CN102342244A - Technology for pre-selecting and breeding new species of apple in early stage - Google Patents
Technology for pre-selecting and breeding new species of apple in early stage Download PDFInfo
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Abstract
The invention relates to a selective breeding procedure of dwarf type apples (comprising dwarf type, semi-dwarf type and dwarf-shoot type apples and having a dwarfing function and a dwarf potential), and the procedure combines isozyme early-stage pre-selection and conventional selective breeding. The isozyme mark is high in reliability, strong in stability and high in accuracy (86.14%) and is used for carrying out early-stage pre-selection on hybrid seedlings of the current year, so that the selection efficiency can be improved and the selective breeding progress can be accelerated. Therefore, a large amount of good individual plants can be obtained in a short period of time. An isozyme early-stage pre-selection special test is used for adopting the selective breeding procedure provided by the invention to 60 hybridized combinations comprising 1535 seedlings so that the selective breeding progress can be obviously accelerated, the selection efficiency is improved, the selection rate of the individual plants is 1.2% and more than 20 good plant systems are bred in more than ten years, wherein 8 species are identified by the specialists. Therefore, the technology provided by the invention has an important meaning for the improvement of an apple breeding procedure.
Description
(1) this invention belongs to apple new variety selective breeding technology field
Crossbreeding and bud mutation breeding are generally adopted in current domestic and international apple breeding; Characteristics are by the form Standard Selection; But apple is a heterozygote, and genetic background is very complicated, is difficult for research; And the tree volume morphing constitutes the influence that is subject to external environment and damage by disease and insect; Apple is perennial seeds in addition, and growth cycle is long, the habit that all kinds of histoorgans of bearing tree grow; Characteristic, being difficult to conclude treelet has increased the breeding work difficulty greatly.Therefore, many scholars both domestic and external extremely pay close attention to the especially short living habit preliminary election of the early stage preliminary election of apple.Just begin broad research has been made in the short living habit and the heredity thereof of apple as far back as the twentieth century KOLapins sixties etc., but because the lability of form sign has a strong impact on the research work progress.This invention is in the isodynamic enzyme sign of research apple development habit in 1979.It is reported that peroxidase enzymes is closely related with growth, in this research, find peroxidase enzymes E9 band and the short living habit related intimate of apple, therefore with the short living habit sign of E9 band as the dwarf form apple.1984, confirm " peroxidase enzymes is as the preliminary election index of 1～2 year living dwarf form apple seedling " through expert appraisal, acquisition Ministry of Agriculture scientific and technological progress third prize in 1988.Nineteen ninety 23 international gardening can on deliver three pieces of papers after, receive colleague's expert favorable comment, and should " Acta Horticaturae " magazine make an arrangement in advance, published in instalments three pieces of papers.
In order to study the application of dwarf form apple isodynamic enzyme sign in breeding, in the period of 1992,1993, select different plain edition and inhomogeneous cultivars such as dwarf form or brachyplast type of ripe phase for use, combine dissimilar combinations.The method that its seedling is combined with conventional seed selection with the early stage preliminary election of isodynamic enzyme sign; Carry out systematic breeding; The a collection of ripe phase that obtained to 1996 is master's a different tree-like elite plant strain with the brachyplast result early, and later emphasis is to color and luster, the further seed selection in quality aspect of fruit.August 2010?
, by the experts through eight lines, agreed that the precocious strain is a rare quality fine lines, should be expanded in the Yellow River and other places to try to grow, to play its role in early advantage.
(2) purpose of this invention
1, one of purpose of this invention is: short living type (comprise short life, semidwarf, compact, brachyplast type have dwarfing characteristics) kind is current domestic and international production development direction.Setting up the early stage preliminary election technology of dwarf form apple, raise the efficiency, accelerate the breeding progress, is this invention purpose.
2, two of the purpose of this invention are: select short life (emphasis is semidwarf, compactness, brachyplast type), precocity, the apple new varieties of high-quality.
3, three of the purpose of this invention is to set up the seed selection program that the early stage preliminary election of apple combines with conventional seed selection, to improve efficiency of selection, reduces the randomness of selecting, and accelerates the breeding progress.
(3) main contents of this invention
1, the isodynamic enzyme sign of dwarf form apple: the reliability of isodynamic enzyme sign, stability, be suitable for extensively, be the successful key of the early stage preselector of decision; Therefore, 1. require method simple and easy, sampling does not injure whole strain; Easy and simple to handle, good reproducibility, and strict operation sequence and the sampling standard of regulation; 2. require this isodynamic enzyme sign to be applicable to the dissimilar kinds of 22 kinds of Malus, stock, hybrid seedling; 3. require this index genetic stability reliable.
2, the seed selection program that combines with conventional seed selection of the early stage preliminary election of apple is a core of the present invention.This invention adopts medium-sized and dwarf form, and 25 of early, middle and late ripe phase different cultivated kinds combine 60 hybrid combinations.To by seedling then, carry out early stage preliminary election, eliminate large quantities of seedlings that do not have the isodynamic enzyme sign, the seedling with isodynamic enzyme sign is carried out conventional seed selection.3 mu of grounds, in 10 years, seed selection obtains a collection of precocity, high yield, the new lines of high-quality.
(4) advantage of this invention and effect
1, method is fast and convenient.Adopt the starch-gel electrophoresis technology, can analyze a large amount of samples every day;
2, sampling does not injure whole strain, gets seedling stem elongation zone cortex or blade 50～100mg then, and original seedling can continue to send out branch growth, does not influence next step conventional seed selection.
3, the popularity of isodynamic enzyme sign: in the period of 1985～1987; 212 types to 22 kinds of apple germ plasm resource; 95 of different stock types; 88 of cultivars; Totally 395 individual plants; Isodynamic enzyme sign and short living habit correlation analysis result, its accuracy rate has certain generality about 86.14%.
4, isodynamic enzyme sign, genetic stability property.Combine 128 of dissimilar hybrid combinations according to cultivar stock by 88 different habit of growth; With Isozyme Analysis 7987 strains give birth to seedlings then; The result shows; The isodynamic enzyme sign---the E9 band is controlled by two pairs of complementary gene; Genetic stability is not subjected to the influence of the age of tree, stock, cultivated area.
5, the seed selection program that combines with conventional seed selection of early stage preliminary election.Breeding cycle is short, efficient is high, only according to 60 hybrid combinations after this procedure Selection, in select 16 combinations, selecting rate in the combination is 27%, by 1535 strain seedlings, in select 19 excellent strains, selecting rate in the individual plant is 1.4%.The apple seed selection program of this invention can improve efficiency of selection significantly, has accelerated the breeding progress, and breeding is significant to apple.
(5) embodiment of detailed description this invention
1, starch-gel electrophoresis:
Different zymoprotein molecules is owing to carried charge, charged different in kind, and direction, speed mobile in the electric field that with the starch gel is holder are different.So different zymoprotein molecule translational speeds are different, different separation bands occurs.With peroxidase substrate dyeing, can show different peroxidase enzymes bands.The Different Organs of apple tree body, tissue, cell peroxidase enzymes kind are different, the different cultivars habit difference of growing, and the kind of peroxidase enzymes is also different.Therefore, the kind of different habit of growth effectively can be separated, the dwarf form apple is held the distinctive mark band, E9 enzyme band, and promptly peroxidase is a kind of, and it only is present in the cortex or corresponding blade of apple stem elongation zone.The present invention is as dwarf form isodynamic enzyme colored belt.
The starch-gel electrophoresis method
1. glue: take by weighing 12g hydrolysis potato starch, pour in the 500ml triangular flask, add 100ml gel buffer liquid, the limit heating; The limit is shaken, and dissolves fully to starch to be transparence, comes to life; Use the negative pressure of 500～600mmHg immediately, bleed, remove the bubble in the gel.Pour subsequently that (Fig. 1 a) covers lastblock mylar and glass plate while hot, presses a weight, cools off 1.5～2.0 hours, gets final product sample introduction in the preprepared mould into.
2. sample preparation: take by weighing the blade 50～100mg of apple seedling stem elongation zone or corresponding site, add sample buffer solution 0.25～0.5ml, produce the homogenate supernatant.
3. sample introduction: shown in figure (1b), with comb at negative electrode gel end 4～6cm place point of contact appearance mouth.Dip in enzyme extract with No. 3 filter papers of Xinhua, insert in the gel otch, cover dacron membrane, glass plate gets final product electrophoresis
4. electrophoresis is poured electrode buffer into electrophoresis tank by shown in Figure 2, does not have the plate washer hole, will advance excellent gel and be placed on the electrophoresis tank, and two ends are put up gauze, immerses electrode solution, covers the terylene glass plate, energized.Electrophoresis is under 1～5 ℃ of condition, to carry out, logical steady current, and every centimetre keeps 2mA, electrophoresis 4～6 hours.
5. cut glue: after electrophoresis finishes, gel is put in the mould with glue, beneathly filled up a glass plate that 1～2mm is thick again, with filament transverse cuts offset plate, offset plate of every cutting-out places in the staining jar.
6. dyeing: 0.5mol/L acetate buffer solution, PH7.050ml, benzidine 50mg; 3%H
2O
210, with the film behind the electrophoresis, be immersed in the dyeing liquor, show blue chromatographic band under the room temperature immediately, spend the night with the running water flushing behind the 30mim, show Chinese red, system for photographing.
2, the stability of isodynamic enzyme sign, reliability
1. the reliability of isodynamic enzyme sign
Dwarf form isodynamic enzyme sign: in Malus, have ubiquity meaning; Malus dissimilar short living habit not of the same race and isodynamic enzyme sign are in close relations; 17 dissimilar resource analyses of kind show according to Malus, and its short living habit is consistent with the isodynamic enzyme sign, accuracy rate is 90.0%; Analyzed 41 dissimilar dwarfing rootstocks, its nanism (dwarfing effect) is with to have an isodynamic enzyme sign consistent, and accuracy rate is 85.37%; Analyzed 51 dissimilar cultivars, its short living habit is with to have an isodynamic enzyme sign consistent, and its accuracy is 81.85%.Mean value is 86.14%.Show that dwarf form isodynamic enzyme sign has extensive applicability in Malus.
2. isodynamic enzyme sign biological stability: dwarf form is not subjected to the influence of the age of tree, tree body developmental stage with enzyme sign down.By state's light, golden hat, Fuji, Red Star, new Red Star; Cultivars such as brachyplast marshal combine seven hybrid combinations, and the age of tree is analyzed respectively relatively respectively in 7～13 years; Isodynamic enzyme colored belt in sharp sprout tillers cortex of its elite stand young sprout or the corresponding site blade, totally 446 strains.Isodynamic enzyme sign in elite stand young sprout and its sprout tillers is in full accord as a result, the influence of not set the body developmental stage;
2 ages and 2～3 monthly age seedlings have been analyzed by state light * marshal 158 combinations; 2 ages, 6 ages and 2～3 monthly age seedlings of Fuji * new Red Star combination; 2 ages, 5 ages and 2～3 monthly age seedlings of state's light * brachyplast marshal combination.The isodynamic enzyme sign of the isodynamic enzyme sign of 2～3 monthly age seedlings 2～6 years living treelets correspondingly is in full accord as a result.
Analyzed by state light * M7, two combinations of state light * M8; Totally 64 strains; Since 2 ages at monthly age to 3; Decide the strain analysis and observation 2 every year; Each individual plant is since 2 ages at monthly age to 3 as a result, and the isodynamic enzyme sign is in full accord in its young sprout cortex or the corresponding site blade, shows; Two monthly age seedlings just can carry out early stage preliminary election, are not subjected to the age of tree and budding the influence.
Dwarf form isodynamic enzyme sign is not subjected to the influence of stock, and rose-red, new Red Star, cigarette green grass or young crops, golden short life, Red Star, Fuji's kind are distinguished grafting at M7, on dissimilar anvils such as MM106, MM111, M27; State's light, gold hat, red hat, Fuji, Red Star kind are distinguished grafting, and tea fruit is on the interstock of root anvil with Malus baccata 22 of MM104, MM106, MM111, M2, M4, M5, M9, M26, M27 etc., forms 48 interstocks/root anvil combination; New Red Star, Red Star, cigarette green grass or young crops, golden short life, short red height are connected on the big tree of red hat.The isodynamic enzyme sign of all different cultivars scion as a result is the same with former kind, the influence that not connect by root anvil, interstock, height.
3. the genetic stability property of isodynamic enzyme sign: hybridize each other with 39 parental breeds; Backcross, reciprocal cross combines 58 combinations; Obtain 3515 F1 for the crossbreed seedling; Make isodynamic enzyme sign compartment analysis; The seedling that the result has the isodynamic enzyme sign has AABB, AaBB, AaBb, four kinds of genotype of AABb, and does not have the seedling of isodynamic enzyme colored belt to have AAbb, Aabb, aaBb, aaBB, five kinds of genotype of aabb.As a result, found out the genotype of 39 parent's isodynamic enzyme signs.For further proving the genetic development of isodynamic enzyme sign; Adopt 88 cultivars; Combine 128 hybrid combinations each other; Wherein plain edition * medium-sized has 38 combinations, plain edition * dwarf form 63 combinations are arranged, dwarf form * dwarf form 27 combinations are arranged; Obtain 7987 strain seedlings altogether; Prove for the separating resulting of seedling isodynamic enzyme sign that by these F1 isodynamic enzyme sign (E9) is to be subjected to two pairs of complementary gene of A_B_ to control genetic stability in the apple seedling.
3, select effect
1. nanism: 1986, qualification result that fruit tea is compared in 1987; 135-1,35-3,41-6,66-11,61-18, the branch analogy of short life of 22-62 autumn excellent strains such as (lifes in 5～9 years), new-tip length with isodynamic enzyme sign, length of internode and with short looks ratio of autumn.The average brachyplast number of result, middle branch number, the long shoot ratio of counting are respectively 59.4%, 24.43%, 16.8%, and on average young sprout is long is 42.63cm, and average internode is long to be 1.42cm branch class ratio, new-tip length, and length of internode is all near with short looks of autumn.Show to have the isodynamic enzyme sign, these strains, kind all have significant short living habit.Hybrid seedling (2～3 age) 160 strains have been identified equally again with isodynamic enzyme sign; The result is short, in, the long shoot ratio is respectively 75.61%, 21.29%, 3.10%; Average new-tip length is 16.63cm; Average internode is long to be 1.25cm; These have its analogy of seedling of isodynamic enzyme sign; Young sprout is long, internode is long, meets short living habit standard.
Above-mentioned examination material, all be earlier after conventional seed selection again with isodynamic enzyme sign screening material.Below with after the early stage preliminary election of isodynamic enzyme sign short living habit being selected effect: by 60 hybrid combinations; 1535 strain seedlings; 2～3 monthly ages; After the early stage preliminary election of isodynamic enzyme; Through conventional 20 individual plant systems of 16 combinations with isodynamic enzyme sign of selecting acquisition, its characteristic all has the brachyplast habit again, and brachyplast is than between 60～80%; With the brachyplast result is main, and fruit-setting rate is high.With the early stage preliminary election of isodynamic enzyme sign to improve short living habit in to select rate be effective.
2. prematurity: to select prematurity is main target, by 35 different parents of ripe phase, combines late * 16 of combinations in evening, and 25 of evening * middle combinations are made up 6 evening * morning, in * make up 5 morning, in * 8 of middle combinations, totally 60 combinations.Obtain hybrid seedling 1535 strains, after the early stage preliminary election of isodynamic enzyme, continue to select by conventional seed selection program again; Select 16 combinations in last; 68 individual plants, these individual plants promptly begin bow structure really at the 2-3 age, in wherein having * 1 strain of combination early; 60 days ripe phases; 30 strains of combination evening * morning, ripe phase 80-95 days, 18 strains of evening * middle combination; Ripe phase 90-120 days, combination in evening * evening; 95～105 days ripe phases of 16 strains; All the other ripe phases of 3 strains were at 150-160 days.With the early stage preliminary election of isodynamic enzyme sign can improve the prematurity individual plant in select rate.
3. efficiency of selection: the program research that the early stage preliminary election of isodynamic enzyme sign combines with conventional seed selection starts from 1992, in the period of 1993; Combine the hybrid seed of 60 combinations with 39 parents, sowed and carried out the early stage preliminary election of isodynamic enzyme in 1993, press morphological index later on again and select; Robust plant was selected in strict screening through 1994, and the short leaf of branch is fertile; Look dark green; Bud is full, individual plant 306 strains of no damage by disease and insect, in the period of 1996～1997 the beginning bow structure fruit; Selected 16 combinations, 68 individual plants best in quality.Obtain 8 of fine combination to final election in 1999, strain is 22, and combination selection efficient is 13%, and the individual plant efficiency of selection is 1.4%.The program that adopts the isodynamic enzyme sign to combine with conventional seed selection only in 7 years, on 3 mu of nursery lots, has been accomplished 60 hybrid combinations, whole selection courses of 1535 strain seedlings, and obtained good selection effect.Adopt this seed selection program can improve apple seed selection progress significantly really, and can save resource.
4, option program: build up the high efficiency selected program (Fig. 3) that is combined with conventional seed selection by the early stage preliminary election of isodynamic enzyme sign, special evidence is characterized in clearly defined objective, and efficiency of selection is high, and the cycle is short, and progress is fast, can improve resource utilization.Result of the test is following:
1. parent's selection and assembly: select for use the high yield level high, wide adaptability, what comprehensive resistance was strong carries kind as the landlord, like Fuji etc.; Select for use to meet the breeding objective requirement, the precocious germplasm that yielding ability is good, short living habit germplasm, the germplasm that the habit of yielding positive results is good, the germplasm that comprehensive resistance is strong selects the strong germplasm of objective trait heritability etc. for use.Select 39 parent materials altogether; Combine 60 hybrid combinations altogether; Wherein there is system of Fuji to account for 20.3% for parent's hybrid combination; Late variety accounts for 38.9% for parent's hybrid combination, and middle seasonal strain accounts for 32.2% for parent's hybrid combination, and early maturing variety accounts for 8.4% for parent's hybrid combination; Obtain 7137 of hybrid seeds; Numbering is given in 1535 strains of improved crossing kind seedling in the period of 1992～1993, lists.
2. early stage preliminary election: according to the requirement of dwarf form isodynamic enzyme sign, to 2～3 month seedling carry out early stage preliminary election, eliminating does not have isodynamic enzyme sign seedling, accounts for 40-50%, to having the seedling of isodynamic enzyme sign, gets into the conventional seed selection stage.
3. conventional seed selection: in the period of 1993～1994 seedling with isodynamic enzyme sign is carried out systematic breeding; Eliminated the individual plant that wild proterties is arranged before this; Like sagging, the long spinosity of branch, overgrow, blade is small and weak, plant strain growth individual plant that do not rectify, that damage by disease and insect is not arranged; Select robust plant, branch is short and thick, blade is loose, look green, bud is big, full, plant strain growth is rectified, individual plant 306 strains of no damage by disease and insect; And grafting and eight rib Malus spectabilis; M26, height are connected to big tree, observe the growth flowering habit.
In the period of the 1995-1997,306 strain elite plant strains of middle choosing are yielded positive results successively, and according to the habit of yielding positive results, proterties such as fruit size, color and luster, quality, taste have been chosen 16 combinations, 68 fine individual plants.
A, in ripe * precocity: combination 41 has 1 individual plant, 95 years beginnings 15 of bow structure fruits, 99 of 96 annual bearings, late June is ripe, and is 65 days ripe phases, special precocious.
B, late-maturing * precocity: 3 combinations are arranged, and 30 individual plants wherein make up 24, and 20 individual plants are arranged, 30 of 95 annual bearings, and 157 of 96 annual bearings, best in quality, ripe down in 7 months, ripe phase 80-90 days; Combination 22 has 2 individual plants, 95 years beginnings 1 of bow structure fruit, yielded positive results 12 in 96 years, fruit is big, look red, and is ripe by the end of June, 75 days ripe phases.Combination 37 has 8 individual plants, 96 years beginnings 22 of bow structure fruits, fruit is big, look red, and is ripe by the end of July, 95 days ripe phases.
C, late-maturing * in ripe: 7 combinations, 18 individual plants.Wherein make up 10,6 individual plants are arranged, 2 of 95 annual bearings, 24 of 96 annual bearings, red entirely, giving off a strong fragrance, ripe at the beginning of 7 months, 70 days ripe phases; Combination 21 has 6 individual plants, 5 fruits of 95 years knots, and 51 fruits of 96 years knots, redness, ripe on July, 75 days ripe phases; Combination 33 has 3 individual plants, and 17 fruits of 96 years knots are red entirely, and is ripe down in 8 months.Make up 18,31,25,23 in addition, select 1 strain in each.
D, late-maturing * late-maturing, 5 combinations, 19 individual plants wherein make up 6, and 9 individual plants are arranged, and 2 fruits of 95 years knots were tied 34 fruits in 96 years, and are red entirely, ripe in 8 months, 115 days ripe phases; Combination 32 has 6 individual plants, and 5 fruits of 95 years knots were tied 78 fruits in 96 years, and are red entirely, and is ripe in 8 months, 105 days ripe phases; Combination 56 has 2 individual plants, and green, succulence, high-quality are ripe on August, 105 days ripe phases.Make up 30,29 in addition, late-maturing, 150～160 days ripe phases.
In the period of 1998～1999, carry out last final election according to yielding ability, yield stability and storage property, require single plant yield more than 6kg, more than the high yield single plant yield 16kg, fruit quality is good, the individual plant of storage tolerance, and the grown place was transplanted in 99 years in selected altogether 22 strains system.2004 annual single plant yield 62.95kg, luffing 50-72.5
5, precocious high quality apple new lines
[0051] program that combines with conventional seed selection according to early stage preliminary election; The precocious high-quality new lines of breeding; More than 20 excellent strains such as No. 1, fragrant garden, No. 2, fragrant garden, No. 3, fragrant garden, No. 5, fragrant garden, No. 6, fragrant garden, No. 7, fragrant garden, No. 8, fragrant garden, No. 12, fragrant garden are arranged, and eight strains in its prostatitis are through expert appraisal.
Description of drawings:
Fig. 1 is preparation starch gel glue die drawing;
Fig. 2 is the electrophoresis tank installation drawing;
Fig. 3 selects the seed selection program sketch that combines for the early stage preliminary election of isodynamic enzyme and conventional.
Fig. 4 is buffer formulation figure.
Claims (2)
1. preamble: theme of the present invention is that the new varieties technology are cultivated in the early stage preliminary election of apple, is characterized in selecting the procedure of breeding to compare with isodynamic enzyme sign (E9 band) to early stage preliminary election of apple hybrid seedling and routine, and efficiency of selection height, progress are fast.
2. characteristic: the characteristic of this invention is
1, the isodynamic enzyme sign (E9) of the early stage preliminary election of apple is closely related with the short living habit of apple, and test operation is easy, reliability is high, good stability; Genetic stability, the test accuracy rate is 1.4%, is suitable for that Malus is not of the same race, kind; Different cultivation conditions have certain generality.
2, early stage preliminary election of apple seedling isodynamic enzyme and the option program that conventional selection combines can reduce the randomness of selecting, and improve efficiency of selection, accelerate the breeding progress.
3, the early stage preliminary election of isodynamic enzyme is selected the special evidence that combines with conventional: by 60 hybrid combinations, 1535 strain seedlings after the isodynamic enzyme preliminary election, select rate to reach 13.0% in the combination, and selecting rate in the individual plant is 1.4%, apparently higher than conventional efficiency of selection.
The requirement of this invention patent comprises that preamble and characteristic characteristics are:
A. the early stage preliminary election sign of hybrid seedling isodynamic enzyme (E9);
B. early stage preliminary election of hybrid seedling isodynamic enzyme and the conventional seed selection program that combines selected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2011100276910A CN102342244A (en) | 2011-01-26 | 2011-01-26 | Technology for pre-selecting and breeding new species of apple in early stage |
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| CN103120119A (en) * | 2013-02-05 | 2013-05-29 | 西北农林科技大学 | Methods for breeding and cultivating qin red apple |
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