| Rapeseed is one of the main oil crops and rapeseed oil accounts for more than50%of domesticvegetable oils. Rapeseed variety identification is the foundation of the seed quality standardization, andcan protect interests of both consumers and breeders. Cultivar identification is also an important methodof seed management and solving the disputes on authenticity of cultivars. SSR marker is the mostwidely used technology in variety identification. SSR fragments are generally analyzed by PAGE andsilver staining at present. But there are some defects in this method such as low-efficiency,low-reproducibility and difficulty of accurate estimation of allele size. Capillary electrophoresis-basedmicrosatellite analysis has been well applied in DNA fngerprinting of rice and maize cultivars, whilst,it is seldom used in rapseed. Therefore, establishment of a highly efficient technology platform based oncapillary electrophoresis with SSR markers is urgently necessary for cultivar identification of rapeseed.In this study evaluation and screening SSR primers, identification of allele-specific capillaryelectrophoresis peaks and methods of multipile markers detection were studied, and a DNA fingerprinting technology platform was established based on the principle of high speed, high accuracy and highreproducity. Furthermore, this platform was used in2010-2013national rapeseed cultivar regional trial.The main research contents and results were as follows:1. Establishment of a technology platform of cultivars identifcation based on capillary electrophoresiswith SSR fluorescence makers. Eight B.napus DH lines were used to evaluate and screen SSR primersfrom87pairs of primers that were selected in previous studies. A total of51pairs of SSR primers wereselected according to the distinguishability of capillary electrophoresis peak and the polymorphism ofthe SSR markers. These51pairs of SSR primers could be detected57SSR loci in our experimentsystem. Hence,96lines from core collection of B. napus were used to further screen the aboveSSRprimers,40pairs which could produce high polymorphism SSR loci with high repoductivity and easydistinguishment were finally selected based on the PIC value and the charater of capillaryelectrophoresis peak. These40pairs of primers could be amplified45loci. By comparing the alleledetection effects of ofmultiplex-PCR products, mixed products of several single-PCR amplifications thelatter was regarded to be the optimal method which could assure the accuracy of detection and save cost.The similarity coeffieciency of the96lines from core collection was analyzed using the40pairs of SSRmarkers, and the results showed their similarity coeffieciency ranged from0.67-0.89, indicating therewere not similar lines among the96lines, which was consistent with the characterization of corecollection. These results also suggested that the DNA fingerpinting plantform established in this studywas competent for identification of the specificationof a cultivars. The technology platform establishedin this study was adopted by an agricultural standard of People’s Republic of China, i.e.,―Protocol forDNA Fingerprinting of Rapeseed (Brassica napus L.) Variety SSR Molecular Marker Method‖(Inapproval).2. Application of the DNA fingerprinting technology platform. Twenty of the40pairs of SSRprimers were used to analyze the DNA fingerprint consistency of the36candidate cultivars in repeated test of2010-2012national rapeseed cultivar regional trial. As a result,35cultivars showed fineconsistency with similarity coeffieciency of90%-100%, and one cultivar had bad consistency withsimilarity coeffieciency lower than90%, which was in consistency with the results of seed qualityanalysis of this cultivar in two years. These results gave robust evidence that the DNA fingerprintingtechnology platform was available and reliable. The40pairs of SSR primers were used to analyze thethe DNA fingerprint of96candidate cultivar of2012-2013national rapeseed cultivar regional trial.Since the DNA fingerprint was digital, we initate construction of DNA fingerprint data library ofrapseed cultivars, and the DNA fingerprint of the96cultivars were imported into this library. Thegenetic diversity of the96cultivars was analyzed.A total of115alleles and178genotypes wereidentified in these cultivars. For the45SSR loci produced by the40pairs of primers, every locus has2.61alleles and4.05genotypes. The similarity coeffieciency of96cultivars ranged from0.63to0.98,and the overall genetic diversity was high except that several pairs of cultivars show high similaritycoeffieciency, which might be suspected to be same cultivars.3. In addition, genetic diversity of Brassica juncea from Tibet Autonomous Region of Chinawereanalyzed in this study. Seventy-three B. juncea accessions from Tibet Autonomous Region of China and35from southwest of China，northwest of China and India were analyzed using8SRAP (sequence－related amplified polymorphism) primers combinations and20phenotypic traits SRAP results showedthat totally139bands were produced with polymorphism rate of24.5%. Clustering result of SRAPshowed that108accessions were divided into4groups, and the accessions in each group were mainlyfrom the same region. Genetic diversity index of China Tibet accessions was higher than others.Variation degree of8important phenotypic traits of Tibet accessions showed the highest variationcoefficiency of41.29%on silique number per plant. Clustering analysis of phenotypic traits showed that108materials were also divided into different groups obviously according to their original regionsexcept of the accessions from Northwest. In conclusion, Tibet B. juncea had abundant genetic diversity.The genetic diversity indexes of the materials from different regions were as following: ChinaTibet>Southwestern China> Northwestern China> India. Genetic differences in of B. juncea were mainlycorrelated with geological and biological conditions.In summary, this study aimed to sovle the problems of low efficiency and poor precision and lowreproductivity when the method of PAGE and silver staining were used, and a technology platform ofcultivars identifcation based on capillary electrophoresis with SSR fluorescence makers was established.This platform was proved to be feasible and reliable by the successful application in the nationalrapeseed cultivar regional trial. This platform could also be used to construction of DNA fingerprintdata library of rapseed cultivars, to identify authenticity of cultivars rapidly and accuratly, which wouldcontribute to protect the interests of the breeder and consumers, and to keep the health development of rapseed market. Besides, the genetic diversity analysis of Brassica juncea from Tibet would provide aimportant reference for utilization of these germplasms. |
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