CN106755289B - Molecular identification method of lotus variety' fodder toming autumn - Google Patents
Molecular identification method of lotus variety' fodder toming autumn Download PDFInfo
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- CN106755289B CN106755289B CN201510804004.XA CN201510804004A CN106755289B CN 106755289 B CN106755289 B CN 106755289B CN 201510804004 A CN201510804004 A CN 201510804004A CN 106755289 B CN106755289 B CN 106755289B
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Abstract
The invention relates to the technical field of agricultural biology, and particularly discloses a molecular identification method for a lotus variety 'fodder tomb and autumn'. The primers are used for PCR amplification and detection to obtain SSR fingerprint data, and whether the lotus variety is 'fodder autumn' can be rapidly and accurately identified by comparing the fingerprint data with the data disclosed by the invention.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a molecular identification method of a lotus variety 'fodder autumn' based on a genome SSR marker.
Background
The lotus (Nelumbo) is also called lotus flower, is a perennial emergent aquatic plant, and is the only aquatic flower in ten traditional famous flowers in China; the method has a cultivation history of more than two thousand years in China, and has extremely high ornamental value and profound cultural background. The lotus has long florescence and ornamental value in the whole growth period, is deeply favored by people due to beautiful shape and unique faint scent, is a theme plant in waterscape garden arrangement, is one of important summer ornamental flowers, and can be used for flower arrangement and bonsai.
The wild lotus resource distributed in China is Chinese lotus, the flower color comprises red, pink and white, the plant is tall and big, the flower type is various, and besides the flower type with few petals, the wild lotus also comprises heavy petals, heavy platforms, thousand petals and the like. The yellow lotus species are still few, and the variety of the yellow lotus species in the flower state and the flower type is not enough. Therefore, the breeding of new species with yellow genes and various flower types by hybridization with the American goldthread has important significance in lotus breeding. An excellent new yellow lotus variety- 'fodder autumn' is cultivated by the plant institute of Chinese academy of sciences in Jiangsu province, university and enterprise. The fodder grass is like chrysanthemum in autumn color, light in initial color, deep in full color, easy to flower, high in planting density, long in florescence period, good in stress resistance and wide in adaptability, and is widely suitable for planting in middle and lower reaches of Yangtze river. This variety has been identified by the agricultural commission of Jiangsu province in 2015.
'ensiling autumn' is a typical new vegetative propagation type lotus variety, and the external morphological characteristics of the lotus variety are easily influenced by environmental conditions and management levels, so that consumers are difficult to distinguish the authenticity of the variety, and the development and utilization of the new variety are influenced. Therefore, it is very important to find a simple and effective identification technique which is real, effective, free from environmental influence and capable of accurately distinguishing different genotypes.
The SSR (simple repeat sequence) molecular marker technology is a DNA polymorphism detection technology based on PCR, has the characteristics of codominance, good stability, high polymorphism and large genetic information amount, has obvious advantages compared with other molecular markers, and is an ideal molecular marker method.
Disclosure of Invention
The technical problem is as follows: the invention provides a method for identifying SSR molecular markers based on lotus genomes, which overcomes the uncertainty of identifying new varieties only according to morphological characteristics, and is quicker, more accurate and more efficient.
The technical scheme is as follows:
the molecular identification method of the lotus variety 'fodder tomb and autumn' is characterized by comprising the following steps of:
1. DNA of 'tommy autumn' young leaves was extracted by CTAB method.
The DNA is amplified by using a fluorescence labeling primer SSRM01 (a forward primer sequence is 5-CATTTTGAAGTTTGGGGACAAAAG-3, and a reverse primer sequence is 5-GACACTAGGCCCAAAGTAGAGGGT-3), an amplification product is detected and analyzed by using a capillary electrophoresis fluorescence detection method, and the size of the fragment of which only the site can be obtained is 198 bp.
The DNA is amplified by using a fluorescence labeling primer SSRM02 (a forward primer sequence is 5-ATTTTCGAATCTCCCTCCCATACT-3, and a reverse primer sequence is 5-GAACAACAAAAAGAATGAGGATCTATTG-3), an amplification product is detected and analyzed by using a capillary electrophoresis fluorescence detection method, and the sizes of the fragments of which only the site can be obtained are 214bp and 238 bp.
The DNA is amplified by using a fluorescence labeling primer SSRM03 (a forward primer sequence is 5-TGAATGTGAAAAGAAAAAGTCGCA-3, and a reverse primer sequence is 5-TGTACTACTTTCCTCGAAGACGGC-3), an amplification product is detected and analyzed by using a capillary electrophoresis fluorescence detection method, and the sizes of fragments of which only the site can be obtained are 136bp and 148 bp.
Meanwhile, the 3 pairs of primers are used for carrying out SSR fluorescence amplification detection on the DNA of a new variety of leaves of the lotus, and the result obtained by each pair of primers is completely consistent with the required SSR fingerprint data, so that the variety is shown to be 'tommy autumn'. Otherwise, the variety is other variety.
2. The amplification and detection method comprises the following steps:
SSR-PCR was performed using 25ul of a reaction system containing 50ng DNA, 2.5. mu.L of 10 × PCR buffer,0.2mmol L-1dNTPs, 1U Taq DNA polymerase, forward and reverse primers 4. mu. mol L each-1。
The PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55-60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10 min at 72 ℃, and storing at 4 ℃.
The amplified products were subjected to automated fluorescence detection using American ABI3730, and the raw data were analyzed using GeneMapper software to obtain SSR fingerprint data.
Has the advantages that:
1. the method disclosed by the invention identifies the DNA sequence of 'fodder tomb and autumn' by directly detecting the DNA sequence, so that the uncertainty of identifying the DNA sequence according to external morphological characteristics is overcome.
2. The invention provides scientific technical support for the protection of new varieties of 'fodder tombing autumn'.
3. The method provides experimental evidence for identification in the popularization and application process of 'fodder and autumn' varieties.
Detailed Description
1. Extraction of DNA and obtaining of identification marker
27 common and representative species of lotus in production were selected, and the 27 species and young leaves of ` tomown autumn ` were collected, and genomic DNA thereof was extracted by the CTAB method. DNA quality and concentration were determined by 0.8% agarose gel electrophoresis and OneDrop spectrophotometer.
The DNA extracted by 16 pairs of SSR primers is amplified, and the SSR-PCR adopts a 25 mu L reaction system which comprises 50ng of DNA, 2.5 mu L of 10 × PCR buffer and 0.2mmol of L-1dNTPs, 1U Taq DNA polymerase, forward and reverse primers 4. mu. mol L each-1. The SSR-PCR amplification program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55-60 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extending for 10 min at 72 ℃, and storing at 4 ℃. The amplification products were subjected to automated fluorescence detection using ABI3730 in USA, and the raw data were analyzed using GeneMapper software to obtain allele data at each locus.
The amplification result shows that the PIC value of three primers of SSRM01, SSRM02 and SSRM03 in 16 pairs of primers is the largest, and the polymorphism is the best (Table 1); the 'toming autumn' and other 27 varieties tested can be completely distinguished by only using the three pairs of primers, and the fingerprint data are shown in table 2.
TABLE 1 three pairs of SSR primer polymorphisms
Fingerprint data of table 228 varieties under 3 pairs of SSR primers
2. Verification experiment for identifying 'fodder autumn' variety by using screened 3 pairs of SSR primers
The 3 pairs of SSR fluorescent primers are used for selecting 16 new lotus varieties newly bred by Chinese academy of sciences and enterprises in Jiangsu province, and the new lotus varieties and the 'fodder autumn' are simultaneously amplified, and the amplification and detection methods are the same as the above. The results show that the 3 pairs of primers can distinguish 'toming autumn' from other varieties, and the 'toming autumn' and the SSR fingerprint data of the 16 varieties are shown in Table 3.
Fingerprint data of 317 varieties under 3 pairs of SSR primers
| Variety of (IV) C | Primer SSRM01 | Primer SSRM02 | Primer SSRM03 |
| Fodder autumn color | 198 | 214/238 | 136/148 |
| XY-N-1 | 198/202 | 244 | 136 |
| XY-N-2 | 198/202 | 224/244 | 136/148 |
| XY-N-3 | 202/216 | 218/244 | 136/142 |
| XY-N-4 | 212 | 222/244 | 136 |
| XY-N-5 | 198/216 | 222/238 | 134/136 |
| XY-N-6 | 198 | 214/222 | 136 |
| XY-N-7 | 188/198 | 224/244 | 136/148 |
| XY-N-8 | 198 | 222/244 | 134/136 |
| XY-N-9 | 202/216 | 218/244 | 136/142 |
| XY-N-10 | 216 | 238/244 | 136/142 |
| XY-N-11 | 212 | 222/238 | 148 |
| XY-N-12 | 212 | 244 | 136/148 |
| XY-N-13 | 216 | 218 | 136 |
| XY-N-14 | 198 | 210/222 | 136/148 |
| XY-N-15 | 198/216 | 214/222 | 136/148 |
| XY-N-16 | 188/198 | 230/238 | 136/148 |
From the verification results, only 1 pair or 2 pairs of SSR primers can be used for distinguishing 'toming autumn' from other 16 new varieties, but considering that more new varieties can appear in the future, the invention proposes to simultaneously detect the 3 pairs of primers for identification scientificity, and if the results obtained by amplifying the 3 pairs of primers are completely consistent with the results disclosed by the invention, the variety is the lotus variety 'toming autumn'.
Claims (1)
1. The molecular identification method of the lotus variety 'fodder tom autumn', is characterized in that: (1) amplifying DNA extracted from leaves of 'fodder grass and autumn' by using a fluorescence labeling primer SSRM01 (a forward primer sequence is 5-CATTTTGAAGTTTGGGGACAAAAG-3, and a reverse primer sequence is 5-GACACTAGGCCCAAAGTAGAGGGT-3), and detecting the amplified product by capillary electrophoresis fluorescence to obtain a fragment with the size of 198bp, wherein the fragment can only be obtained from the site; (2) amplifying DNA extracted from 'fodder autumn' leaves by using a fluorescence labeling primer SSRM02 (a forward primer sequence is 5-ATTTTCGAATCTCCCTCCCATACT-3, and a reverse primer sequence is 5-GAACAACAAAAAGAATGAGGATCTATTG-3), and carrying out capillary electrophoresis fluorescence detection on an amplification product to obtain fragments with the sizes of 214bp and 238bp, wherein the fragments only can be obtained from the sites; (3) carrying out amplification products on DNA extracted from 'fodder autumn' leaves by using fluorescence labeling primers SSRM03 (forward primer sequence 5-TGAATGTGAAAAGAAAAAGTCGCA-3, reverse primer sequence 5-TGTACTACTTTCCTCGAAGACGGC-3) through capillary electrophoresis fluorescence detection to obtain fragments with the sizes of 136bp and 148bp only at the site; meanwhile, the 3 pairs of primers are used for carrying out SSR fluorescence amplification on a certain variety of the lotus, and the result obtained by each pair of primers is completely consistent with the required SSR fingerprint data, so that the variety is shown to be 'tommy autumn'.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455175A (en) * | 2008-12-29 | 2009-06-17 | 江苏省中国科学院植物研究所 | Mint hybridization method |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455175A (en) * | 2008-12-29 | 2009-06-17 | 江苏省中国科学院植物研究所 | Mint hybridization method |
Non-Patent Citations (4)
| Title |
|---|
| Comparative analysis of genetic diversity of lotus (Nelumbo) using SSR and SRAP markers;Mei Yang等;《Scientia Horticulturae》;20120525;第142卷;185-195 * |
| 基于EST_SSR标记的莲属种质资源遗传多样性分析;徐玉仙等;《植物分类与》;20151102;第37卷(第5期);595-604 * |
| 秣陵秋色;丁跃生;《花卉园艺》;20130831(第8期);4 * |
| 荷花遗传多样性的ISSR标记分析;刘艺平等;《西北农林科技大学学报（自然科学版）》;20130430(第4期);139-146 * |
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