CN102499056A

CN102499056A - Method for breeding new line of Brassica napus by utilizing biotechnology

Info

Publication number: CN102499056A
Application number: CN2011103270174A
Authority: CN
Inventors: 黄镇; 徐爱遐; 班元元; 张学贤
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2011-10-25
Filing date: 2011-10-25
Publication date: 2012-06-20
Anticipated expiration: 2031-10-25
Also published as: CN102499056B

Abstract

The invention provides a method for breeding a new line of Brassica napus by utilizing a biotechnology. The method comprises the steps of: selecting each individual from separation colonies BC1F1, BC2F1, BC3F1, BC4F1 and BC5F1 utilizing a codominant mark CB10022, selecting individuals with Bb genotype and eliminating non-target individuals in combination with field inspection; at the same time, carrying out background selection on the target individuals by utilizing AFLP primers, carrying out quality analysis on the target individuals in each separation colony to select double-low offspring, finally selecting three individuals with highest similarity with recurrent parent Xu102 and carrying out backcrossing; and carrying out foreground selection in BC5F2 by utilizing SSR mark CB10373 and SCAR mark to select individuals with bb genotype, and then carrying out selfing on each generation to obtain the stable Brassica napus line. By adopting the method, the breeding time of the Brassica napus line is greatly shortened and the workload is reduced.

Description

Utilize the method for biotechnology seed selection yellow seed rape new lines

Technical field

The invention belongs to agricultural technology field, relate to the research of interspecific cross initiative yellow-seeded brassica napus, be specifically related to the heredity of the yellow seed proterties of landrace juncea yellow seed rape, molecular labeling and the method for utilizing molecular selection yellow seed rape new lines.

Background technology

Yellow seed rape is compared with black seed rape, has the oil content height, and protein content is high, plants proterties advantages such as skin is thin, fiber content is low, and oil is as clear as crystal, quality is good.After the sixties in last century, professor Liu Houli found at first that at home the oil content of yellow seed rape is higher than non-yellow seed rape, the research of rape yellow seed gene and utilization were the focuses of rape research field always.Many research proof yellow seed rapes can improve rape oil content and protein content simultaneously, improve rape oil and grouts quality, so the yellow seed rape breeding are considered to one of rapeseed breeding approach the most efficiently than its black seed rape with background.Yet yellow seed breeding is carried out in decades, and the kind of the yellow seed rate of breeding 100% does not almost have, and the yellow seed germplasm that can supply in the world wide to utilize is very limited.The yellow seed gene-based of China's cabbage type rape is originally from Chinese cabbage type yellow seed rape; Yet in turnip type rape the performance genetic stability yellow seed gene once transformation in cabbage type rape; Its yellow seed proterties just can not genetic stability; Scientist has carried out years of researches to this lability both at home and abroad, and its internal mechanism is still not clear so far.

Mustard type rape is allotetraploid aggregate species similar in appearance to cabbage type rape, on kind of skin pigment is synthetic, possibly have similar mechanism.The yellow mustard in northern Shensi is the local germplasm of the distinctive yellow seed of China, and research shows that its yellow seed gene is controlled by a pair of major gene resistance only, and yellow seed character inheritance is simple and can genetic stability; Simultaneously, the yellow mustard in northern Shensi has drought-enduring, barren-resistant; Characteristics such as heat-resisting, cold resistance is strong, and adaptability is strong; Extensively planting in western arid area of China and highlands, is the yellow seed heredity of research mechanism, the good material of initiative yellow-seeded brassica napus germplasm and yellow seed rape breeding.Can the yellow seed gene of the yellow mustard in northern Shensi be imported to and breed the yellow-seeded brassica napus new lines in the cabbage type rape, widen yellow-seeded brassica napus germplasm basis, help the breeding of yellow-seeded brassica napus.Yet the breeding method that traditional phenotype is selected receives environmental influence, has many shortcomings, and the efficient of selection is low, the time limit is longer, and the pool heredity of yellow-seeded brassica napus kind color of the leather is extremely unstable in addition, and it is bigger influenced by environment and extraneous factor.The use of molecular labeling can address this problem; Because the genotype of molecular labeling can be discerned, can directly select, therefore can carry out directly the genotype of yellow seed proterties and selection apace by molecular labeling; Have quick, economical, advantage such as efficient is high, accuracy is strong.Molecular marker assisted selection comprises to the foreground selection of target gene or claims that forward is selected and also claim the negative sense selection to the selection of genetic background that background is selected to accelerate the genetic background resume speed, shortening the breeding cycle and the effect that alleviates chain burden.In the breeding process molecular marking technique is combined with back cross breeding, can be apace with the chain gene transfer of molecular labeling in another material.

In mustard type rape, Negi etc. found 3 AFLP marks and brown seed proterties close linkage in 2000, and one of them mark is converted to the SCAR mark; Sabharwal equal to find in 2004 15 with the grain color base because of chain AFLP mark, 11 AFLP marks are by on collection of illustrative plates to two linkage group, mark density is respectively 6.0 and 3.6cM.Lakshmi in 2005 etc. identify 3 SSR marks and grain color base because of chain, and these three marks are navigated on two linkage groups.These work are that molecular marker assisted selection seed selection yellow seed rape kind is laid a good foundation.But utilize these marker assisted selection (MAS) yellow-seeded brassica napus also not appear in the newspapers.

In sum, there is following shortcoming in the prior art:

1, in the cabbage type rape, yellow seed blastogenesis basis is narrower, and the heredity of yellow seed proterties is unstable, is difficult to be used.

2, traditional backcross transformation method efficiency of selection is low, take time long and be difficult to the yellow seed proterties of cabbage type rape is effectively improved.

The genetic linkage maps of the yellow seed proterties of 3, being set up is comparatively coarse, is difficult in yellow seed gene clone and the yellow seed breeding to use.

Summary of the invention

In order to solve the problems of the technologies described above; The present invention provides a kind of method of utilizing biotechnology seed selection yellow-seeded brassica napus new lines; The yellow seed proterties of the yellow mustard in the mustard type rape that yellow seed proterties is stable-northern Shensi imports in the cabbage type rape; Formulate the yellow-seeded brassica napus new germ plasm, be expected to form the yellow-seeded brassica napus new lines of genetic stability; Set up the highdensity genetic linkage maps of the yellow seed proterties of mustard type rape; Obtain closely linked dominance and codominant marker; In the backcross transformation process, yellow seed proterties is carried out foreground selection, utilize the AFLP mark that improved progeny is carried out background simultaneously and select, accelerate the seed selection process of yellow seed proterties.

The present invention utilizes the yellow mustard in northern Shensi (true yellow seed) and Central Shanxi Plain leaf mustard (brown seed) hybridization, its F ₁Obtain a F for selfing ₂Colony, each F ₂For individual plant selfing, according to the seed color judgment F on the selfing individual plant ₂The phenotype of plant.Utilize AFLP to combine with group's analytical method (BSA) method and filter out and the closely linked molecular labeling of yellow seed proterties, and these marks are changed into more stable, easy-operating SCAR mark with the SSR technology.The result shows has 21 AFLP marks, 2 SSR marks and brown seed gene close linkage, and wherein SSR mark CB10022 is the codominant marker.The nearest molecular labeling in brown seed gene both sides is EA02MC08 (0.3cM) and P03MC08 (0.5cM); Mark EA06MC11, EA08MC13, P11MG01, P15MG15, P16MC02, P09MC12 are converted into the SCAR mark, and EA08MC13 is converted into and is the codominant marker.

With the yellow mustard in northern Shensi is yellow seed genetic donor; Cabbage type rape strain Xu102 (black seed) to be provided by Xibei Univ. of Agricultural & Forest Science & Technology rape germ plasm resource research department is an acceptor; Utilize hybridize-repeatedly to backcross-method of selfing; The yellow seed gene of the yellow mustard in northern Shensi is imported among the Xu102 the two low yellow-seeded brassica napus new lines of seed selection.Concrete scheme is following:

At segregation population BC ₁F ₁, BC ₂F ₁, BC ₃F ₁, BC ₄F ₁, BC ₅F ₁In utilize codominant marker CB10022 that each individual plant is selected, select Bb genotype individual plant, combine the field economical character to investigate, superseded non-target individual plant.Utilize the AFLP primer that the target individual plant is carried out background simultaneously and select, in each segregation population the target individual plant is carried out attributional analysis, select two low offsprings, select and remain at last 3 strains and the highest individual plant of recurrent parent Xu102 similitude are backcrossed.At BC ₅F ₂In utilize SSR mark CB10373 and SCAR mark (being transformed) to carry out foreground selection by EA06MC11, select bb genotype individual plant, later per generation selfing obtains stable Brassica type strain.

Beneficial effect of the present invention:

The present invention has set up the highdensity genetic linkage maps of the yellow seed proterties of mustard type rape; Obtain closely linked dominance and codominant marker; In the backcross transformation process, yellow seed proterties is carried out foreground selection, utilize the AFLP mark that improved progeny is carried out background simultaneously and select, shortened the time of cabbage type rape yellow seed strain breeding greatly; Reduced workload, a seed selection cabbage type rape yellow seed strain effective way is provided.

Description of drawings

Fig. 1 is the genetic linkage maps of the yellow seed gene of the yellow mustard in northern Shensi;

Fig. 2 is the yellow seed proterties molecular marker assisted selection seed selection scheme among the Xu102 that imports to of backcrossing;

Fig. 3 is SSR mark CB10022 expanding effect figure;

Fig. 4 is SCAR mark (EA06MC11 conversion) expanding effect figure.

Embodiment

Below in conjunction with accompanying drawing and embodiment method of the present invention is done explanation in further detail.

With reference to Fig. 1, Fig. 2, the present invention utilizes the yellow mustard in northern Shensi (true yellow seed) and Central Shanxi Plain leaf mustard (brown seed) hybridization, its F ₁Obtain a F for selfing ₂Colony, each F ₂For individual plant selfing, according to the seed color judgment F on the selfing individual plant ₂The phenotype of plant.Utilize AFLP, SSR technology to combine with group's analytical method (BSA) method and filter out and the closely linked molecular labeling of brown seed gene, and these marks are changed into more stable, easy-operating SCAR mark.The result shows has 21 AFLP marks, 2 SSR marks and brown seed gene close linkage, and wherein SSR mark CB10022 is the codominant marker.The nearest molecular labeling in yellow both sides, seed site is EA02MC08 (0.3cM) and P03MC08 (0.5cM); Mark EA06MC11, EA8MC13, P11MG01, P15MG15, P16MC02, P09MC12 are converted into the SCAR mark, and EA08MC13 is converted into the codominant marker.

With the yellow mustard in northern Shensi is yellow seed genetic donor; With good cabbage type rape strain Xu102 (black seed) is acceptor; Utilize hybridize-repeatedly to backcross-method of selfing, the yellow seed gene of the yellow mustard in northern Shensi is imported among the Xu102, the yellow seed of seed selection is two to hang down good cabbage type rape strains.

At segregation population BC ₁F ₁Utilize codominant marker CB10022 that each individual plant is selected in (165 individual plants); Select Bb genotype individual plant, investigate, stayed 72 target individual plants in conjunction with the field economical character; Utilize simultaneously 20 pairs of polymorphisms preferably the AFLP primer target individual plant carried out background select; Coamplification has gone out 167 polymorphism bands, and through similarity analysis, select and remain 3 strains and the highest individual plant of recurrent parent Xu102 similitude are backcrossed again.At BC ₂F ₁, BC ₃F ₁, BC ₄F ₁, BC ₅F ₁In utilize identical method, obtain 3 strain target individual plants, selfing has obtained BC again ₅F ₂Colony's (152 individual plants) utilizes SSR mark CB10373 and SCAR mark (being transformed by EA06MC11) to carry out foreground selection, has selected 3 strain bb genotype individual plants, and later per generation selfing obtains stable Brassica type strain.

Claims

1. method of utilizing biotechnology seed selection yellow seed rape new lines, its concrete steps are:

1) the yellow mustard in northern Shensi (true yellow seed) and Central Shanxi Plain leaf mustard (brown seed) hybridization, its F ₁Obtain a F for selfing ₂Colony, each F ₂For individual plant selfing, according to the seed color judgment F on the selfing individual plant ₂The phenotype of plant;

2) utilize AFLP, SSR technology to combine with group's analytical method (BSA) method and filter out and the closely linked molecular labeling in yellow seed site; And these marks are changed into the SCAR mark; The result shows has 21 AFLP marks, 2 SSR marks and brown seed gene close linkage; Wherein SSR mark CB10022 is the codominant marker; The nearest molecular labeling in yellow both sides, seed site is EA02MC08 (0.3cM) and P03MC08 (0.5cM), and mark EA06MC11, EA8MC13, P11MG01, P15MG15, P16MC02, P09MC12 are converted into the SCAR mark, and EA08MC13 is converted into the codominant marker;

3) being yellow seed genetic donor with the yellow mustard in northern Shensi, be acceptor with cabbage type rape strain Xu102 (black seed), utilizes hybridize-repeatedly to backcross-method of selfing, and the yellow seed gene of northern Shensi Huang mustard is imported among the Xu102, and seed selection obtains the yellow-seeded brassica napus strain.

2. the method for utilizing biotechnology seed selection yellow seed rape new lines according to claim 1 is characterized in that, at segregation population BC ₁F ₁, BC ₂F ₁, BC ₃F ₁, BC ₄F ₁, BC ₅F ₁In utilize codominant marker CB10022 that each individual plant is selected, select Bb genotype individual plant, investigate in conjunction with the field economical character; Eliminate non-target individual plant; Utilize the AFLP primer that the target individual plant is carried out background simultaneously and select, in each segregation population the target individual plant is carried out attributional analysis, select two low offsprings; Select and remain at last 3 strains and the highest individual plant of recurrent parent Xu102 similitude backcrossed, at BC ₅F ₂In utilize SSR mark CB10373 and SCAR mark to carry out foreground selection, select bb genotype individual plant, later per generation selfing obtains the Brassica type strain.

3. the method for utilizing biotechnology seed selection yellow seed rape new lines according to claim 2 is characterized in that the SCAR mark is transformed by EA06MC11.