Research On Cultivar Identification And DNA Fingerprinting Of Crops Based On Molecular Markers

Seed was the most basic means of agricultural production. Fast, accurate and high efficient crop cultivar identification and DNA fingerprinting methods and their application were of great importance in seed quality control. Combining suitable molecular markers with detection techniques to invent simple, low cost, accurate and high efficient technical system for cultivar identification and fingerprinting was urgent need to crack down on counterfeit and shoddy seeds, protect the legitimate rights and interests of the breeder, seed company and farmers, standardize seed market and improve the international competitiveness of Chinese seed industry. In present study, rice, cotton, wheat, maize and tobacco were selected to study the cultivar identification and fingerprinting methods. The achieved results were as follows:1. Uniform design was applied to optimize ISSR-PCR reaction system for rice. cotton, tobacco, wheat and maize, and the optimal ISSR-PCR reaction systems for each crop were established, respectively.24rice cultivars.12tobacco cultivars.10cotton cultivars.8wheat cultivars and18maize cultivars were identified using the optimal PCR reaction systems, respectively. The results showed that variations of Mg2-, dNTPs. primer, and Taq polymerase concentrations changed the fingerprinting patterns; each crop had different optimal ISSR-PCR reaction system. Combination of2ISSR primers (UBC900and UBC825) was able to identify24rice cultivars.3ISSR primers (UBC848. UBC841and UBC830) were able to identify12tobacco cultivars. Combination of3ISSR primers (UBC807. UBC811and UBC820) could identify10cotton cultivars. ISSR primer UBC849was able to discriminate8close-related wheat cultivars. A group of3ISSR primers (UBC807. UBC811and UBC822) were able to identify18maize cultivars. The results showed that uniform design was an efficient method for optimization of ISSR-PCR reaction systems. ISSR molecular markers could be used in rapid cultivar identification of rice, cotton, tobacco, wheat and maize, which might be a potential technique for crop cultivar identification and fingerprinting.2. New method for crop variety identification and fingerprinting based on SSR and ISSR molecular markerswas constructed. The new method included the following steps:extracting DNA of seedlings with improved CTAB method; SSR and ISSR PCR amplification with the genomic DNA as the template; detecting PCR products by agarose gel electrophoresis (staining with GelRed) and take pictures in the automated gel imaging system under UV light:analyzing the data of electrophoresis bands. DNA fingerprinting constructed by the new method was based on cultivar number, type of amplified bands, band number and’0’and’1’array as the basic information. DNA fingerprinting was prepared for each tested cultivars based on each SSR or ISSR primers. The method was prior to the previous methods, which was more convenient, faster and more suitable for the rapid identification of crop cultivar.3. Comparing the discriminating efficiency between SSR and ISSR markers on rice fingerprinting and cultivar identification based on PIC (polymorphic information content), Ibav (average band informativeness), R (Resolving Power). MI (Marker Index), D (Discriminating Power Calculated) and D□(Discriminating Power Estimated). The results showed that average PIC and Ibav values of SSR (0.33,0.47) were higher than ISSR (0.28.0.41). respectively, and the average D, DL, R and MI values of ISSR (0.642,0.636,2.41.1.63) were higher compared with SSR (0.614,0.606,1.03.0.72), respectively. It was indicated that both SSR and ISSR molecular markers were suitable for rapid rice cultivar identification, and ISSR might have a higher discriminating efficiency in rice cultivar identification compared with SSR marker system. Combination of SSR and ISSR markers might be a capable strategy in fingerprinting and cultivar identification of rice in further study. However, the stability of ISSR technique might be improved in further study, and SSR molecular markers were much steadier and more suitable for automatic analysis. So, SSR molecular markers were selected as the ideal marker for further study of crop cultivar identification and fingerprinting in present paper.4. DNA fingerprinting of the48main rice cultivars in Zhejiang province were constructed based on SSR molecular markers. A total of42bands were produced with the18polymorphic SSR primer pairs,41(97.6%) of which were polymorphic. The number of polymorphic bands detected by each SSR primer pair ranged from1(RM210) to4(RM16), with an average of2.28bands per primer pair. The number of band patterns produced by each primer pair ranged from2to5, with an average of2.88. DNA fingerprinting of48rice cultivars based on each primer pair were constructed, which were able to identify40out of the48rice cultivars.7of the8groups of close-related cultivars were able to be identified based on the fingerprinting. UPGMA clustering results based on18polymorphic SSR primer pairs divided the48rice cultivars into two groups: the first group consists of34indica rice cultivars, and the second group gathered14japonica rice cultivars. And the primer pairs RM249and RM250could identify japonica from indica rice cultivars. The results showed that combination of SSR molecular markers and agarose gel electrophoresis (staining with GelRed) detection method could be used for convenient, economy rice cultivar identification, and japonica and indica germplasm identification.5. TP-M13-SSR technique was a high throughput and low cost analysis method of SSR product based on fluorescence detection of capillary electrophoresis. DNA fingerprinting of130upland cotton were constructed with TP-M13-SSR method.130cotton cultivars from10countries were selected as the test materials to analyze the100SSR primer pairs which showed good polymorphic among upland cotton in previous study. Among the100groups of TP-M13-SSR primers,60sets of primers showed steady, clear and polymorphic amplification results, and the length of produced fragments ranged from102bp to388bp.50sets of steady and polymorphic TP-M13-SSR primers were selected for fingerprinting of130cotton cultivars. A total of368DNA fragments were produced with the50sets of primers, with an average of7.36. DNA fingerprinting database of130cotton cultivars were constructed using the cultivar identification system software (V1.0) based on the number and length information of the nucleotide fragment produced.130cotton cultivars were able to be identified based on the fingerprinting. The fingerprinting database could be used for fingerprinting data query and match between tested material and standard cultivar. The results showed that TP-M13-SSR method was suitable for standard fingerprinting and cultivar identification of upland cotton, especially for a large number of samples. Combination of TP-M13-SSR technique, conventional gel electrophoresis. and DNA fingerprinting database of crop cultivars might be an efficient strategy for rapid and accurate crop cultivar identification based on molecular markers.6. High resolution melting curve analysis (HRM) was a new genotyping method developed based on real-time fluorescence quantitative PCR technology, and its principle was that the melting temperatures of nucleotide fragments were different base on their different base composition or sequence. SSR molecular markers combining high resolution melting curve analysis method were used to construct the DNA fingerprinting of3main cultivated rice hybrids and their parents in Zhejiang province. The results showed that the method based on combination of SSR molecular markers with high resolution melting curve analysis (SSR-HRM) was suitable for rice cultivar identification and had the characteristics of high throughput, high sensitivity, automatic, fast, and intuitive.7. The advantages and disadvantages of agarose gel electrophoresis, TP-M13-SSR capillary fluorescence detection and SSR high resolution melting curve analysis were compared. A suitable strategy for crop cultivar identification and fingerprinting was proposed. Firstly, high concentration agarose gel electrophoresis was used to detect the sizes, numbers and amplification effect of the produced fragments to screen out SSR primers with clear, stable and polymorphic bands. Secondly, standard DNA fingerprinting was constructed by the TP-M13-SSR capillary electrophoresis with fluorescence detection method. Finally, the fingerprinting was used for cultivar identification. If the difference among fragment sizes of the primer was greater than10bp. high concentration of agarose gel electrophoresis detection method could be selected. If the difference among fragment sizes was smaller than10bp. capillary electrophoresis with fluorescence detection or high resolution melting curve analysis should be selected if there were a large number of tested cultivars. or high resolution melting curve analysis should be given priority with only a small number of samples.