CN104585018A - Cultivating method of wheat-agropyron elongatum FHB (Fusarium head blight)-resistant 7E chromosome long arm translocation line - Google Patents

Abstract

The invention relates to a cultivating method of a wheat-agropyron elongatum FHB (Fusarium head blight)-resistant 7E chromosome long arm translocation line. The cultivating method comprises the following steps: processing F2-generation seeds obtained by hybridizing a wheat-agropyron elongatum 7E substitution line and Yangmai 16 by <60>Co radiation, and in a propagation process of radiated offspring, firstly, performing field FHB resistance screening to select individual plants which are high in FHB resistance and have related features of agropyron elongatum; and based on this, detecting the obtained individual plants by using an agropyron elongatum 7E chromosome long arm specific marker, and finally, confirming the wheat-agropyron elongatum FHB-resistant translocation line by virtue of cytology. By combining field phenotype screening, molecular marker identification and cytology confirmation, the wheat-agropyron elongatum FHB-resistant 7E chromosome long arm translocation line is created; the obtained FHB-resistant translocation line provides a new germplasm or base material for wheat variety improvement and genetic research, and can be used as a parent to participate in cultivation of the FHB-resistant wheat.

Description

The breeding method of wheat-E. elongata anti gibberellic disease 7E chromosome long arm translocation line

Technical field

The invention belongs to field of crop genetic breeding, this research and utilization ⁶⁰co (Co 60) irradiation treatment wheat-E. elongata 7E substitution line (DS7E/7B) with raise F2 that wheat 16 hybridizes for seed, in the reproductive process in generation after irradiation, first field scab resistance screening is carried out, scab resistance is selected better to have again the single-strain planting of E. elongata correlated characteristic, utilizing E. elongata 7E chromosome long arm specific mark to detect being selected individual plant on this basis, carrying out wheat-E. elongata anti gibberellic disease translocation line finally by cytology and confirming.Utilize the screening of variable rate technology type, molecular markers for identification and cytology to confirm that three kinds of methods combine, create wheat-E. elongata anti gibberellic disease 7E chromosome long arm translocation line.

Background technology

1 wheat breeding target and its wild relatives

Wheat is the cereal crops that cultivated area is maximum, gross yield is only second to corn in the world.Nearly half a century, the gross yield of world wheat has more than tripled, and wherein, excellent wheat breed has played conclusive effect to raising wheat yield.Current wheat breeding research mainly concentrates on four aspects: improve wheat yield, reduce production cost; Molecular mark; Resistance of Wheat To Adversity breeding; Nutritional quality of wheat (He Zhonghu etc., 2006).For completing above breeding objective, the genetic resources of wheat itself is obviously inadequate, and has in many wildlife species of Characterization with wheat and there is unexistent advantageous genetic resource in a large amount of wheat.Therefore, the fine genes in Wheat relatives is utilized to cultivate new variety of wheat one of target becoming wheat breeding.

Common wheat (AABBDD, 2n=42) be allohexaploid plant, belong to grass family Triticum common wheat kind (Triticum aestivumL.), the genus near with Triticum Relationship Comparison has Hordeum (Hordeum), Elymus (Elymus), Hedgehog grass belongs to (Asperella), thin smooth wheat belongs to (Sitanion), Psathyrostachys (Psathyrostachys), rib axle grass belongs to (Crithopsis), band Chinese silvergrass belongs to (Taeniatherum), Agropyron (Agropyron), Dasypyrum (Haynaldia), Secale (Secale), abnormity Pittosporum (Heteranthelium), awnless brome belongs to (Henrardia), Eremopyrum (Eremopyrum), Aegilops (Aegilops).Containing a large amount of excellent genes in these wild relatives plants, is a huge genetic storehouse with potential value.The excellent genes of this gene pool, as utilized, will have important value to genetic improvement of wheat, can not only improve its output, improvement quality, more can proceed in wheat by abundant disease-resistant, adversity gene, the disease resistance of improvement wheat and resistance.The kind of having carried out successful cross at present with wheat has Secale (Sando et al, 1953), Dasypyrum (Sears et al, 1953; Hyde et al, 1953), Genus Agropyron (Dvor á k et al, 1974), Aegilops (Sears et al, 1956; Chapmnaet al, 1970), Hordeum (Kruse et al, 1973; Islam et al, 1975) etc.The success of these distant hybridization is the drought resisting of Wild related germplasm, the excellent genes of Salt And Alkali Tolerance and disease and insect resistance carries out transfer to Wheat volatiles and established solid foundation, makes to find or open up new important gene resource from wheat relative belongs to become possibility.

The scab resistance of 2 wheats and E. elongata

The disease received much attention in Wheat Production is wheat scab (Fusarium head blight, FHB), it is one of global Major Diseases causing wheat yield reduction and quality to decline, and is also the Major Diseases (Lu Weizhong etc., 2001) affecting Wheat in China production.The head blight time disease tassel yield that is very popular reaches 50% ~ 100%, and the underproduction is up to 10% ~ 40% (Yao Jinbao etc., 2000).This disease is caused by multiple sickle-like bacteria, multiple mycotoxin is produced after pathogen infection wheat seed, these toxin are to harmful (the Bottalico et al of people and animals, 1998), particularly deoxynivalenol (DON) toxin wherein, under the concentration of 50mg/kg, just can suppress the activity of human T cells 80%, the health (Song Fengying etc., 2005) of the mankind and domestic animal in serious threat.Winter Wheat Area, China middle and lower reach of Yangtze River is multiple district and the severely afflicated area of wheat scab, and Yellow River-Huai River region and Central Shanxi Plain Mai Qu wheat scab occur also to be on the rise in recent years, affect the grain security of domestic wheat main producing region.Along with global climate warms and the increase of corn-wheat rotation system, during the nearly last ten years wheat scab in North America and Europe also large area occur, cause serious output and economic loss (Bai et al, 2004).Therefore, lot of domestic and international scholar has carried out research work widely around wheat scab.China was studied wheat scab the mid-1970s from last century, result of study thinks that wheat scab resistance has hereditary basis, and cultivate the good quality and high output new variety of wheat of a collection of better resistance, wherein Sumai 3 has become the head blight high resistance kind (Yao Jinbao etc., 2000) that the whole world is generally acknowledged at present.

Wheat Breeding For Scab Resistance research for many years shows, in Wheat Species, the resource of anti gibberellic disease is little, and the Resistance resource had been found that is difficult to effective utilization due to the existence of other character genes in Resistance resource background in wheat breeding practical application.Make a general survey of breeding for scab resistance both domestic and external, anti-source used is mainly limited to Sumai 3 and some landrace (as Wangshuibai, the wheat that crosses over a hill, the red Buddhist monk in Wenzhou etc.), the new middle length of Japan, Brazilian Frantana etc., the economical character in these anti-sources itself is just poor, utilize these anti-sources and Derivative line thereof to carry out breeding for scab resistance decades, but there are not only high resistance but also the kind of getting bumper crops incubation (Jin Shanbao, 1996) on producing so far.From late 1970s to the beginning of the eighties, China had once carried out the national screening operation of scab resistance to more than 30,000 parts of collection materials, screen a great deal of landrace to head blight tool stable resistance, but the kind matter of these preciousnesses overwhelming majority never obtains suitable utilization in Wheat Breeding For Scab Resistance.From near isogenic wheat line, open up the anti-source of new head blight is Critical policies in Wheat Breeding For Scab Resistance, and carry out a large amount of correlative studys, and successfully from the kindred plant of wheat as searched out some new anti-sources (Oliver R Eet al, 2005) couchgrass, Dalaicao, roegneria kamoji, Roegneria Ciliaris and perennial barley wild species.But how these outer rim resistant genes are stably incorporated in common wheat is gone, and can be utilized effectively in breeding and should become the emphasis of research.

Genus Agropyron (Thinopyrum) is grass family barley race (Horseae), herbaceos perennial in wheat subtribe (Triticeae), comparatively near with the Characterization of common wheat, about there are 50 kinds in world wide, are mainly distributed in temperate zone and refrigerant latitudes.This genus adaptability and fertility are comparatively strong, and have many excellent genes and proterties, as disease-resistant, cold-resistant, drought resisting, Salt And Alkali Tolerance and fringe are spent more greatly, are one of near isogenic wheat line having using value in genetic improvement of wheat.There is the E. elongata of 3 types in occurring in nature, i.e. Diploid Thinopyron elongatum grass (Th.elongatum, 2n=14), Tetraploid Elytrigia (Th.elongatum, 2n=28) and Thinopyrum ponticum (Th.ponticum, 2n=70).Diploid Thinopyron elongatum grass has E chromosome set, and E chromosome set is the basic chromosome set (Dewey et al, 1984) of Genus Agropyron polyploid species.The acquisition of a whole set of China spring-E. elongata alien addition lines, substitution line has established good basis (Dvor á k J et al, 1974) for furtheing investigate E. elongata.There are some researches show 1E (Liu Dengcai etc., 2001 at E. elongata; Jauhar PP et al, 2009), 7E (Somers DJ et al, 2003; Shen X R et al, 2004; Xu Guohui etc., 2009; Zhang X L et al, 2011) chromosome may have good scab resistance gene, therefore, E. elongata has become one of the hereditary basis of improvement common wheat, the important wild near edge species improving scab resistance (Wang Liming etc., 2005).

The establishment of 3 translocation lines and qualification

Exogenous chromosome imports common wheat and can directly utilize Common Wheat Varieties and Genus Agropyron species to carry out hybridizing and backcrossing, and cultivating wheat alien addition line, substitution line and translocation line is the important channel utilizing exogenous germplasm to improve wheat.Because while external source channel genes, the unfavorable proterties of alien species is also brought into thereupon.Minimum in order to make adverse effect drop to, the ideal style that foreign gene utilizes is seed selection wheat-Alien Chromosome Translocation Induced system, especially carries the Small piece transposition of target gene or middle insert type transposition.So translocation line imports the advantage of the desirable fragment of chromosome of excellent target proterties with it, demonstrate it than addition line and the meaning of substitution line in plant breeding more great (Liu Houli, 1993).Chromosomal transposition between common wheat and its Spherical scanning often can be there is in Tribe Triticeae; from Sears (Sears E R; 1972) since within 1956, createing first wheat-Aegilops umbelluata translocation line; many desirable genes that wheat relative belongs to are (as the gene resistant to stripe rust Yr9 in rye; leaf rust resistance gene Lr26; anti-stem rust gene Sr3; mildew-resistance gene Pm8 and anti-aphides gene Gb (Jiming Jiang et al, 1995)) to transfer in common wheat and to have obtained many wheat alien translocation lines.These excellent breeding materials are widely used in wheat genetic breeding, cultivated many improved seeds, whole world wheat increase yield serves immeasurable effect.

The creation method of 4.1 translocation lines

Wheat-nearly edge species-foreign translocation line is just to the chromosome segment that wheat has imported xenogenesis, different genus contains target gene, subsidiary other bad gene introduced is less, there is good genetic stability, play a part very large to the developing of Wheat Germplasm Resources and breeding of new variety.The selection of inducing wheat-alien translocation line best approach depends primarily on the distance of exogenous chromosome and corresponding chromosome of wheat Characterization.

4.1.1 spontaneous transposition

In distant hybrid progeny, the chromosome that in same cell, wheat and xenogenesis belong to sometimes can rupture simultaneously, then wrong reclosing and spontaneous generation transposition, but the frequency that this translocation line produces is very low, and transposition fragment mostly is whole arm.The most outstanding advantage of spontaneous transposition is that genetic stability is good.The wheat-rye 1BL/1RS translocation line of extensive use in wheat breeding is exactly (Mettin D et al, 1973) that formed by this way.

4.1.2 utilize Ionizing Radiation-induced chromosome translocation

A kind of effective means that translocation line carries out genetic improvement is created in ionizing radiation, and ionizing radiation can make to be produced primary ionization and secondary ionization by irradiated material, and its character is changed.Light then the molecular structure of gene can be caused to change and produce gene mutation, heavy then chromosome random fracture can be caused, chromosome generation reclosing, it is the most popular method of bringing out chromosome translocation, the transposition of its induction is not confined between homoeologous chromosomes, and transposition effect is by the impact of nearly edge species and wheat Characterization distance and target gene position on chromosome.The requirement of ionization radiation induction chromosome of wheat transposition is also few, and material is a lot of sample also, the seed of such as alien addition line and alien substitution, amphidiploid, distant hybrid F1, premeiotic young fringe, meiosis stage strain etc.Since Sears utilizes the method for x-ray bombardment by the leaf rust resistance gene transposition of Aegilops umbelluata on chromosome of wheat, and name this transposition to be " Transfer " (Sears, 1956), wheat and haynaldia villosa (ChenP D et al has been obtained at present by radiation, 2005), Dalaicao (Wang Linsheng, 2008), rye (Sun Guangzu etc., 1990) translocation line and between the species such as Thinopyrum intermedium (Li Wen waits quietly, 2014) more than 30.

4.1.3 by regulating the effect of Ph gene to bring out chromosome of wheat transposition

Chromosome of wheat pairing is that the Ph1 gene on wheat 5B is long-armed is the gene of a control section homogenetic association by genetically controlled.When it exists, pairing is strictly limited between homologous chromosome; But when its disappearance or when undergoing mutation, also can match between homoeologous chromosomes.Therefore, by eliminating 5B chromosome, utilizing ph1b to suddenly change or utilizing height pairing gene (PhI) from Ae.speltoides, promote that the pairing of chromosome of wheat and exogenous chromosome in wheat distant hybrid is recombinated.Utilize a suitable external source substitution line or aneuploid, the chromosome segment of specific wheat homoeologous group is on purpose replaced with exogenous sequences, translocating to exogenous chromosome fragment on chromosome of wheat can the chromosome of wheat fragment of compensating missing in heredity, and produces best interaction.Utilize the method that inducing moiety homologous chromosome matches, obtained at least 20 translocation lines, comprise (Sears, 1973 such as E. elongata, Thinopyrum intermedium, Ae.speltoides; Dvorak, 1977; Wang, 1977).

4.14 utilize Plant Tissue Breeding to bring out transposition

Plant species, bigener after cultured in vitro, the heredity between parental set of chromosome exchanges to be increased, and regeneration strain there will be the chromosomal structural variation such as transposition (LaptinaNLV et al, 1984; Hu Han etc., 1999), therefore, the amphiploid of wheat-nearly edge species, alien addition line or alien substitution are hybridized as parent, Hybrids F1 is regeneration plant after cultured in vitro, likely from regeneration strain offspring seed selection to the alien translocation line of wheat-nearly edge species.Therefore, the method by group training obtains chromosome translocation to distant hybrid offspring and alien addition line or alien substitution.Larkin proposes the imagination being imported foreign gene by somaclonal variation to wheat the earliest, and by tissue culture technique, the gene against meloidogyne on rye 6R chromosome is imported wheat (Larkin et al, 1981).This technology is utilized the mildew-resistance gene of the anti gibberellic disease gene of the anti-wheat cecidomyiia gene of the anti-barley yellow dwarf gene of Thinopyrum intermedium, rye, Dalaicao, haynaldia villosa to be imported wheat (Banks PM et al, 1995; Old filial piety etc., 1996).

4.1.5 gametocidal chromosome is utilized to bring out transposition

In recent years, the gene transfer of some species of goatweed is finding by people in common wheat and durum wheat process, some goatweed is with gametocidal chromosome, when this chromosome is in hemizygous condition or heterozygous state, do not have the chromosome in the female and male gametophyte of gametocidal chromosome random fracture can occur, high-frequency induction generation can comprise the chromosomal structural variation such as transposition, disappearance.Compared with all kinds of physics, chemical means, its action character has high efficiency, multidirectional and stability etc.The research being brought out transposition by gametocidal chromosome is started late, but applies this approach and created the translocation line (Endo TR, 1990 that relate to leymus, Secale, Dasypyrum; Endo TR, 1985).

The qualification of 4.2 translocation lines

Genetic marker is the form of expression being easy to identify particular inheritance material.Utilize various genetic marker identify exogenous chromosome in Wheat Background or fragment existence, this selects significant to there being object to carry out wheat alien translocation line.Genetic marker mainly contains GISH, FISH etc. that morphological markers, cytological marker, biochemical marker, molecular labeling and cytological marker combine with molecular labeling, and they detect the Main Means importing exogenous chromosome in Wheat Background or chromosome segment.

4.2.1 morphological markers, cytological marker and biochemical marker

Morphological markers (Morphological markers) refers to the surface that clearly can characterize plant genetic polymorphism, as aristiform, shell look, grain look, fringe type, grain type etc.The morphological markers of broad sense, except above morphological characteristic, also comprises some development characters (as shooting stage, heading stage, maturing stage etc.), yield traits (as grain number per spike, thousand kernel weight etc.).Morphological markers is simple, cost is low and visual in image, is the genetic marker that people use in breeding process.But the plant characteristics limited amount of morphological markers can be used as, and polymorphism is low, is easily subject to the impact of the season of growth and environmental condition.Utilize these morphology to mark to carry out selecting mostly to need long Breeding Process, and require that breeder has abundant experience in breeding.

Cytological marker (Cytological markers) refers to the cytologic characteristic that clearly can show genetic polymorphism, conventional cytological marker has chromosomal architectural feature and quantative attribute, and they are respectively from structure with quantitatively reflect chromosomal genetic polymorphism.Chromosome morphology structure comprises chromosomal caryogram (number of chromosome number, chromosome absolute size and relative size, satellite number, secondary constriction and position, centric position etc.) and banding pattern, and (chromosome is after the aobvious band of specific stain, the shade be with, width and sequence of positions etc., can reflect euchromatin and heterochromatic distributional difference on chromosome thus, conventional banding technique has C band, G band and N band etc.).

Biochemical marker (Biochemical markers) refers to the technology utilizing the biochemical component acquired a special sense in vegetable metabolic process or product to carry out variety identification and genetic diversity mark.At present, isodynamic enzyme and seed storage protein are differentiation Wheat Species, belong to good biochemical marker, and they can provide larger different information, wheat heterologous gene can be detected more accurately.In addition, biochemical marker have cost less, fast, the advantage such as technical operation is relatively simple, be therefore widely used the Analysis and Identification of chromosome nonhomologous in common wheat.

4.2.2 molecular labeling

Molecular labeling arranges as mark with nucleotides sequence specific on chromosomal DNA.Due to the base type of DNA and the difference of number, the nucleotide that result in its composition is different.And nucleotide type and the difference put in order determine the difference that gene is formed.Therefore can utilize test at 2 or three point test whether chain with this distinguished sequence to detect certain proterties, if chain, that gene controlling this proterties is just positioned at this section chromosomal naturally.Molecular labeling has the superiority of the incomparable uniqueness of other several marks: the difference that (1) marks directly manifests with the form of DNA, plant individual Time and place all there is expression, therefore respectively organize plant, organ and developmental stage thereof all can detect, neither by season environmental limitations, there is not expression whether problem yet; (2) polymorphism is high, and plant corpus has existed many allelic variations under natural situation, avoids special creation particular inheritance material; (3) quantity is many, and mark is uniformly distributed in genome; (4) do not affect the expression of objective trait, and not with the bad linkage of characters; (5) major part mark shows as codominance (codominance), and isozygotying and heterozygosis of energy sldh gene type, provides complete hereditary information.Current molecular marking technique has been widely used in the research of Crop Genetic Breeding, and the most frequently used molecular labeling type mainly contains: RAPD, SSR, AFLP etc.

Along with developing rapidly of modern molecular biology technique, the progress of molecular marking technique, the chromosome segment that can detect is also more and more less, and the polymorphism detected is more and more higher, and molecular marking technique is used for the chromatinic detection of outer rim and becomes more simple and convenient.Therefore, will more and more new molecular labeling be had to be applied to the detection of wheat genetic variation and the qualification of allos new germ plasm.

4.2.3 in situ hybridization

In situ hybridization (In situ hybridization, ISH) be that one utilizes base pair complementarity principle, by there being the DNA hybridization of the strand on the DNA probe of radioactivity or nonradioactive labeling and chromosome after sex change, direct-detection alien chromatin and identify chromosomal technology.Utilize the DNA hybridization on DNA probe and target chromosome, on target chromosome, the molecular marking technique detected directly is carried out by detection system, utilize this technology traditional cytogenetics can be combined with modern molecular genetics, thus define an emerging cross discipline-molecular cytogenetics (moleeulareytogeneties).Hybridization in situ technique is from generation (Gall andPardue, 1969; John et al.1969) only have time of three, 40 years till now, become the important technical detecting heterochromatin (body) and gene or DNA sequence dna are carried out to physical mapping till now; Hybridization in situ technique is widely used in the research of the Characterization between the Origin and evoluation of homeologous relation in allopolyploid species between each chromosome set and species and species, has also played great function analyzing in the location of heterochromatin in different genetic background, behavior, expression and structure etc.Different according to the probe used in situ hybridization, 2 classes can be divided into: a kind of is using chromosome set or genome DNA as genomic in situ hybridization (the Genomic in situhybridization of probe, GISH) technology, a kind of is take cloned DNA fragments as fluorescence in situ hybridization (Fluorescence in situhybridization, the FISH) technology of probe.

The genomic in situ hybridization (GISH) that Durnam equals exploitation in 1985 is a hybridization in situ technique being intended to identify Heterologous genetic material.Be widely used in the current qualification at alien chromosome addition lines, substitution line and Introgressed line, gene location and chromosome identification etc.

Schwarzacher etc. (1992) utilize genomic in situ hybridization technology to identify the common wheat containing chromosome nonhomologous, prove from Leymus multicaulis (L.multicaulis) chromosome nonhomologous and the existence from common rye (S.cereale) chromosome arm.Chen Q etc. (1994,1996) utilize genomic in situ hybridization technology for detection to arrive in common wheat and carry couchgrass and the chromatinic composition of haynaldia villosa.Jia etc. (2002) genomic in situ hybridization and RFLP technology filter out the material that 15 strains contain Leymus multicaulis chromosome or chromosome segment in the filial generation of wheat-Leymus multicaulis (L.multicaulis).

Secale (the Friebe relating to wheat relative has been created at present by methods such as radiation, homologous chromosome pairing, tissue cultures and gametocidal chromosomes, et al.1991), Capra (Sears, 1956), Dasypyrum (Chenet al, 1995) and Genus Agropyron (Friebe etal, 1991; Sharmaet al.1997; Ohm et al, 2005; Zhang et al, 2004; Hao Weiwei etc., 2012) multiple translocation line such as.But the research major part for Genus Agropyron is all Thinopyrum intermedium and Thinopyrum ponticum, but seldom see the research report about Diploid Thinopyron elongatum grass translocation line.This experiment utilizes the method for radiation to obtain wheat Diploid Thinopyron elongatum grass translocation line first, and on the chromosome of wheat, the transposition chromosomal fragment of Diploid Thinopyron elongatum grass 7E, this fragment may be carried gene wheat scab to resistance.

Summary of the invention

The object of this invention is to provide the breeding method of one grow wheat-E. elongata anti gibberellic disease 7E chromosome long arm translocation line, the method utilizes ⁶⁰co irradiation treatment wheat-E. elongata 7E substitution line and Y16 hybridize F ₂for seed, the method screening wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line combined by form, molecular labeling and fluorescence in situ hybridization three.

The breeding method of wheat of the present invention-E. elongata 7E chromosome long arm anti gibberellic disease translocation line, comprises the following steps:

1) China spring-E. elongata 7E7B substitution line with raise wheat 16 and hybridize, hybridization F ₁f is obtained for seed selfing ₂for seed, by F ₂carry out for seed ⁶⁰co (Co 60) radiation, i.e. M ₁;

2) M is planted ₁for seed, select there is E. elongata morphological feature and scab resistance higher than negative control An8455 and China spring, the individual plant results of raising wheat 16, namely gather in the crops M ₂seed;

3) by M ₂for seed selfing, individual plant extracts M ₂for plant genomic DNA, first PCR detection is carried out with E genome molecules mark SM1-7E_1, SM1-7E_2, P7E_No.32 and P7E_No.36, select to have at least an E genome molecules mark to occur the sample of band, identify with E. elongata 7E long-armed specific molecular marker P7E_No.7, P7E_No.20, P7E_No.24, P7E_No.31, P7E_No.37, P7E_No.49, P7E_No.52, P7E_No.53 and P7E_No.56 again, select and wherein at least detect 1 long-armed specific molecular marker plant of 7E and carry out scab resistance detection; Results scab resistance higher than An8455, China spring, raise the individual plant of wheat 16; Namely M is gathered in the crops ₃seed;

4) by M ₃for seed selfing, select the same step 3) of authentication method, individual plant results M ₄seed; Namely successively carry out E genome molecules mark, the long-armed specific molecular marker of 7E and plant and carry out scab resistance detection, results scab resistance higher than An8455, China spring, raise the individual plant of wheat 16;

5) indoor germination M ₄for seed, get the tip of a root and carry out cytology film-making, the sectioning cells chosen carries out genomic in situ hybridization (GISH); In conjunction with the long-armed specific molecular marker qualification result of E. elongata 7E, filter out wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line.

In concrete practice, obtain 3 wheats-E. elongata 7E chromosome long arm anti gibberellic disease translocation line (W-7EL1, W-7EL2, W-7EL3) by the inventive method; 9 strains of existing 3 translocation lines.Obtain that wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line has isozygoty stable, can be used for the location of scab resistance gene in E. elongata, can as parent in the genetic breeding of wheat and quality-improving, the breeding of wheat-resistance to scab can be participated in, also can continue irradiation treatment screening and obtain wheat-E. elongata 7E chromosome long arm anti gibberellic disease Small piece transposition system.

This research and utilization 60Co (Co 60) irradiation treatment wheat-E. elongata 7E substitution line with raise F2 that wheat 16 hybridizes for seed, in the reproductive process in generation after irradiation, first carry out field scab resistance screening, select scab resistance height to have again the plant single-strain planting of E. elongata correlated characteristic.Utilizing E. elongata 7E chromosome long arm specific mark to detect being selected individual plant on this basis, carrying out wheat-E. elongata anti gibberellic disease translocation line finally by cytology and confirming.Utilize the screening of variable rate technology type, molecular markers for identification and cytology to confirm that three kinds of methods combine, create wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line.It is that wheat breed improvement and genetic research provide new germ plasm or basic material that this research institute obtains anti gibberellic disease transposition, can be used as the cultivation that parent participates in wheat-resistance to scab.In the filial generation that this translocation line participates in, the selection of economical character can be carried out routinely, and scab resistance early in generation, can by molecular marker assisted selection, retain the good and individual plant with E. elongata 7E chromosome segment of economical character to breed, and the inoculated identification of head blight is carried out again offspring, can save early for head blight inoculated identification, and obtain the High-yield Wheat Varieties with E. elongata anti gibberellic disease kind matter.In addition, the whole arm translocation system that this research institute obtains, utilize this germplasm materials to continue radiation and selection, the wheat-E. elongata 7E chromosome long arm Small piece transposition system of anti gibberellic disease will be obtained, in Genus Agropyron, material foundation has been established in the further investigation of anti gibberellic disease gene.

Accompanying drawing explanation

Fig. 1. the stability of molecular labeling and specificity identification result

M is that marker2501,1-6 are respectively 1:DA7E 2:DA7ES 3:2X 4:DA7EL 5:CS 6:Y16

Fig. 2 .M ₂for molecular markers for identification result

M is marker2501,1-18 is M ₂for each individual plant, positive control 2X and negative control Y16

Fig. 3 .M ₃for molecular markers for identification result

M is marker2501,1-21 is M ₃for each individual plant, positive control position 2X, negative control CS and Y16

Fig. 4 .M ₄for genome breeding qualification result

Embodiment

Embodiment 1

One, experiment material

Common wheat China spring (CS), Diploid Thinopyron elongatum grass (EE) (2X), China spring-E. elongata disome substitution line: DS7E7A, DS7E7B; China spring-E. elongata disomic addition line: DA7E, DA7EL, DA7ES (above 5 materials are so kind as to give by the Dr.Fedax of Ministry of Agriculture Canada, and material original is " Disomic and ditelosomic additions ofdiploidAgropyron elongatum chromosomesto Triticum aestivum "); Raise wheat 16 (Y16, namely raising 0-126 is that the institute of agricultural sciences of Lixiahe region in Jiangsu is with raising 91F138 and raising 90-30 cross breeding), above-mentioned material is provided by the lane housing institute of agricultural sciences of Jiangsu Province, all has commercially available; Peace agriculture 8455 (An8455, Anhui academy of agricultural sciences is bred as), Soviet Union wheat 3 (Su3, the Suzhou District institute of agricultural sciences utilizes Funo and Taiwan wheat to be bred as in nineteen sixty-eight) are number for having kind by oneself in laboratory).

Two, the extraction of genomic DNA

For examination Material growth to two leaf one heart stage, extract genomic DNA with SDS method.Its step is as follows:

(1) get young leaflet tablet (about 0.1g), shred in the centrifuge tube loading 2ml, be placed in liquid nitrogen and cool, be crushed to grinding rod Powdered;

(2) centrifuge tube is positioned over room temperature and slightly cools, and adds the buffer A of 700 μ l, mixes gently, then 65 DEG C of water-bath 20min, and period turns upside down mixing once every 5min;

(3) taking-up is slightly cooled to room temperature, adds phenol and each 350 μ l of chloroform, turns upside down, fully mix, extracting 5min;

(4) 12000rpm, centrifugal 10min, be drawn in a new centrifuge tube by supernatant;

(5) add about 750 μ l chloroforms, turn upside down, fully mix, extracting 5min;

(6) 12000rpm, centrifugal 10min, be drawn in a new centrifuge tube by supernatant;

(7) add about 560 μ l isopropyl alcohols, treat mixing without proper respect, room temperature places 10min, visible flocculent deposit;

(8) 12000rpm, centrifugal 10min, abandoning supernatant;

(9) ethanolic solution adding 500 μ l 70% washes twice, and each 2-3min. room temperature is dried;

(10) DNA dried is dissolved in 20 μ l TER, and 37 DEG C of temperature are bathed 45min.-20 DEG C and saved backup.

Three, chromosome molecular labeling stability and specific detection

Molecular labeling for carrying out chromosome or chromosome arm qualification comes from early-stage Study (the Chen et al.The Developmentof7E Chromosome-Specific Molecular Markers for Thinopyrum elongatum Based on SLAF-seq Technology.PlosOne of this research department, 2013,8 (6): 1-8; Qin Shuwen etc., based on exploitation and the application of the E. elongata SCAR mark of TRAP. wheat crops journal, 2014,34 (12): 1595-1602).The molecular labeling that strong and Qin Shuwen develops to the old generation in this laboratory carries out stability and specific detection.Material for detecting is DA7E, DA7EL, DA7ES, 2X, CS, Y16.If DA7E, DA7EL, DA7ES, 2X all occur band, then this molecular labeling is E genomic marker; If band appears in DA7E, DA7EL, 2X, then this molecular labeling is the long-armed specific molecular marker of 7E; If band appears in DA7E, DA7ES, 2X, then this is labeled as 7E galianconism specific molecular marker; All there is not band in above-mentioned CS, Y16.

The system that PCR detects and program are in table 1 and table 2, and the molecular labeling of screening is in table 3, and stability and specificity identification the results are shown in Figure 1.

Table 1:PCR reaction system

Table 2:PCR response procedures

Table 3: identify 7E chromosome specific molecular marker used

To M ₁13 portions of wheats for individual plant results carry out individual plant results and plant plantation.Extract M in 13 plants ₂individual plant DNA, molecular marker screening contains the chromosomal individual plant of E. elongata 7E, part the selection result as shown in Figure 2: the M in this time detecting representated by 7,8 and No. 12 swimming lanes ₂there is band for Wheat DNA through the long-armed specific molecular marker detection of E. elongata 7E, fragment long-armed containing E. elongata 7E in the chromosome of these wheats is described.Qualification result is positive and the strain of individual plant results wheat 7.

To M ₂withhold acquisition 7 portions of wheats and carry out individual plant results and plant plantation.Extract M in 7 plants ₃individual plant DNA, molecular marker screening contains the chromosomal individual plant of E. elongata 7E, part the selection result as shown in Figure 3: the M in this time detecting representated by 5,6,7,10,11,12,13,18,20,21 swimming lanes ₃there is band for Wheat DNA through the long-armed specific molecular marker detection of E. elongata 7E, fragment long-armed containing E. elongata 7E in the chromosome of these wheats is described.Qualification result is positive and the strain of individual plant results wheat 21.

Four, scab resistance detects

M is selected in the annual wheat flower initial stage in April ₁, M ₂, M ₃radiation Progenies of Wheat and positive control SU3, negative control An8455, CS, Y16 are using the left base portion little Hua of the 5th small ear from top to bottom as inoculation unit, adopt single flower inoculation method inoculation gibberella spore suspension 5 μ l, pathogen is provided by the Lixiahe District institute of agricultural sciences, for local velogen strain F4, F5, F17, F34 (Zhao Han etc., 2002), bacterial concentration is visible about 20 free spores under 10 × 10 fields of microscope.Bagging three days or timing every day water spray after inoculation, with the humidity keeping fringe portion higher, inoculate latter 21 days investigation incidences.The investigation of morbidity result, in units of individual plant, gathers the result that each per plant on average can be used as full line.Scab resistance is to represent with the percentage of sick spikelet number divided by total spikelet number, and the material percentage that scab resistance is good is little.The resistance of all being carried out to head blight each generation after radiation is selected, the M1 individual plant Fusarium-resistance selected is different, but have the feature of E. elongata, the later Single-plant selection of M2 requires both had the amplification of chromosome specific molecular marker, and Fusarium-resistance is good again.

The mark of table 4. wheat-E. elongata translocation line pedigree and different generations detects and head blight qualification

Five, the specific molecular marker of experiment material detects

To M ₂, M ₃radiation self progeny, individual plant extracts the PCR detection that genomic DNA carries out specific molecular marker.PCR system and program are in table 1 and table 2, and molecular labeling used is in table 3.The wheat plant that continuous 2 years E. elongata 7E Markers for Detection go out is in theory containing the chromosomal fragment of E. elongata 7E.

Six, cytology film-making

Continuous 2 years long-armed specific molecular markers of E. elongata 7E are detected and the M4 that gathers in the crops higher than the individual plant of negative control An8455 and CS, Y16 of scab resistance for wheat seed, carry out cytological detection.

(1) tip of a root process

1. seed is soaked in water about 2h be placed on bottom be covered with in the culture dish of wetting filter paper, after 25 DEG C of incubators are cultured to and put into 4 DEG C of refrigerator dark processing 1-2 days when showing money or valuables one carries unintentionally, continue to cut the tip of a root being about 0.5mm when 25 DEG C of incubators grow to the tip of a root long about 1-2cm.

2. the tip of a root is put into 1.5ml dactylethrae, topped up with water is placed in ice and puts refrigerator (4 DEG C) about 22h.Effect: make chromosome pyknosis, be convenient to chromosome number inspection.

3. the taking-up tip of a root is put in another dactylethrae and adds Kano fixer (absolute ethyl alcohol 3:1 acetic acid) 1ml.The tip of a root can be placed by longer-term at 4 DEG C.

4. about 24h fixed by Kano fixer, in culture dish, the tip of a root rinsed 2 times with clear water, each 5 minutes.

5. the flushed tip of a root is put into new 1.5ml dactylethrae, adds 2% aceto-camine (acetocarmine), 4 DEG C of refrigerator 24h make the tip of a root dye.

(2) direct tablet compressing

1. cut Root apical meristem district during film-making on slide, add 45% acetic acid, cover cover glass, knock, make organization of root tips broken and to surrounding diffusion, disperse as seen with material with the tweezers point contrast tip of a root, it is degree that chromosome remains in same cell substantially.

2. alcolhol burner baking, not overheated, then spread filter paper, with thumb align material compressing tablet, whether strength is according to Chromosome spread and determine at same plane.

3. microscopic examination, the film-making that Chromosome spread is good is put in ultra low temperature freezer, and at least 24h takes off cover glass, then-20 DEG C of Refrigerator stores (ultra low temperature freezer can be preserved for a long time) after 70%, 85%, 100% Gradient elution using ethanol.

Seven, genomic in situ hybridization (GISH)

(1) mark of probe

1. 1ug template DNA (i.e. China spring genomic DNA) is dissolved in 16ul sterilizing deionized water, or draws the concentration known DNA of a certain amount of (ul), make DNA quality be equivalent to 1ug.

2. press cumulative volume 20ul, add 4ul Dig-Nicktranslation Mix, mixing, simply once centrifugal.

3.15 DEG C of reaction 90min.

4. add 1ul 0.5M EDTA (pH8.0), cessation reaction, 65 DEG C, reaction 10min.

5.-20 DEG C Refrigerator store.

(2) kit is utilized to carry out the detection of label probe

1. get 1ul contrast Dig-DNA (standard DNA be equipped with inside kit) and utilize digoxin (Dig) kit to mark China spring genomic DNA at hybond membrane (10X3cm, quantity according to probe determines hybond membrane size), more than 80 DEG C of half an hour be fixed after drying at room temperature.

2. be placed in culture dish by hybond membrane, add 10ml cleaning solution (Washingbuffer), room temperature washing 10 minutes, can shake at a slow speed.

3. add 50ml blockade working solution (Blocking solution) washing, 2 times, each 15 minutes.

Washing 30 minutes in 4.10ml antibody response liquid (Anti-body solution).

Wash 2 times in 5.10-20mlWashingbuffer, each 15 minutes.

6.20ml Detectionbuffer neutral equilibrium 5 minutes.

Carry out staining reaction (dark, not rock) in the variable color matrix liquid (color substrate solution) of the fresh configuration of 7.10ml, a few minutes to 16 hour, observation color is carried out, to occur that namely color terminates in midway.Hybond membrane is put into the distilled water 5 minutes of 50Ml, taking-up is dried.

(3) fluorescence in situ hybridization step

1, taken out by the slide at low temperature refrigerator, soaked in absolute ethyl alcohol 30min-2h under room temperature, dries.

2, preparing hybrid fluid component (40ul), operates (table 5) on ice.

Table 5: hybridization solution formula

3, the slide dried is placed in the formamide of 70%, sex change 70S in 70 DEG C of water-baths, takes in 70%, 90%, 100% alcohol immediately ,-20 DEG C of 5min that dewater successively.

4, gas is done, and drip hybridization solution, covered, 37 DEG C of temperature bathe at least 6h ~ 24h.

5, wash by table 6.

Table 6: slide washing methods

6, slide adds 50ulAnti-Dig-Rhodamine-Fab working solution (concentration 1ug/ml, get 2ul mother liquor and add 198ul 1%blocking solution, blocking solution maleate buffer is prepared), covered, put in 37 DEG C of wet boxes, temperature bath 60min.(requiring after this step in the dark to operate)

7, take out slide room temperature 1 × TNT wash-out 3 times, each 5min, is placed in dark place and dries.

8,15ul DAPI is dripped, covered, fluorescence microscopy Microscopic observation.

To M ₄in situ hybridization partial results is carried out as shown in Figure 4 for wheat seed; Be divided into 3 strain W-7EL1, W-7EL2, W-7EL3.W-7EL1 and W-7EL2 is the long-armed whole arm translocation system of E. elongata 7E, and isozygotys; And the W-7EL3 Small piece transposition that to be E. elongata 7E long-armed, 2 fragments of transposition are respectively on 2 chromosomes of wheat.

Agents useful for same formula in above-mentioned steps seven:

1) Dig-Nick translation Mix (article No.: 11745816910); , anti-dig-fluoresence-solution for being Roche Holding Ag's kit, containing 160ul Dig-Nick translation Mix.

2) Anti-Dig-rhodamine-Fab (No:11207750910), these commodity are 200ug powder, the 1%Blocking solution of 2ml is utilized to be mixed with mother liquor (or other solution of specification recommendation) during use, concentration is 100ug/ml, i.e. 100ng/ul, packing can be stored in-70 DEG C, avoid repeatedly melting when using.

3) EDTA (PH=8.0): Na2H ₂o 186.1g, ddH ₂o is settled to 1L, regulates PH to 8.0

4) DS formamide: 20g dextran sulfate (DS) is dissolved in formamide stoste, sealing is heated to 60-65 DEG C and stirs evenly, and continues to add heat shock to dissolving, and is settled to 100ml, rapid packing ,-20 DEG C of preservations.

5) 20 × SSC: sodium chloride 175.32g, trisodium citrate 88.23g, adds ddH ₂o is settled to 1L, regulates PH to preserve to 7.0 sterilizings.

6) 70% formamide: formamide 70ml, 20 × SSC10ml, ddH ₂o 20ml.

7) 50% formamide: formamide 50ml, 20 × SSC10ml, ddH ₂o 10ml.

8) 10 × TNT:Tris-Base 121.1g, concentrated hydrochloric acid 49ml, NaCL 87.8g, Tween205ml, ddH ₂o is settled to 1L, regulates PH to 7.5 sterilizing to preserve.

9) maleate buffer (Maleic acid buffer): 500ml:Maleic acid (maleic acid, maleic acid Mr=116) 5.8g; NaCl 4.3875g;

DdH2O is settled to 500ml.

10) to blockade liquid:

Mother liquor (10%): 10g blocking reagent adds 100ml Maleic acid buffer, heating for dissolving.

Working solution (1%): utilize 10ml blocking reagent to add 90ml Maleic acid buffer and carry out diluting (prevention unspecific binding sites).

11) concentration of the anti-color fading agent (1000H) containing DAPI: DAPI is 100ug/ml.

Claims (1)

1. the breeding method of wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line, comprises the following steps:

1) China spring-E. elongata 7E7B substitution line with raise wheat 16 and hybridize, hybridization F ₁f is obtained for seed selfing ₂for seed, by F ₂carry out for seed ⁶⁰co (Co 60) radiation, i.e. M ₁;

2) M is planted ₁for seed, select there is E. elongata morphological feature and scab resistance higher than negative control An8455 and China spring, the individual plant results of raising wheat 16, namely gather in the crops M ₂seed;

3) by M ₂for seed selfing, individual plant extracts M ₂for plant genomic DNA, first PCR detection is carried out with E genome molecules mark SM1-7E_1, SM1-7E_2, P7E_No.32 and P7E_No.36, select to have at least an E genome molecules mark to occur the sample of band, identify with E. elongata 7E long-armed specific molecular marker P7E_No.7, P7E_No.20, P7E_No.24, P7E_No.31, P7E_No.37, P7E_No.49, P7E_No.52, P7E_No.53 and P7E_No.56 again, select the plant wherein at least detecting 1 long-armed specific molecular marker of 7E and carry out scab resistance detection; Results scab resistance higher than An8455, China spring, raise the individual plant of wheat 16; Namely M is gathered in the crops ₃seed;

4) by M ₃for seed selfing, select the same step 3) of authentication method, individual plant results M ₄seed;

5) indoor germination M ₄for seed, get the tip of a root and carry out cytology film-making, the sectioning cells chosen carries out genomic in situ hybridization; In conjunction with the long-armed specific molecular marker qualification result of E. elongata 7E, filter out wheat-E. elongata 7E chromosome long arm anti gibberellic disease translocation line.