Method for identifying purity of cucumber hybrid and InDel primer combination used by method

Abstract

The invention discloses a method for identifying the purity of a cucumber hybrid and an InDel primer combination used by the method. The InDel primer combination provided by the invention is composed of four primer groups; each primer group consists of two primer sequences and is used for amplifying an InDel site; the nucleotide sequences of the primers are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 8. The InDel primer combination can be used for early identification in the seed or seedling stage of the cucumber hybrid, so that the purity of the hybrid is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for seed quality management of cucumber varieties. The method provided by the invention has the advantages of being accurate, low in cost, simple to operate, clear in result, capable of saving manpower and material resources and the like, and has a very wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for identifying the purity of cucumber hybrids and an InDel primer combination used therein; hybrids refer to hybrids of the first generation, which may have mixed parents. Background Art

[0002] Cucumber, as an indispensable member of my country's vegetable industry, is of great importance. It has not only won the favor of consumers with its unique taste and rich nutritional value, but also played a pivotal role in ensuring the stable supply of my country's vegetable market because of its characteristics of being cultivated in all seasons and supplied all year round. According to statistics, the cultivated area of ​​cucumber has reached 1.396 million hectares, accounting for 6.2% of the total vegetable cultivated area in the country. This figure fully demonstrates the important position of cucumber in my country's agricultural economy. However, with the continuous advancement of cucumber breeding technology and the continuous emergence of new varieties, we are also facing unprecedented challenges. Due to the relatively small scale and scattered distribution of cucumber breeding enterprises in my country, seed quality management has become an urgent problem to be solved. It is common to see counterfeit and substandard cucumber seeds on the market, which not only damages the vital interests of farmers, but also seriously affects the healthy development of the cucumber industry.

[0003] In order to regulate the cucumber seed market and ensure seed quality, my country officially implemented the "Registration Measures for Non-Major Crop Varieties" in April 2017. The introduction of this policy has greatly promoted the standardized management of cucumber varieties. Up to now, the number of cucumber varieties that have applied for registration nationwide has reached 1,607, of which more than 93% are hybrids (referred to as hybrids). Hybrids have a dominant position in cucumber production due to their excellent genetic characteristics and production performance. However, hybrids must undergo strict purity identification before entering the market. According to the current crop seed inspection procedures (GB / T 3543.1-3543.7-1995), the purity of cucumber hybrids must reach more than 98%. This standard is of great significance for ensuring seed quality and maintaining market order. However, the traditional field purity identification method has the disadvantages of high cost and long time consumption, which is difficult to meet the needs of modern seed industry development. Against this background, DNA-based molecular detection technology came into being. With its advantages of low cost, time saving and high efficiency, this technology has quickly become a new favorite for identifying the purity of cucumber hybrids. By screening and obtaining a molecular marker combination for identifying the purity of cucumber hybrids based on DNA detection technology, we can complete the purity identification work more accurately and quickly, thereby effectively curbing the circulation of counterfeit and inferior seeds, protecting the legitimate rights and interests of farmers and the healthy development of the cucumber industry. Therefore, it is particularly important to screen and obtain a molecular marker combination for identifying the purity of cucumber hybrids based on DNA detection technology.

[0004] InDel (Insertion-Deletion) markers are insertion-deletion markers, which refer to the differences in the whole genome sequences of two parents. Specifically, relative to one parent, a certain number of nucleotides are inserted or deleted in the genome of the other parent. This insertion-deletion marker is widely present in the genomes of different organisms. Usually, specific InDel marker primers are designed based on the sequences on both sides of the insertion-deletion site. Compared with SNP, InDel is faster to apply and can be quickly detected using a common electrophoresis technology platform. It has simpler requirements for facilities and methods and lower requirements for sample DNA quality. Some scholars have compared it with the results of SSR amplification and found that its stability and separation effect are better than SSR. It has the characteristics of good stability, wide distribution, high polymorphism, strong versatility, simple typing system and low cost. Summary of the invention

[0005] The purpose of the present invention is to identify the purity of cucumber hybrids.

[0006] The present invention first protects an InDel site combination, including four InDel sites in the cucumber genome; the four sites are as follows: the InD_Cu1 site is located at the 400412th nucleotide on chromosome 4; the InD_Cu2 site is located at the 18170370th nucleotide on chromosome 6; the InD_Cu3 site is located at the 183336th nucleotide on chromosome 7; and the InD_Cu4 site is located at the 3748553rd nucleotide on chromosome 5.

[0007] The InDel site combination specifically consists of the above-mentioned 4 InDel sites.

[0008] The present invention also protects 4 pairs of InDel primer combinations, including primer set 1 for amplifying InD_Cu1; primer set 2 for amplifying InD_Cu2; primer set 3 for amplifying InD_Cu3; and primer set 4 for amplifying InD_Cu4.

[0009] Among the four pairs of InDel primer combinations, the primer set 1 consists of the forward primer F1 shown in SEQ ID NO: 1 and the reverse primer R1 shown in SEQ ID NO: 2. The primer set 2 consists of the forward primer F2 shown in SEQ ID NO: 3 and the reverse primer R2 shown in SEQ ID NO: 4. The primer set 3 consists of the forward primer F3 shown in SEQ ID NO: 5 and the reverse primer R3 shown in SEQ ID NO: 6. The primer set 4 consists of the forward primer F4 shown in SEQ ID NO: 7 and the reverse primer R4 shown in SEQ ID NO: 8.

[0010] In the above primer set, the molar ratio of the primer containing "F" in its name to the primer containing "R" in its name can be specifically 1:1.

[0011] Any of the above-mentioned InDel primer combinations may specifically consist of the primer set 1, the primer set 2, the primer set 3 and the primer set 4.

[0012] The method for identifying the purity of the cucumber hybrid to be tested protected by the present invention may specifically include the following steps:

[0013] (1) obtaining genomic DNA of N strains of cucumber hybrids to be tested;

[0014] (2) using the genomic DNA of 8 to 12 strains (e.g., 8 to 10 strains, 10 to 12 strains, 8 strains, 10 strains, or 12 strains) of the cucumber hybrids to be tested obtained in step (1) as templates, and using four primer sets in the InDel primer combination to perform PCR amplification to obtain corresponding PCR amplification products;

[0015] (3) After completing step (2), perform agarose gel electrophoresis and use an instrument to view the results and count the number of strains with heterozygous bands of the four primer sets; the primer set with more and clearer heterozygous bands is the target primer set;

[0016] (4) using the genomic DNA of the N strains of cucumber hybrids to be tested obtained in step (1) as templates, respectively, and using the target primer sets to perform PCR amplification to obtain corresponding PCR amplification products;

[0017] (5) After completing step (4), use an instrument to check the bands of each PCR amplification product, and obtain the purity of the cucumber hybrid to be tested based on the number of heterozygous bands.

[0018] In the above method, the method of "obtaining the purity of the cucumber hybrid to be tested according to the number of heterozygous bands" can be: counting the number of plants showing heterozygous bands and the number of plants without bands for each target primer set, calculating the purity respectively, and then calculating the average value;

[0019] Purity = number of strains showing heterozygous bands with the target primer set / (N-number of strains without bands with the target primer set)×100%.

[0020] In the above method, the reaction system of PCR amplification can be specifically: 5μl template DNA (50ng / μl), 10μl 2×Mix, 1μl forward primer and 1μl reverse primer (20μmol / L), 3μl ultrapure water. The reaction procedure of PCR amplification can be specifically: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s; 58℃ annealing for 30s; 72℃ extension for 1min, 35 cycles; 72℃ extension for 10min.

[0021] In the above method, the cucumber hybrid to be tested can be Zhongnong 15, Zhongnong 9, Jinyou 12, Jinchun 4, Jinyou 2, Jinyou 35, Zhongnong 26, Bomei 11, Jinyou 48, Jingyan 107, Jinyou 108, Huanai H1104, Jinlu 3, Bomei 5032, Bomei 517, Jinyou 10, Deruite D19, Bomei 10, Bomei 74, Zhongnong 50, Jinyou 308, Deruite 4, Bomei 6913, Deruite 79, Jinyou 335, Jinyou 316, Jinyou 358, Jinchun 3, Zhongnong 37, Qiande 2, Qiande 117, Jingyan 403 ... Any of the following: Beijing Yan 207, Beijing Yan 402, Beijing Yan 1568, Beijing Yan 15163, Beijing Yan 108, Beijing Yan 118, Beijing Yan Green Arrow, Beijing Yan Green Arrow 2, Beijing Yan 407, Beijing Yan 109, Hu Za 6, Tian Jiao 7, Tian Jiao 8, Beijing Yan Han Bao 5, Ning Jia 3, Zhong Nong 19, Shen Lu 72, Shen Lu 64, Jasper, Ning Yun 3, Don Nong 806, Beijing Yan Mini 1, Beijing Yan Cui Yu Mini 2, Beijing Yan Mini 2, Qian De 1217, Beijing Yan Mini White, Beijing Yan Yu Tian 156, Beijing Yan Mini White 1, Beijing Yan Mini 5, Beijing Yan Jing Bao, Beijing Yan Mini 8, Beijing Yan Mini 9, Beijing Yan Mini 10, Sheng Yan Mini.

[0022] In the above-described method, the larger the value of N is, the higher the accuracy of identifying the purity of the cucumber hybrid to be tested is.

[0023] It should be noted that the present invention focuses on the purity assurance of cucumber hybrids, specifically first-generation hybrids, and the core challenge is to avoid parental mixing rather than mechanical mixing, that is, not a simple mixture of multiple cucumber hybrids.

[0024] The InDel primer combination provided by the present invention is intended to achieve early identification of cucumber hybrids at the seed or seedling stage, thereby ensuring the purity of the hybrids, effectively safeguarding the legitimate rights and interests of producers and breeders, and providing technical support for seed quality management of cucumber varieties. The method provided by the present invention has the advantages of high throughput, accuracy, low cost, simple operation, saving manpower and material resources, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The InDel typing effects of the four primer sets in some tested cucumber hybrids.

[0026] Figure 2 The figure shows the distribution of heterozygous loci of four primer sets in 66 cucumber species.

[0027] Figure 3 This is the band distribution effect of primer set 2 in 96 Jingyan 118 heterozygous strains. DETAILED DESCRIPTION

[0028] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0030] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0031] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0032] Example 1: Obtaining an InDel primer combination for identifying the purity of cucumber hybrids.

[0033] 1. Discovery of four InDel sites

[0034] Based on the resequencing data of 182 representative cucumber resources, the present invention develops cucumber whole genome InDel markers and uses them for variety purity identification to obtain 4 InDel sites. These 182 cucumber resources are rich in types, covering North China type, South China type, European fruit type and other types, basically including the main ecological types and agronomic traits of cucumber, reflecting as much germplasm representativeness as possible, and having high genetic diversity.

[0035] Specifically, the screening criteria for InDel sites are as follows: first, the dimorphic InDel sites are screened out on the whole genome chromosomes, and the InDel sites with 200 conserved wings and >30bp are selected. Then, the InDel sites with Miss <0.2, MAF >0.2, and He <0.05 are screened. Subsequently, the InDel sites with MAF >0.2 of the two populations are screened. Finally, blast specific analysis is performed and primers are designed.

[0036] The basic information of the four InDel sites is detailed in columns 1 to 4 of Table 1. The positions of the InDel sites on the chromosome are determined based on the comparison of the cucumber 9930 reference genome sequence, the version number of the cucumber 9930 reference genome sequence is V2 (download address: http: / / cucurbitgenomics.org / organism / 2 ).

[0037] Table 1. Basic information of the four InDel sites

[0038]

[0039] 2. Obtaining InDel primer combinations for identifying the purity of cucumber hybrids

[0040] Based on the four InDel sites found in step 1, the inventors of the present invention developed an InDel primer combination with high polymorphism for identifying the purity of cucumber hybrids.

[0041] The InDel primer combination consists of 4 primer sets, and the name of each primer set is shown in the second column of Table 2. Each primer set consists of 2 primer sequences and is used to amplify one InDel site. The nucleotide sequence of each primer in the 4 primer sets is shown in the fourth column of Table 2.

[0042] Table 2

[0043]

[0044]

[0045] Example 2, Validation of the InDel Primer Combination Developed in Example 1

[0046] The basic information of the 66 cucumber hybrids tested in this example is shown in Table 3. The 66 cucumber hybrids tested are all common high-quality hybrids or hybrids introduced from abroad.

[0047] Table 3. Basic information of 66 tested cucumber hybrids

[0048]

[0049]

[0050]

[0051] 1. Obtaining genomic DNA of test cucumber hybrids (DNA extraction by magnetic bead method)

[0052] Take an appropriate amount of leaf samples from each cucumber material and collect them in a centrifuge tube, add steel beads, and freeze them in liquid nitrogen, then use an ultra-high throughput grinder for grinding; add 800μl of SDS extract to each sample after grinding; place in a 60℃ oven for 30-40min, invert and mix 3 times during the period; add 240μl (i.e. 0.3 times the volume of SDS extract) of 3M potassium acetate solution to each sample, invert and mix thoroughly 5 times, and place in a 4℃ refrigerator for 20min; centrifuge at 4000 rpm for 10 minutes; prepare a new centrifuge tube or deep-well plate in advance, add 600μl of magnetic bead isopropanol to it (make sure the volume is equal to the supernatant), note that the operation order is to add magnetic bead isopropanol first, then add supernatant, and the volume of supernatant absorbed should not exceed 2 / 3 of the total volume; invert and mix, let stand at -20℃ until the magnetic beads are precipitated for 30 minutes, invert and mix again, adsorb with a magnetic stand, discard the supernatant, and invert on absorbent paper to pick up the residual liquid; add 200μl of 75% ethanol solution to the sample, invert and mix thoroughly, and let stand until the magnetic beads are completely precipitated; invert and mix again, adsorb with a magnetic stand, discard the supernatant, and invert on absorbent paper to pick up the residual liquid; after the magnetic beads are completely air-dried, add 100μl of pure water to it, invert and mix thoroughly to ensure that the DNA is completely dissolved; use Nanodrop (micro spectrophotometer) to take 2μl of DNA extract for detection, determine its OD260 / 280 and OD260 / 230 ratios (the ideal range is 1.8-2.0), and record the DNA concentration. Subsequently, the DNA concentration was adjusted to 50ng / μl for subsequent use.

[0053] 2. Using the genomic DNA of 66 test cucumber hybrids as templates, 4 primer sets were used for PCR amplification to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name and the primer containing "R" in the name was 1:1.

[0054] The reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min, 35 cycles; and extension at 72°C for 10 min.

[0055] 3. After completing step 2, perform agarose gel electrophoresis on the PCR amplification product, take photos and read the results through a gel imaging system. The genotype of the 66 test cucumber hybrids based on each InDel site is determined by the band situation. The specific judgment principle is as follows: If a test cucumber hybrid shows a heterozygous band based on a certain InDel site, the genotype of the test cucumber hybrid based on the InDel site is heterozygous; if a test cucumber hybrid shows a single band based on a certain InDel site, the genotype of the test cucumber hybrid based on the InDel site is homozygous.

[0056] Some results can be found in Figure 1 The results showed that each primer set could obtain good typing effect in the tested cucumber hybrids.

[0057] 4. Distribution of heterozygous sites and efficiency evaluation

[0058] (1) Based on the genotypes of 66 test cucumber hybrids based on four InDel loci, the number of heterozygous loci in each test cucumber hybrid was counted.

[0059] The distribution results of the number of heterozygous loci of 66 tested cucumber hybrids based on four primer sets are shown in Figure 2 The results showed that the four primer sets could make each of the tested cucumber hybrids have at least one heterozygous site.

[0060] (2) Hybrid purity identification can be carried out by using multiplex PCR to reduce the workload.

[0061] The results showed that the coverage rate of heterozygous sites in 66 tested cucumber hybrids by multiplex PCR (4 primer sets) reached 100%.

[0062] It can be seen that the InDel primer combination developed in Example 1 can be applied to the purity identification of cucumber hybrids.

[0063] Example 3: Using the InDel primer combination developed in Example 1 to detect the purity of Jingyan 118 hybrid

[0064] 1. Using the InDel primer combination developed in Example 1 to detect the purity of Jingyan 118 hybrid

[0065] 1. Obtaining genomic DNA of Jingyan 118 hybrid

[0066] (1) Planting 200 seeds of Jingyan 118 hybrid seedlings to obtain Jingyan 118 hybrid seedlings.

[0067] (2) Leaves or roots of 96 Jingyan 118 hybrid seedlings were randomly selected and genomic DNA was extracted using the magnetic bead method to obtain 96 genomic DNA samples of Jingyan superior hybrids.

[0068] 2. Screening of primer sets

[0069] (1) Using 8 genomic DNAs of the Jingyan 118 hybrid as templates, PCR amplification was performed using 4 primer sets in the InDel primer combination developed in Example 1 to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name to the primer containing "R" in the name was 1:1.

[0070] The reaction system of PCR amplification can be specifically: 5 μl template DNA (50 ng / μl), 10 μl 2×Mix, 1 μl forward primer and 1 μl reverse primer (20 μmol / L), and 3 μl ultrapure water.

[0071] The specific reaction procedure of PCR amplification can be: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 58°C for 30 seconds; extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 10 minutes.

[0072] (2) After completing step (1), perform agarose gel electrophoresis, and use a gel imaging system to take pictures of the electrophoresis results to obtain the results. Compare the number of strains at the heterozygous sites of the four primer sets, and the primer set with the largest number of strains at the heterozygous sites is the selected primer set.

[0073] The results showed that the number of strains at the heterozygous site of primer set 2 was the largest, which was 8. Therefore, primer set 2 was the selected primer set for subsequent experiments.

[0074] 3. Obtain the purity of Jingyan 118 hybrid

[0075] (1) Using genomic DNA of four Jingyan 118 hybrids as templates, PCR amplification was performed using four primer sets in the InDel primer combination developed in Example 1 to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name to the primer containing "R" in the name was 1:1.

[0076] The reaction system of PCR amplification can be specifically: 5 μl template DNA (50 ng / μl), 10 μl 2×Mix, 1 μl forward primer and 1 μl reverse primer (20 μmol / L), and 3 μl ultrapure water.

[0077] The specific reaction procedure of PCR amplification can be: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 58°C for 30 seconds; extension at 72°C for 1 minute, 35 cycles; extension at 72°C for 10 minutes.

[0078] (2) After completing step (1), perform agarose gel electrophoresis and use a gel imaging system to photograph the electrophoresis results to obtain the results.

[0079] InDel typing results are shown in Figure 3 .

[0080] (3) After completing step (2), the number of plants showing heterozygous sites and the number of plants without heterozygous sites in primer set 2 are counted; the purity of the Jingyan 118 hybrid is calculated according to the following formula; and the average value is further calculated to obtain the average purity.

[0081] Purity = number of strains showing heterozygous sites with the primer set / (96 - number of strains with no bands with the primer set) x 100%.

[0082] The results showed that the number of strains showing heterozygous bands in primer set 2 was 96, the number of strains without bands was 0, and the purity was 96 / (96-0)×100%=100%.

Claims

1. An InDel site combination, comprising 4 InDel sites in the cucumber genome; the 4 sites are as follows: the InD_Cu1 site is located at the 400412th nucleotide on chromosome 4; the InD_Cu2 site is located at the 18170370th nucleotide on chromosome 6; the InD_Cu3 site is located at the 183336th nucleotide on chromosome 7; and the InD_Cu4 site is located at the 3748553rd nucleotide on chromosome 5.

2. InDel primer combination, including primer set 1 for amplifying InD_Cu1; primer set 2 for amplifying InD_Cu2; primer set 3 for amplifying InD_Cu3; primer set 4 for amplifying InD_Cu4.

3. The InDel primer combination according to claim 2, characterized in that: The primer set 1 consists of a forward primer F1 shown in SEQ ID NO: 1 and a reverse primer R1 shown in SEQ ID NO: 2; The primer set 2 consists of a forward primer F2 shown in SEQ ID NO: 3 and a reverse primer R2 shown in SEQ ID NO: 4; The primer set 3 consists of a forward primer F3 shown in SEQ ID NO: 5 and a reverse primer R3 shown in SEQ ID NO: 6; The primer set 4 consists of a forward primer F4 shown in SEQ ID NO: 7 and a reverse primer R4 shown in SEQ ID NO:

8.

4. A method for identifying the purity of a cucumber hybrid to be tested, comprising the following steps: (1) obtaining genomic DNA of N strains of cucumber hybrids to be tested; (2) using the genomic DNA of 8 to 12 strains (e.g., 8 to 10 strains, 10 to 12 strains, 8 strains, 10 strains, or 12 strains) of the cucumber hybrids to be tested obtained in step (1) as templates, and using four primer sets in the InDel primer combination to perform PCR amplification to obtain corresponding PCR amplification products; (3) After completing step (2), perform agarose gel electrophoresis and use an instrument to view the results and count the number of strains with heterozygous bands of the four primer sets; the primer set with more and clearer heterozygous bands is the target primer set; (4) using the genomic DNA of the N strains of cucumber hybrids to be tested obtained in step (1) as templates, respectively, and performing PCR amplification using the target primer sets to obtain corresponding PCR amplification products; (5) After completing step (4), use an instrument to check the bands of each PCR amplification product, and obtain the purity of the cucumber hybrid to be tested based on the number of heterozygous bands.

5. The method according to claim 4, characterized in that: The method of "obtaining the purity of the cucumber hybrid to be tested according to the number of heterozygous bands" can be: counting the number of plants showing heterozygous bands and the number of plants without bands for each target primer set, calculating the purity respectively, and then calculating the average value; Purity = number of strains showing heterozygous bands with the target primer set / (N-number of strains without bands with the target primer set)×100%.

6. The method according to claim 5, characterized in that: The cucumber hybrids to be tested are Zhongnong 15, Zhongnong 9, Jinyou 12, Jinchun 4, Jinyou 2, Jinyou 35, Zhongnong 26, Bomei 11, Jinyou 48, Jingyan 107, Jinyou 108, Huanai H1104, Jinlu 3, Bomei 5032, Bomei 517, Jinyou 10, Deruite D19, Bomei 10, Bomei 74, Zhongnong 50, Jinyou 308, Deruite 4, Bomei 6913, Deruite 79, Jinyou 335, Jinyou 316, Jinyou 358, Jinchun 3, Zhongnong 37, Qiande 2, Qiande 117, Jingyan 403, Jingyan 207 , Beijing Research Institute 402, Beijing Research Institute 1568, Beijing Research Institute 15163, Beijing Research Institute 108, Beijing Research Institute 118, Beijing Research Institute Green Arrow, Beijing Research Institute Green Arrow No. 2, Beijing Research Institute 407, Beijing Research Institute 109, Shanghai Miscellaneous No. 6, Tianjiao No. 7, Tianjiao No. 8, Beijing Research Institute Hanbao No. 5, Ningjia No. 3, Zhongnong No. 19, Shenlu 72, Shenlu 64, Jasper, Ningyun No. 3, Dongnong 806, Beijing Research Institute Mini No. 1, Beijing Research Institute Cuiyu Mini No. 2, Beijing Research Institute Mini No. 2, Qiande 1217, Beijing Research Institute Mini White, Beijing Research Institute Yutian 156, Beijing Research Institute Mini White No. 1, Beijing Research Institute Mini No. 5, Beijing Research Jingbao, Beijing Research Institute Mini No. 8, Beijing Research Institute Mini No. 9, Beijing Research Mini No. 10, Shengyan Mini.

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