Development and application method of solanum melongena L. EST-SSR marker
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An application method, eggplant technology, applied in the field of breeding, can solve the problems of low marker polymorphism, limited sequence information, weak research foundation, etc.
Active Publication Date: 2013-05-15
SHANGHAI JIAO TONG UNIV
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However, within eggplant species, the polymorphism of markers is relatively low, and more markers need to be developed in order to construct a high-density linkage map
[0006] At p...
Method used
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specific Embodiment 1
[0072] 1. Experimental material selection: Eggplant materials numbered 1-48 as shown in Table 1 were taken and planted in the experimental greenhouse.
[0073] 2. Get the tender green leaves from the field material and extract its total DNA. The specific steps are as follows:
[0074] (1) Put 100-200mg frozen leaves or fresh leaves of tender green leaves into a pre-cooled mortar, add liquid nitrogen to grind. Transfer the ground powder into a 2ml centrifuge tube, add 800ul freshly prepared extraction buffer (0.35M Glucose, 0.1M Tris.HCl, 5mM Na.EDTA, 2%PVP, 1%(V / V)β-Me, dd Water), vortexed and kept in ice bath for 10 min. Centrifuge at 10000rpm for 15min (4°C), discard the supernatant;
[0075] (2) Add 700ul 65°C preheated lysis buffer (1.4M NaCl, 0.1M Tris.HCl, 20mM Na.EDTA, 2%CTAB, 2%PVP, 1%(V / V)β-Me, ddWater), and use a toothpick to loosen, vortex to mix, 65 ℃ water bath for 30min;
[0076] (3) Add 750ul of chloroform: isoamyl alcohol (24:1) mixture, invert more than 50...
specific Embodiment 2
[0096] 1. The eggplant parents numbered 44 and 41, F 1 and its F 2 Group planted in greenhouses;
[0097] 2. Take tender green leaves from the field materials and extract their total DNA.
[0098] 3. Use the developed primers for analysis: the specific steps are as follows:
[0099] 3.1 Use the 43 pairs of markers developed by this marker to analyze;
[0100] The PCR reaction system of SSR is: the total volume of the system is 10 μL, the template DNA is about 20 ng., the front primer and the back primer are respectively 0.1 μM, 2.5 mM MgCl 2 , 0.2mM dNTPs, 1×Taq buffer and 1U Taq DNA Taq polymerase (Shanghai Promega). The PCR reaction was performed on a 96-well PCR machine (Eppendorf AG6321, Eppendorf). The reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 0.5 min, renaturation at 55°C for 1 min, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min and storage at 4°C.
[0101] The amplified product undergoes non-denaturin...
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Abstract
The invention relates to a development and application method of a solanum melongena L. EST-SSR marker. The development and application method comprises the following steps of: (1) obtaining an EST sequence from a NCBI database, processing and detecting the SSR; (2) designing the sequences of the primers of an SSR marker; (3) obtaining the data of the solanum melongena L. No.1-48 in a table 1 and the light green leaves of the solanum melongena L. in an solanum melongena L. group F2, and extracting the total DNA (deoxyribonucleic acid); (4) amplifying the data of the solanum melongena L. No.1-12 in the table 1 by using the designed sequences of the primers of the SSR marker; (5) obtaining a different EST-SSR marker in the data of the solanum melongena L. No.7-12 in the table 1, wherein the sequence of the EST-SSR marker is showed in a sequence table; (6) amplifying the data of the solanum melongena L. No.7-12 in the table 1 and the solanum melongena L. group F2 by using the obtained EST-SSR marker; (7) calculating the content of the polymorphism information of each pair of primers, and analyzing the genetic diversity of the data of the solanum melongena L. No13-48 in the table 1 in a clustering way; and (8) carrying out linkage mapping and QTL positioning to the solanum melongena L. group F2 obtained in the step (6). The results show that the G249 primer is linked to the solanum melongena L. fruit length and the fruit solanum melongena L. fruit character to explain 4% and 5.1% of phenotypic variation, and the G192 primer is linked to the solanum melongena L. calyx size and the solanum melongena L. calyx character to explain 5.3% of phenotypic variation.
Description
technical field [0001] The present invention relates to a plant breeding method, in particular to a method for developing eggplant EST-SSR markers, and the application of the markers to evaluate eggplant genetic diversity, construct molecular genetic linkage maps, and locate QTLs for important traits of eggplant The method belongs to the technical field of breeding. Background technique [0002] SSR markers, also known as microsatellite DNA, are composed of short sequences repeated in tandem, and the repeat unit length is generally 1-6 bp. SSR markers play an important role in genetic analysis due to their high polymorphism, co-dominance, abundant quantity, wide genome coverage distance, high resolution, easy detection, and simple operation (Powell et al., 1996 ; Stagel et al., 2008). [0003] Eggplant (Solanum melongena L.), belonging to the family Solanaceae, is an important vegetable crop in many countries. Eggplant is rich in minerals and vitamins, and polyphenols con...
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