CN113249514B - Cucumber female line character related SCAR marker and application thereof - Google Patents

Abstract

The invention discloses a cucumber female line character related SCAR marker and application thereof, wherein the marker is positioned in a promoter region of a coding cucumber ethylene synthetase gene CsACS1G on a No. 6 chromosome, female line breeding can be carried out by utilizing the SCAR marker, the breeding time can be effectively shortened, the material planting area is reduced, and the breeding process of the female line is greatly accelerated. The method replaces the traditional field phenotype observation and identification method, can be used for identifying in seedling stage and indoors, is more rapid and efficient than the method for identifying in field phenotype observation in flowering stage, is suitable for screening breeding materials on a large scale, and can save land and labor cost.

Description

Cucumber female line character related SCAR marker and application thereof

Technical Field

The invention relates to a cucumber female line character related SCAR marker and application thereof, belonging to the field of molecular biology.

Background

Cucumber is one of the major vegetable crops in the world, according to the statistics of a world food and agriculture organization public database (FAOSTAT), the cultivation area and the yield of the cucumber in China in 2019 reach 125.8 million hectares and 7033.9 million tons, which respectively account for 56.4 percent and 80 percent of the total amount of the world, and the significance of the cucumber in the production of the China is highlighted. The main cultivated species currently produced in China is the North China stichopus japonicus selenka type cucumber, and the yield and the quality of the cucumber are limited by the number of female flowers. The female line variety has more female flowers and more melons, so that the yield can be effectively improved, meanwhile, the emasculation is not needed, the seed production procedure can be greatly simplified under the condition of ensuring the purity of the hybrid seeds, and the efficiency and the yield of fine breed breeding can be effectively improved. However, female line varieties bred in the current production are mostly bred through multi-generation backcross transformation, the breeding period is long, time and labor are wasted, partial female line materials are influenced by the environment, and the field selection accuracy is limited. The existence of the problems greatly restricts the screening of cucumber female line materials and the breeding of female line varieties.

Sequence-specific amplified region (SCAR) is a technology for specifically amplifying a certain specific fragment by designing a primer on the basis of sequence sequencing, and has the characteristics of rapidness, simplicity and low cost in application due to strict PCR reaction conditions, stable result and strong repeatability, so that the SCAR becomes a preferred marker in various molecular markers. The female line material widely used in the production at present is mainly a material with an F gene locus, the conservation of the F gene determining the female line character is caused by 1-3 CsACS1G gene copies caused by the repetition of a segment of 30.2kb of chromosome 6, and the unequal exchange of CsACS1G region CNV influences the stability of the female line. Therefore, the efficient and stable SCAR marker can be developed in the promoter region of the CsACS1G gene recombination, is used for screening female line materials and auxiliary selection of backcross transformation, can effectively shorten breeding time, reduce material planting area, greatly accelerate the breeding process of female lines, and has great production value.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides the SCAR marker related to the cucumber female line character and the application thereof, the marker is positioned in the promoter region of the coding cucumber ethylene synthetase gene CsACS1G on the No. 6 chromosome, female line breeding can be carried out by utilizing the SCAR marker, the breeding time can be effectively shortened, the material planting area can be reduced, and the breeding process of the female line can be greatly accelerated.

An SCAR marker related to cucumber female line traits, wherein the sequence of the SCAR marker is shown as SEQ ID NO. 3.

The method for screening the cucumber female line material by the SCAR marker related to the cucumber female line character comprises the following steps:

1) Extracting the genome DNA of the cucumber to be detected;

2) Taking the genome DNA of the cucumber to be detected as a template, and carrying out PCR amplification reaction by using a marker primer;

3) Detecting the amplification product, marking the amplification result to judge the variety to be identified, wherein the material with 356bp band is female material, and the material without band is non-female material.

Further, the labeled primer in the step 2) comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.

Further, the reaction system of the PCR amplification reaction in step 2) is 20 μ l, which is as follows:

mu.l of 10 ng/. Mu.l template DNA, 1. Mu.l of 10 pmol/. Mu.l upstream primer, 1. Mu.l of 10 pmol/. Mu.l downstream primer, 10. Mu.l of 2X M5HiPer plus taq HiFi PCR mix, and 6. Mu.l ultrapure water.

Further, the amplification procedure of the PCR amplification reaction in step 2) above is:

pre-denaturation at 95 ℃ for 1min; then denaturation at 95 ℃ is 15s, annealing at 55 ℃ is 15s, extension at 72 ℃ is 30s, and 30 cycles are counted; finally, extension is carried out for 10min at 72 ℃.

The SCAR marker related to the cucumber female line character is applied to screening of cucumber female line materials and auxiliary selection of backcross transformation.

The cucumber female line character related SCAR marker is applied to the cucumber molecular marker assisted breeding.

Has the beneficial effects that:

(1) The melon female line character related SCAR marker can be used for female line breeding, the method replaces the traditional field phenotype observation and identification method, identification can be carried out in seedling stage and indoor, and the method is quicker and more efficient than the method that identification is carried out in flowering stage through phenotype observation and identification in field, is suitable for large-scale screening of breeding materials, and can save land and labor cost.

(2) The marker is a dominant marker, 12 single plants can be selected for marker detection in the seedling stage of each strain of the early generation, and the homozygous lines which are all provided with strips can be considered to be planted in the field. The identification method has the theoretical accuracy rate of about 97 percent, is greatly higher than that of the identification method for field phenotype observation, can effectively shorten the breeding time, reduce the planting area of materials and accelerate the breeding process of cucumber female line varieties

Drawings

FIG. 1 shows the electrophoretic patterns of different cucumber material markers.

FIG. 2 shows the electrophoretic patterns of F3 family markers derived from the crossing of female and non-female lines.

Detailed Description

In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.

Example 1

Planting 14 North China type materials and 14 south China type materials in the field respectively, taking leaves at the seedling stage to extract DNA, then carrying out PCR amplification and molecular marker detection analysis, and investigating the sex type of each material at the flowering stage. The method comprises the following specific steps:

1) DNA extraction:

1. taking cucumber leaves with proper size of 1cm 2;

2. adding steel balls and 500. Mu.L of extraction buffer (12.1g of Tris,28.1g of NaCl,18.6g of EDTA, adding water to a constant volume of 1L), grinding for 3min, and centrifuging at 12000rpm for 10min;

3. sucking 400-450 μ L of supernatant to 1.5mL centrifuge tube, adding equal volume of isopropanol (or 2 times volume of glacial ethanol) for precipitation, standing at-20 deg.C for about 30min, and centrifuging for 5min;

4. pouring off ethanol, adding 500 μ L of 75% ethanol, soaking and washing for 10min, pouring off 75% ethanol, and washing repeatedly for 1 time;

5. after air drying, adding 100 mu L ddH2O to dissolve for about 1h to obtain the cucumber sample DNA solution.

2) And (3) PCR amplification:

taking the prepared cucumber sample DNA solution for PCR amplification, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2, and the reaction system of the PCR amplification reaction is 20 mu l, which is specifically as follows:

mu.l of 10 ng/. Mu.l template DNA, 1. Mu.l of 10 pmol/. Mu.l upstream primer, 1. Mu.l of 10 pmol/. Mu.l downstream primer, 10. Mu.l of 2X M5HiPer plus taq HiFi PCR mix, and 6. Mu.l ultrapure water.

PCR amplification procedure: pre-denaturation at 95 ℃ for 1min; then denaturation at 95 ℃ is carried out for 15s, annealing at 55 ℃ is carried out for 15s, extension at 72 ℃ is carried out for 30s, and 30 cycles are counted; finally, extension is carried out for 10min at 72 ℃.

3) And (3) detecting the amplification result of the product:

the PCR amplified product was loaded on 1% agarose gel containing gelred nucleic acid dye at a concentration of 1% by mass, electrophoresed for 15min at a constant voltage of 180V, and the labeling results were archived and analyzed by photographing using a gel imaging system, and the results are shown in FIG. 1. The Marker used in FIG. 1 is DL2000, 4 bands are shown in the figure, the sizes are 750bp,500bp,250bp and 100bp in sequence, the band corresponding to the amplified product is 356bp, and the band with 356bp is female line.

The upper panel in fig. 1 is the result of amplification of the marker in a south china type cucumber, where female line material is: 1, new jade star; the upper panel shows the results of the amplification of the marker in a south China cucumber type, where the female line material is: 1, new jade star; 2, grinding into jade; 3, carrying out blue grinding on the mixture to obtain 1;5, carrying out blue and white grinding for 2;7, grinding the mixture into powder; 8, grinding the mixture into 4 parts; 9, grinding the mixture into 5 parts; 10, grinding the mixture into 6 parts; 13, grinding the mixture into powder and 7. The non-female material is 4, and the wild white fur of the Haiyang is 1;6, breeding 2 places of the white skin of the Haiyang; 11, local breeding of the cucumber in Laisiidiu 1;12, local species of the cucumber in Laisiidiu 2;14, local species of cucumber leixi di 3.

The lower panel in fig. 1 is the result of amplification of the marker in north China Spanish type cucumber, where the female line material is: 1, kerun 99;3, jin you 385;4, jin you 626;5, dride 10;6, bomei 8;11, bomei 9;12, de-let L108;13, jin Sheng 103. Non-female line materials were: 2, luhuang No. 9; 7, 1-2 parts of wintersweet; 8, 1-3 parts of wintersweet; 9, jin you 35;10, zhongnong No. 26; 14, xintaimi thorn.

Example 2

In order to further verify the effectiveness of the method, the method is adopted to detect the F3 families derived by the hybridization of female lines and non-female lines in the transformation process of 6 female line materials planted in the field, each family detects 12 single plants, and the genotype of the family is judged according to the detection result. The method comprises the following specific steps:

1) DNA extraction:

1. taking cucumber leaves with proper size of 1cm 2;

2. adding steel balls and 500 μ L of extraction buffer (12.1g Tris,28.1g NaCl,18.6g EDTA, adding water to a constant volume of 1L), grinding for 3min, and centrifuging at 12000rpm for 10min;

3. sucking 400-450 μ L of supernatant into a 1.5mL centrifuge tube, adding equal volume of isopropanol (or 2 times volume of glacial ethanol) for precipitation, standing at-20 deg.C for about 30min, and centrifuging for 5min;

4. pouring off ethanol, adding 500 μ L of 75% ethanol, soaking and washing for 10min, pouring off 75% ethanol, and washing repeatedly for 1 time;

5. after air drying, 100 mu L ddH2O is added to dissolve for about 1h, and then the cucumber sample DNA solution can be obtained.

2) And (3) PCR amplification:

taking the prepared cucumber sample DNA solution for PCR amplification, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer is shown as SEQ ID NO.2, and the reaction system of the PCR amplification reaction is 20 mu l, which is specifically as follows:

mu.l of 10 ng/. Mu.l template DNA, 1. Mu.l of 10 pmol/. Mu.l upstream primer, 1. Mu.l of 10 pmol/. Mu.l downstream primer, 10. Mu.l of 2X PCRMIX, and 6. Mu.l ultrapure water.

PCR amplification procedure: pre-denaturation at 95 ℃ for 1min; then denaturation at 95 ℃ is 15s, annealing at 55 ℃ is 15s, extension at 72 ℃ is 30s, and 30 cycles are counted; finally, extension is carried out for 10min at 72 ℃.

3) And (3) detecting the amplification result of the product:

the PCR amplified product was loaded on 1% agarose gel containing gelred nucleic acid dye at a concentration of 1% by mass, electrophoresed for 15min at a constant voltage of 180V, and the labeling results were archived and analyzed by photographing using a gel imaging system, and the results are shown in FIG. 2. In FIG. 2: the Marker is DL2000, 6 bands are shared in the figure, the sizes are 2000bp,1000bp,750bp,500bp,250bp and 100bp in sequence, the band corresponding to the amplification product is 356bp,12 single plants all have the band and are considered as homozygous female line, 12 single plants all do not have the band and are considered as non-female line, and in other cases, the heterozygous female line is adopted

The three families A, D and E in FIG. 2 are heterozygote female lines

Two families B and C in FIG. 2 are homozygous female lines

Family F is a non-female line.

SEQUENCE LISTING

<110> institute of vegetables and flowers of academy of agricultural sciences of Shandong province

<120> cucumber female line character related SCAR marker and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

ggaaagtgtc tccgaccaga 20

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

tatctaggct gccactggag 20

<210> 3

<211> 356

<212> DNA

<213> sequence

<400> 3

tatctaggct gccactggag cgtgccctca ctgaacaaaa tcaaactggc ctattttcct 60

ttcttttttt gtttttcatt aaaataatca ctccttcatt gatatgcaac ttagcaaaat 120

atataaatac tagataagaa aacccaaatc aagagaagca acgacaaaaa cagggtggtt 180

taagtattta actacttcgg acgggcatag ttatgagcaa cattaattat gttttcccct 240

aacaaattaa cccccaaatc attaacattt ttcattatcc cattcatttt tgtttcataa 300

tccccatttt catcttccat atcaaaccat acctgctctg gtcggagaca ctttcc 356

Claims (4)

1. A method for screening cucumber female line material by SCAR marker related to cucumber female line character is characterized by comprising the following steps:

1) Extracting the genome DNA of the cucumber to be detected;

2) Taking the genome DNA of the cucumber to be detected as a template, and carrying out PCR amplification reaction by using a labeled primer; the labeled primers comprise an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2;

3) Detecting the amplification product, marking the amplification result to judge the variety to be identified, wherein the material with 356bp bands is a female material, and the material without bands is a non-female material; the sequence of the SCAR marker is shown as SEQ ID NO. 3.

2. The method for screening female cucumber line material as claimed in claim 1, wherein the reaction system of the PCR amplification reaction in step 2) is 20 ul, and specifically the following:

mu.l of 10 ng/. Mu.l template DNA, 1. Mu.l of 10 pmol/. Mu.l upstream primer, 1. Mu.l of 10 pmol/. Mu.l downstream primer, 10. Mu.l of 2X M5HiPer plus taqHiFi PCR mix, and 6. Mu.l ultrapure water.

3. The method for screening cucumber female line material as claimed in claim 1, wherein the amplification procedure of the PCR amplification reaction in step 2) is as follows:

pre-denaturation at 95 ℃ for 1min; then denaturation at 95 ℃ is 15s, annealing at 55 ℃ is 15s, extension at 72 ℃ is 30s, and 30 cycles are counted; finally, extension is carried out for 10min at 72 ℃.

4. Use of primers for SCAR markers related to cucumber female line traits as defined in claim 1 for screening cucumber female line material and for assisted selection for backcross transformation.

Images