Gel Electrophoresis of Proteins for the Identification of Crop Varieties

With the development of the international trade and agricultural science and technology, especially after the execution of the rules on protection of new plant varieties, considerable emphasis has been placed on variety identification. Many evidences have suggested that gel electrophoresis have great influence on this area. This paper reviewed study status of various gel electrophoresis, including development of the meth-

Differential distribution of authenticity results of different varieties of refined honey based on stable isotope ratio method

Objective Using the stable isotope ratio method for the authenticity identification and variety identification of refined honey.Methods In this paper,a total of 17 samples of different varieties of refined honey were used to obtain refined honey proteins by precipitation with sodium tungstate solution and sulphuric acid solution.The isotope mass spectrometer was used to simultaneously detect theδ^(13)C values of refined honey proteins and refined honey as well as theδ^(18)O andδ^(2)H values of refined honey,processed of the results obtained,analysed the authenticity of the samples and conduct a variety identification study.Results Tested of the resulting honey samples,the results showed that four batches of refined honey did not up to standard,two batches of C-4 vegetable syrup were detected as adulterated,and two batches of protein were not detected.Theδ^(18)O andδ^(2)H values of refined honey were also found to be effective in distinguishing the varietal origin of refined honey to a certain extent.Conclusions The stable isotope ratio method is useful in the authenticity identification of refined honey,and provides new ideas to further promote the authenticity of refined honey and variety identification research.

Study and Identification of Gaultheria yunnanensis Leaves from Different Regions

[Objective] In order to identify and comparatively study leaves of ethnic medicine Gaultheria yunnanensis from different regions of Guizhou.[Method] Characteristics,microscopic,physical and chemical identification were studied on Gaultheria yunnanensis.[Result] There was no obvious difference in characteristics and microscopic sections among Gaultheria yunnanensis leaves from different regions.Blue fluorescence spots were observed clearly by TLC（Thin Layer Chromatography） test.[Conclusion]The study provided a basis for improvement of the quality standard of the Gaultheria yunnanensis.

Purity Identification of Xinshikui 6 Using SSR Marker Technique

[Objective] SSR molecular marker technique was used to determine the purity of sunflower seed with the aim to provide accurate, convenient method for the identification of the purity of hybrid seeds in production and processing. [Method] With the DNA of Xinshikui 6 and its parents as template, about 100 pairs of SSR molecular markers were screened after DNA extraction, PCR amplification and electrophoresis production. [Results] SSR polymorphic primer marker 532 produced a specific band of 469 bp in the female parent, and a specific band of 451 bp in the male parent; primer marker 574 produced a specific band of 364 bp in the female parent, and a specific band of 384 bp in the male parent. The indoor molecular purity identification and field purity identification were consistent with each other. The primer marker 532 and 574 could be obtained from the SSR molecular marker method to distinguish the male parent, female parent and hybrid of Xinshikui 6, and both of the 2 primer markers can effectively identify the purities of the hybrid seeds of Xinshikui 6, as well as the authenticity of the seeds. [Conclusion] The proposed method was simple, fast, accurate to operate with the advantages of high reproducibility, and it had become the major method in the identification of sunflower varieties.

Application of Computer Vision Technique to Maize Variety Identification

Variety identification is important for maize breeding, processing and trade. The computer vision technique has been widely applied to maize variety identification. In this paper, computer vision technique has been summarized from the following technical aspects including image acquisition, image processing, characteristic parameter extraction, pattern recognition and programming softwares. In addition, the existing problems during the application of this technique to maize variety identification have also been analyzed and its development tendency is forecasted.

Genetic relationship and pedigree of Chinese watermelon varieties based on diversity of perfect SNPs

Watermelon(Citrullus lanatus)is one of the world’s most important fruit crops,and China produces the most watermelons in the world.Recently,a watermelon variome consisting of 414 key resequenced accessions was reported.However,the genetic relationships and pedigree of Chinese watermelon varieties in the seed market remain unclear.In this study,241 evenly distributed perfect single nucleotide polymorphisms(SNPs)derived from the watermelon variome were selected for variety identification.The diversity of 247 Chinese watermelon varieties was identified based on their SNP genotypes.The 247 watermelon varieties were clustered into five subpopulations:the East Asian ecotype,intermediate ecotype,small fruit with red flesh ecotype,small fruit with yellow flesh ecotype,and American ecotype.We further established the pedigree of four subpopulations,of which JingXinNo.1,ZaoChunHongYu,HuangXiaoYu and XiaoLan,and Sugarlee were the main doner of the East Asian ecotype,small fruit with red flesh ecotype,small fruit with yellow flesh ecotype,and American ecotype,respectively.Thirty-two core SNPs were selected and applied in watermelon variety identification.They were also validated by the Kompetitive allele-specific PCR(KASPar)platform.The present study furthered our understanding of the genetic relationships and pedigree of watermelon varieties in China,and will help to manage the plant variety protection in watermelon.

Comparison of DUS testing and SNP fingerprinting for variety identification in cucumber

Variety identification plays an important role in protecting the intellectual property of varieties,ensuring seed quality,and encouraging breeding innovation.Currently,morphological evaluation in the field,such as distinctness,uniformity,and stability(DUS)testing,and DNA fingerprinting in the laboratory using molecular markers are two dominant methods used for variety identification.Few studies have compared the results of these approaches,and the relationship between the two methods is obscure.In this study,134 dominant cucumber varieties were evaluated using 50 DUS testing traits and genotyped by 40 single nucleotide polymorphisms(SNPs).The 40 SNPs were developed in our previous study and arewell suited for variety identification.In the DUS testing,significant positive or negative correlations among 50 DUS traits were observed,and 20 core traits,including 15 fruit traits,were further selected to increase field inspection efficiency.This suggested that fruit shape plays an important role in variety identification.The ratio of fruit length/diameter was themost important trait,explaining 9.2%of the phenotypic variation.In the DNA fingerprinting test,the 40 SNPs were highly polymorphic and could distinguish all of the 134 cucumber varieties,and 14 core SNPs were selected to improve the identification rate.Interestingly,the population structure analysis of 134 cucumber varieties by phenotypic data in the DUS test was in accordance with the genotypic data from the DNA fingerprinting,indicating that all varieties could be divided into the same four subgroups:European type,North China type,South China type,and hybrids of the North China and South China types.Moreover,linear correlativity of distinguishment for each pair of varieties was observed between the DUS test and the DNA fingerprinting.These results indicated that these two methods have good application in future research,especially for the scaled-up analysis of hundreds of varieties.

Establishment and application of an SNP molecular identification system for grape cultivars

We aimed to develop a set of single nucleotide polymorphism(SNP) markers that can be used to distinguish the main cultivated grape(Vitis L.) cultivars in China and provide technical support for domestic grape cultivar protection, cultivar registration, and market rights protection. A total of 517 high-quality loci were screened from 4 241 729 SNPs obtained by sequencing 304 grape accessions using specific locus amplified fragment sequencing, of which 442 were successfully designed as Kompetitive Allele Specific PCR(KASP) markers. A set of 27 markers that completely distinguishes 304 sequenced grape accessions was determined by using the program, and 26 effective markers were screened based on 23 representative grape cultivars. Finally, a total of 46 out of 48 KASP markers, including 22 markers selected by the research group in the early stage, were re-screened based on 348 grape accessions. Population structure, principal component, and cluster analyses all showed that the 348 grape accessions were best divided into two populations. In addition, cluster analysis subdivided them into six subpopulations. According to genetic distance, V. labrusca, V. davidii, V. heyneana, and V. amurensis were far from V. vinifera, while V. vinifera×V. labrusca and V. amurensis×V. vinifera were somewhere in between these two groups. Furthermore, a core set of 25 KASP markers could distinguish 95.69% of the 348 grape accessions, and the other 21 markers were used as extended markers. Therefore, SNP molecular markers based on KASP typing technology provide a new way for mapping DNA fingerprints in grape cultivars. With high efficiency and accuracy and low cost, this technology is more competitive than other current identification methods. It also has excellent application prospects in the grape distinctness, uniformity, and stability(DUS) test, as well as in promoting market rights protection in the near future.

Construction and Testing of a Primary Microsatellite Database of Major Rice Varieties in China

Sixty-three major inbred varieties and parental lines of major F1 hybrids used in the commercial rice production in China were assayed with rice microsatellites screened in a previous study and additional microsatellites on four chromosomes. A set of 24 markers was selected and proposed for its application in the variety identification of rice, which are distributed on all the 12 rice chromosomes with 2 markers on each chromosome. The 63 major varieties and parental lines, as well as 41 major F1 hybrids, were genotyped with the markers. Alleles detected in each line at each marker locus were verified. By matching marker genotypes of corresponding F1, maternal and paternal lines of hybrid rice, high reliability of the maternal lines was verified, data on the paternal lines were modified, and a false hybrid was removed. A database containing genotype data of 103 major rice vadeties and parental lines at the 24 marker loci was constructed and analyzed.

Identification of variety of rice and determination of content of each rice in mixtures of two rices by nonlinear chemical analysis

Fingerprints of two varieties of rice and their mixtures were investigated by a nonlinear chemical reaction system consisting of rice components,sodium bromate,manganese sulfate,sulfuric acid and acetone.The variety of rice was identified by the visual characteristic of fingerprint and system similarity pattern recognition,and the content of each variety of rice in the mixture was determined by the quantitative information of fingerprint.The results show that nonlinear chemical analysis may be used to exactly identify the variety of pure rice and to accurately determine the content of each variety of rice in the mixture,indicating the method is simple and convenient.

AFLP Fingerprint and SCAR Marker of Watermelon Core Collection

The identification of germplasm is an important step for effective utilization of the available germplasm. In previous studies, isoenzyme, RAPD and SSR techniques had been used to conduct the genetic identification of watermelon ( Citrullus lanatus (Thunb.) Mansf.), but their effectiveness was limited due to the extremely narrow genetic background among watermelon genotypes. In this research, amplified fragment length polymorphism (AFLP), which was reported as a reliable technique with high efficiency in detecting polymorphism, was used to conduct genetic analysis and variety identification of thirty genotypes of watermelon core collection that represent a wide range of breeding and commercially available germplasm. As a result, a DNA fingerprint based on 15 bands amplified with four primer combinations was developed. In this fingerprint, each genotype has its unique fingerprint pattern and can be distinguished from each other. Furthermore, in or der to facilitate the utilization of AFLP marker in practice, one specific AFLP band of genotype 'PI296341' coming from fragment amplified by primer combination E-AT/M-CAT was successfully converted into a sequence characterized amplified region (SCAR) marker.

A Set of SCAR Markers Efficiently Differentiating Hybrid Rice

Molecular markers have been widely used in crop genetic improvement, seed test and genetic mapping. Of which, sequence characterized amplified region （SCAR） markers are particularly popular for its diversity, stable reproducibility, and suitability for analyzing large number of samples. In this study, 500 random amplified polymorphic DNA （RAPD） primers were tested, and a set of SCAR markers comprising 37 pairs of loci-specific primers were developed from the DNA fragments ranging from 300 to 1000 bp which correspond to the stable, distinctive RAPD banding patterns. Using these SCAR markers, 59 hybrid rice combinations were assessed and distinguished into 58 subgroups at the similarity coefficient of 0.97 in a genetic clustering tree based on the allele diversities of the SCAR markers. Furthermore, 13 hybrid rice combinations were reassayed with 40 randomly selected simple sequence repeat （SSR） markers to evaluate the effectiveness of these SCAR markers. SSR markers produced similar results to SCAR markers as the 13 hybrid rice combinations were completely separated at the similarity coefficient of 0.91 in the clustering tree established from SSR patterns. Taken together, SCAR markers prove to be effective tools for identifying and differentiating hybrid rice combinations.

Identification of maize seed varieties based on near infrared reflectance spectroscopy and chemometrics

False seeds can often be seen in the maize seed market,leading to a serious decline in maize yield.Those existing variety identification methods are expensive,time consuming,and destructive to seeds.The aim of this study is to develop a cheap,fast and non-destructive method which can robustly identify large amounts of maize seed varieties based on near-infrared reflectance spectroscopy(NIRS)and chemometrics.Because it is difficult to establish models for every variety in the market,this study mainly investigated the performance of models based on a large number of samples(more than 40 major varieties in the market).The reflectance spectra of maize seeds were collected by two modes(bulk kernels mode and single kernel mode).Both collection modes can be applied to identification,but only the single kernel mode can be applied to purity sorting.The spectra were pretreated with smoothing,the first derivative and vector normalization;and then principal component analysis(PCA),linear discriminant analysis(LDA)and biomimetic pattern recognition(BPR)were applied to establish identification models.The environmental factors such as producing areas and years have a significant influence on the performance of the models.Therefore,the method to improve the robustness of the models was investigated in this study.New indexes(correct acceptance degree(CAD),correct rejection degree(CRD)and correct degree(CD))were defined to analyze the performance of the models more accurately.Finally,the models obtained a mean correct discrimination rate of over 90%,and exhibited robust properties for samples harvested from different areas and years.The results showed that NIR technology combined with chemometrics methods such as PCA,LDA,and BPR could be a suitable and alternative technique to identify the authenticity of maize seed varieties.

Advance of Study on SSR Molecular Marker in Citrus and Its Close Relatives

Simple sequences repeat （SSR） molecular maker, as a new type of DNA molecular marker, the second generation based on the polymerase chain reaction （PCR）, is valuable and of great potential as genetic markers for its characteristics of abundant quantity, high polymorphic, reproducibility, specific site amplification, high occurring frequency, and co-dominant inheritance etc. This paper outlined its principles and characteristics, and introduced its application to variety identification, phylogenetic relationship analysis, genetic diversity analysis, DNA fingerprinting and linkage map constructing etc. in recent years in Citrus and its close relatives.

Differentiation of Indica-Japonica rice revealed by insertion/deletion(InDel)fragments obtained from the comparative genomic study of DNA sequences between 93-11(Indica)and Nipponbare (Japonica)

DNA polymorphisms from nucleotide insertion/deletions(InDels)in genomic sequences are the basis for developing InDel molecular markers.To validate the InDel primer pairs on the basis of the comparative genomic study on DNA sequences between an Indica rice 93-11 and a Japonica rice Nipponbare for identifying Indica and Japonica rice varieties and studying wild Oryza species,we studied 49 Indica,43 Japonica,and 24 wild rice accessions collected from ten Asian countries using 45 InDel primer pairs.Results indicated that of the 45 InDel primer pairs,41 can accurately identify Indica and Japonica rice varieties with a reliability of over 80%.The scatter plotting data of the principal compo-nent analysis(PCA)indicated that:(i)the InDel primer pairs can easily distinguish Indica from Japonica rice varieties,in addition to revealing their genetic differentiation;(ii)the AA-genome wild rice species showed a relatively close genetic relationship with the Indica rice varieties;and(iii)the non-AA genome wild rice species did not show evident differentiation into the Indica and Japonica types.It is con-cluded from the study that most of the InDel primer pairs obtained from DNA sequences of 93-11 and Nipponbare can be used for identifying Indica and Japonica rice varieties,and for studying genetic relationships of wild rice species,particularly in terms of the Indica-Japonica differentiation.