CN102757959A

CN102757959A - Gene fragment tightly linked with cucumber hairiness gene and application of gene fragment

Info

Publication number: CN102757959A
Application number: CN2012102601314A
Authority: CN
Inventors: 杨瑞环; 李淑菊; 王惠哲; 管炜
Original assignee: Kerun Agricultural Science & Technology Co Ltd Tianjin
Current assignee: Kerun Agricultural Science & Technology Co Ltd Tianjin
Priority date: 2012-07-25
Filing date: 2012-07-25
Publication date: 2012-10-31
Anticipated expiration: 2032-07-25
Also published as: CN102757959B

Abstract

The invention discloses a gene fragment tightly linked with a cucumber hairiness gene and the application of the gene fragment. The gene fragment is a sequence shown in SEQ ID No.1. Through carrying out hairiness character evaluation on constructed F2-generation segregation population and cucumber germplasm resources by using the gene fragment tightly linked with the cucumber hairiness gene, the coincidence rate of the result to the phenotype identification reaches 100%, and resource hairiness character selection and utilization in cucumber breeding have great application value. On the basis of the sequences, hairiness character molecular marker auxiliary selecting and breeding can be carried out, and the foundation can be provided for carrying out cucumber hairiness gene positioning, plotting and cloning and improving the character of cucumber breeds through a gene engineering.

Description

With cucumber closely linked gene fragment of hair gene and purposes are arranged

Technical field

The invention belongs to technical field of biological genetic engineering, relate to the method and technology of the breeding of cucumber conventional hybridization, molecular mark, particularly the closely linked gene fragment of hair gene is arranged with cucumber.

Background technology

In 45 strain breed cucumber materials, found the two mutants of 3 strain tools " nothing hair " proterties in autumn in 1999, genetic research shows not have the hair proterties and be the recessive character by a pair of nuclear gene control, and is consistent with the result of study of Cao Chenxing (1999).Cucumber does not have the chalaza variant to be compared with common cucumber, and the various proterties of seedling all do not have considerable change, and whole strain does not all have short bristle Anywhere, is smooth to the touch, and does not have the sensation of pricking the hand, and these characteristics just can be distinguished with common cucumber seedling when seedling cotyledon flattens.

Cucumber does not have the chalaza variant the earliest by Robinson and Mishanec (1964) report (producing through radioinduction); Show as stem, leaf, tendril, calyx, ovary and all do not have epidermal hair; Fruit surface is not fruit thorn and fruit knurl also, and is glabrous (gl) with this recessive mutation unnamed gene.Whelan (1973), Cao Chen Xinghe Guo Hong rue (1999) also successively find that cucumber does not have the chalaza variant.The blade of Inggamer&Ponti (1980) report does not have chalaza variant (gl-2) cucumber NCG-042 and shows as sparse fine hair is arranged on carpopodium, anthocaulus, the calyx, and rare fruit knurl is arranged on the fruit, but smooth nothings of stem, leaf and petiole mao.Research shows that there is the recessive epistasis effect in hairless gene to the fruit tumor gene, participates in the formation (Cao Chenxing etc., 2001) of cucumber fruit knurl.

Guan Yuan (2008) utilizes cotton and Arabidopis thaliana TTG1 gene pairs cucumber to carry out homologous clone; Obtain a cucumber TTG 1-like gene---CsTTG1; Functional verification shows the phenotype that CsTTG1 can complementary Arabidopis thaliana ttg1 two mutants, but the expression analysis proof causes that the gene of gl sudden change is not a cucumber TTG 1-like gene.Relaxation etc. (2009) are the parent with Cao Chen Xinghe Guo Hong no chalaza variant and the common hairiness cucumber of finding that rue, and have obtained two SRAP mark ME6EM5 and ME23OD15 with gl gene linkage, and genetic distance is respectively 3.6cM and 12.9cM.It is the examination material that Miao Han etc. (2011) (P1) (P2) (contain blade hairless gene gl-2) with no chalaza variant ' NCG-042 ' with cucumber hairiness agriotype ' 9110Gt '; The clear and definite hairiness of cucumber, do not have a hair proterties (gl-2) and control by a pair of nuclear gene, hairiness mao is a dominance to not having.And with F ₂Be mapping population, made up the SSR linkage group of the gl-2 gene that comprises 18 SSR marks, and with this assignment of genes gene mapping on the 2nd karyomit(e) of cucumber, the nearest linked marker in both sides is SSR10522 and SSR13275, genetic distance is respectively 0.6cM and 3.8cM.

Because not having the hair proterties is the recessive character by a pair of nuclear gene control, be suitable as very much the mark property of morphological markers, purity evaluation.Therefore, cucumber is not had the research of mao proterties, particularly confirm have the closely linked SSR fragment of hair gene that cucumber is produced and breeding all has important theory and practice significance with cucumber.

Summary of the invention

The object of the present invention is to provide a kind of and cucumber that the closely linked gene fragment of hair gene is arranged.

Second purpose of the present invention provides the purposes that a kind of and cucumber have the closely linked gene fragment of hair gene.

Technical scheme of the present invention is summarized as follows:

A kind of have the closely linked gene fragment of hair gene with cucumber, and said gene fragment is the described sequence of SEQ ID No.1.

The said gene fragment is being carried out the hairiness character screening or cucumber hairiness proterties is being carried out the purposes of molecular mark to cucumber germ plasm resource.

Utilize of the present invention and cucumber that the F of the closely linked gene fragment of hair gene to making up arranged ₂Carry out the evaluation of hairiness proterties for segregating population and cucumber germ plasm resource, its result and phenotypic evaluation coincidence rate reach 100% (data are seen table 1), in breed cucumber, carry out resource hairiness character screening and have very big using value with utilizing.With these sequences is foundation, can carry out hairiness trait molecular marker assisted Selection, breeding; Can be for carrying out the assignment of genes gene mapping of cucumber hairiness, mapping and clone, the proterties that improves cucumber variety through genetically engineered lays the foundation.

Description of drawings

Fig. 1 is the acquisition collection of illustrative plates that of the present invention and cucumber have the closely linked gene fragment of hair gene: wherein A (M) is the Marker of indication DNA standard band; B (P ₁) be maternal F80 hairiness gene fragment; 1-5 is the hairiness individual plant; 6-10 is not for there being the hair individual plant, and M is the DNA standard.

Fig. 2 is F80 * F80WM filial generation F ₂For the separation case collection of illustrative plates: wherein A (M) is the DNA standard; C is F ₂Hairiness gene fragment in the colony, 1～32,42～53,78～93 is the hairiness individual plant, 33～41,54～77 for there not being the hair individual plant.

Embodiment

The present invention be to cucumber hairiness parent F80 with do not have hair parent F80WM and filial generation F thereof ₁, F ₂, BC ₁Colony carries out manual observation and identifies, obtaining cucumber does not have on the basis of mao germplasm, adopt the SSR technical point from the gene fragment of cucumber hairiness gene linkage, check order, analyze, for screening, the molecular mark of germ plasm resource lays the foundation.

In order to realize above purpose, the present invention adopts SSR (Simple sequence repeat) molecular marking technique, adopts the BSA method that the hairiness genes involved of cucumber germplasm F80 and the genes involved of cucumber germplasm F80WM have been carried out molecule marking research.Obtaining has 1 of the closely linked gene DNA fragment of hair gene with cucumber, and behind this sequencing fragment, its size is 164bp (the described nucleotide sequence of SEQ ID No.1).

What the present invention obtained has the concrete operations step of the closely linked gene order of hair gene following with cucumber:

1.F ₂The structure of segregating population and phenotypic evaluation:

Utilize cucumber hairiness parent F80 and do not have hair parent F80WM hybridization acquisition F ₁, F ₁Selfing obtains F ₂

Field planting F ₂Colony, normal management.Visual inspection and by microscopic examination F during the 2nd true leaf ₂The individual plant separation case.

2.DNA extract and the ssr analysis system

The tender leaflet of clip cucumber children adopts CTAB method individual plant to extract DNA ,-20 ℃ of preservations.

The PCR reaction system is 20 μ l, cucumber gene group DNA20ng, each 50ng of SSR primer, dNTP0.2mM, Mg2+1.5mM, 1 times PCR damping fluid, Taq archaeal dna polymerase 1.6 units; Reaction conditions is 94 ℃ of preparatory sex change 300 seconds, 94 ℃ of sex change 30 seconds, and 55 ℃ of annealing 30 seconds, 72 ℃ were extended 60 seconds, 35 circulations, 72 ℃ were extended 350 seconds again.

3.SSR primer screening and checking

1164 pairs of SSR primers of screening and 29 pairs of EST-SSR primers between anti-sense parent produce 15 pairs of the primers of polymorphum between parents, wherein primer SSR01647 is at two parents and F ₂Extreme individual individual plant carries out pcr amplification, has obtained a dominance SSR mark, uses this combination of primers respectively to organizing other outer F ₂Hairiness height in individual plant and breeding and the production increases for self-mating system, further confirms the mark that is screened.The result shows that hairiness parent and hairiness individual plant amplify the specific fragment of 164bp, does not have the hair parent and does not amplify this specific fragment with a nothing hair individual plant.Through outer all the other F of group ₂Individual plant and breeding self-mating system are analyzed, and mark that is screened and target gene---cucumber hairiness trait related gene close linkage, coincidence rate are 100%.The base sequence of used SSR primer SSR01647 is:

P1：5'-CACCCTCTCACTTGTAGCCC-3＇(SEQ?ID?No.2)

P2：5'-CGATCAGAAGGGGGAAAAA-3＇(SEQ?ID?No.3)

4.PCR the recovery of product and order-checking

Adopt 5% denaturing polyacrylamide gel to carry out electrophoretic separation amplified production; In amplified production, add the denaturant gel sample-loading buffer; In 95 ℃ of sex change 300 seconds; Be splined on the sex change polypropylene amine gel of 30 minutes prerunnings, the permanent power electrophoresis of 100W to bromjophenol blue has just arrived the other end of gel, and gel is through observing under visible light behind the cma staining.

Adopt boiling method that the purpose fragment is dyed glue from silver and reclaim, increase, deliver to Beijing three rich polygala root bio-engineering corporations and check order.

5. sequential analysis

According to sequencing result, there is the closely linked specific fragment of hair gene that 164 bases (shown in the SEQ ID No.1) are arranged with cucumber.

Below in conjunction with embodiment the present invention is further described.

Embodiment 1

Adopt the BSA method, with cucumber hairiness parent F80 and nothing hair parent F80WM and 184 strain F thereof ₁Be the examination material for segregating population, adopt the CTAB method to extract cucumber gene group DNA.Carry out pcr amplification with 1164 pairs of SSR primers and 29 pairs of EST-SSR primers, according to specific DNA fragment at F ₂For the performance situation in the segregating population, the artificial phenotype qualification result in seedling stage that contrast is above, obtain with cucumber have hair gene closely linked SSR mark SSR01647 and with the closely linked dna fragmentation of hair gene (shown in the SEQ ID No.1) is arranged.

Embodiment 2

With the hairiness cucumber germ plasm resource W43 that is stored in Tianjin Ke Run cucumber institute breeding one Room, G5, XL6-1-2, Q12, P57-1, P59-1-1, P60-1-1, P62-1-1,66, F51, Q6, Q21 etc.With cucumber the closely linked SSR special primer of hair gene SSR01647 is arranged according to what embodiment 1 obtained, above-mentioned cucumber material genomic dna is carried out pcr amplification, the material of the specific fragment of 164bp appears in all amplified productions, is hairiness cucumber resource; All materials that does not amplify 164bp product specific fragment are not for there being hair cucumber resource.

Table 1F ₂Individual plant phenotype and mark separate statistics

Annotate: 1: hairiness; 0: do not have hair; +: band is arranged;-: no band

By finding out in the table, do not exchange individual plant, so the rate of coincideing is 184/184 * 100%=100%.

Claims

One kind with cucumber the closely linked gene fragment of hair gene is arranged, it is characterized in that said gene fragment is the described sequence of SEQ ID No.1.
2. the said gene fragment of claim 1 is being carried out the hairiness character screening or cucumber hairiness proterties is being carried out the purposes of molecular mark to cucumber germ plasm resource.