. method according to claim 1 is characterized in that, can choose individual plant and carry out described evaluation.

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