Manual Cultivar Identification Diagram Research Articles

Novel and efficient approach to identify hop cultivars (Humulus lupulus L.) using cultivar identification diagram strategy based on fingerprint of flavonol di- and tri-O-glycosides

The profile of flavonoids in hops (Humulus lupulus L.) showed a significant cultivar diversity applicable for distinguishing cultivars. The aims of this study were (1) to identify and describe flavonol di- and tri-glycosides in hops; (2) to analyze the profile of flavonol glycosides in various hop cultivars; and (3) to develop a new approach for identifying hop cultivars using a manual cultivar identification diagram that consisted of the ratios between specific flavonol di- and tri-glycosides. The total of 22 different flavonol O-glucosides were detected and tentatively identified using liquid chromatography (RP-HPLC) coupled with high-resolution mass spectrometry (HR-MS/MS). The identified O-flavonol glycosides, mainly containing aglycones, quercetin, kaempferol and myricetin, were categorized into five groups according to their sugar moieties. Furthermore, fourteen characteristic peaks with a low sample-to-sample variation and acceptable coefficient of variation (< 20%) throughout growing seasons were selected for chemometric analysis. Hierarchical cluster analysis (HCA) was applied to two data sets to characterize the similarity and differences among samples: one data set contained five hop cultivars common in the Czech Republic and the second one consisted of 17 hop cultivars from around the world. The results of the HCA of flavonol di- and tri-glycosides proved the genotypic effect on their profile and allowed to select compounds primarily responsible for clustering hop cultivars into groups. These compounds were used for developing an efficient cultivar identification strategy based on the manual cultivar identification diagram. The functionality of the identification diagram was successfully tested and allowed, based on the specific ratios between flavonol glucosides, reliable discrimination among the five most common hop cultivars planted in the Czech Republic. Compared to the genetic or chemotaxonomic methods, the newly developed approach for the hop cultivar identification provided reliable results even if the tested materials were hop pellets or beer.

Development of SNP markers based on transcriptome sequences and their application in germplasm identification in radish (Raphanus sativus L.)

Single nucleotide polymorphism (SNP) is the most frequent form of DNA variation in the genome. The cleaved amplified polymorphic sequence (CAPS) and derived CAPS (dCAPS) are co-dominant and cost-effective markers for SNP genotyping. Recently, the large-scale transcriptome sequencing has greatly facilitated SNP detection, and the applications of CAPS/dCAPS markers have been successfully carried out in various plant species. However, SNP detection via transcriptome sequencing and development of CAPS/dCAPS markers remain limited in radish. In this study, 56,089 SNPs were detected in the radish transcriptome, and 4941 high-confidence SNPs were further filtered out and analyzed, which could alter the recognition sites of 222 restriction enzymes. The available frequency of different enzymes varied a great deal, and ten enzymes were selected for marker development. A total of 262 pairs of CAPS and 15 pairs of dCAPS primers were synthesized, among which 148 and six could generate polymorphic bands after enzyme digestion, respectively. Furthermore, the polymorphism information content (PIC) values of 154 SNP markers ranged from 0.0739 to 0.3750. In addition, 24 radish genotypes were gradually distinguished by six CAPS markers with the manual cultivar identification diagram (MCID) approach, indicating the feasibility of CAPS/dCAPS markers in genetic diversity analysis and germplasm identification. These newly developed SNP markers could enrich the types and numbers of functional markers, and could be valuable in facilitating gene tagging, genetic mapping, comparative genomic studies and marker-assisted selection (MAS) of important horticultural traits in radish.

EFFICIENT IDENTIFICATION OF 46 IMPORTANT STRAWBERRY CULTIVARS USING RAPD MARKERS WITH A MANUAL CULTIVAR IDENTIFICATION DIAGRAM STRATEGY

DNA markers are potentially useful for plant cultivar identification. However, its use is limited due to various drawbacks, especially how to use marker information to efficiently identify cultivars. In this study, we applied a very new approach, called the manual cultivar identification diagram (MCID), to use RAPD fingerprints in distinguishing 46 strawberry cultivars after the optimization of RAPD through choosing 11 nt primers and strict screening PCR annealing temperatures. The advantage of this MCID strategy includes use of few primers in the separation of the strawberry cultivars and the real utility of cultivar identification (CID) in practical strawberry cultivar identification by the corresponding primers marked on the diagram. The MCID approach can serve strawberry cultivar identification efficiently and provide information to separate the groups of cultivars studied. The reliability and efficiency of this method was checked and verified. To the best of our knowledge, this new approach is the most efficient and reliable strategy using the power of DNA markers in identification of existing plant cultivars, identification of new plant cultivars, protection of cultivar-rights and also beneficial for the nursery industry for early identification of seedlings.

Identification of grapevine rootstock cultivars using expressed sequence tag-simple sequence repeats.

Grapevine (Vitis) rootstock varieties or cultivars are used to confer resistance and tolerance to insect and disease pests, unfavorable soil conditions, and other environmental conditions to cultivars that are susceptible to these conditions but otherwise have desired properties. The need to genotype and thoroughly identify grapevine rootstock varieties in the grape industry has become increasingly critical as more and more varieties are bred or selected. Although DNA markers have advantageous applications in plant identification, markers developed from classic DNA fingerprint analysis methods are not practical for plant cultivar identification. The manual cultivar identification diagram (MCID), which was previously developed in our research group, has been shown to select DNA markers that are relatively more exploitable in identifications of genotyped plant individuals. Using this MCID strategy and expressed sequence tag-simple sequence repeat (EST-SSR) markers, we identified 22 grapevine rootstock cultivars of diverse origin. All cultivars were clearly separated by fingerprints of seven pairs of EST-SSR primers and the grapevine rootstock CID (V-R-CID) generated is both practical and referable for the identification of any grapevine rootstock cultivars studied here. Furthermore, fewer primers can be used to distinguish all cultivars using this approach since the fingerprint from each primer pair could be used several times once it is generated. This initial version of V-R-CID can be made more informative with the identification and incorporation of more cultivars, thus providing better service to the grape industry.

Rapid identification of red-flesh loquat cultivars using EST-SSR markers based on manual cultivar identification diagram strategy

Manual cultivar identification diagram is a new strategy for plant cultivar identification based on DNA markers, providing information to efficiently separate cultivars. We tested 25 pairs of apple EST-SSR primers for amplification of PCR products from loquat cultivars. These EST-SSR primers provided clear amplification products from the loquat cultivars, with a relatively high transferability rate of 84% to loquat; 11 pairs of primers amplified polymorphic products. After analysis of 24 red-fleshed loquat accessions, we found that only 7 pairs of primers could clearly separate all of them. A cultivar identification diagram of the 24 cultivars was constructed using polymorphic bands from the DNA fingerprints and EST-SSR primers. Any two of the 24 cultivars could be rapidly separated from each other, according to the polymorphic bands from the cultivars; the corresponding primers were marked in the correct position on the cultivar identification diagram. This red-flesh loquat cultivar identification diagram can separate the 24 red-flesh loquat cultivars, which is of benefit for loquat cultivar identification for germplasm management and breeding programs.

Efficient identification of ornamental peach cultivars using RAPD markers with a manual cultivar identification diagram strategy

One of the most important uses of DNA markers is cultivar identification. However, no DNA fingerprint analysis strategy is available for making DNA markers helpful in practical plant cultivar identification, especially for the identification of a large number of cultivars. We developed a manual cultivar identification diagram strategy for efficient identification of plant cultivars, from which a cultivar identification diagram (CID) of genotyped plant individuals can be constructed manually. This CID could be used as a reference for quick identification of plant cultivars of interest. We used 11-mer RAPD primers to amplify DNA samples of 32 ornamental peach genotypes; all the cultivars were well distinguished by fingerprints from 6 primers. The utility of this CID was verified by identification of three randomly chosen groups of cultivars among the 32 ones that we selected. This CID generated will be useful for the identification of commercially important ornamental peach cultivars.

Genetic diversity and relationships among different tomato varieties revealed by EST-SSR markers

The genetic diversity and relationship of 42 tomato varieties sourced from different geographic regions was examined with EST-SSR markers. The genetic diversity was between 0.18 and 0.77, with a mean of 0.49; the polymorphic information content ranged from 0.17 to 0.74, with a mean of 0.45. This indicates a fairly high degree of diversity among these tomato varieties. Based on the cluster analysis using unweighted pair-group method with arithmetic average (UPGMA), all the tomato varieties fell into 5 groups, with no obvious geographical distribution characteristics despite their diverse sources. The principal component analysis (PCA) supported the clustering result; however, relationships among varieties were more complex in the PCA scatterplot than in the UPGMA dendrogram. This information about the genetic relationships between these tomato lines helps distinguish these 42 varieties and will be useful for tomato variety breeding and selection. We confirm that the EST-SSR marker system is useful for studying genetic diversity among tomato varieties. The high degree of polymorphism and the large number of bands obtained per assay shows that SSR is the most informative marker system for tomato genotyping for purposes of rights/protection and for the tomato industry in general. It is recommended that these varieties be subjected to identification using an SSR-based manual cultivar identification diagram strategy or other easy-to-use and referable methods so as to provide a complete set of information concerning genetic relationships and a readily usable means of identifying these varieties.

Novel and useful genic-SSR markers from de novo transcriptome sequencing of radish (Raphanus sativus L.)

Simple sequence repeats (SSRs) are highly polymorphic and co-dominant markers, providing an important genomic resource for genetic research. Recently, large-scale transcriptome sequencing has become a reliable and efficient approach for the identification and development of new genic-SSR markers and has been successfully conducted in a few important plant species. However, SSR development based on transcriptome sequencing remains limited in radish (Raphanus sativus L.). In the present study, from a total of 73,084 unigenes and 150,455 contigs which were assembled from 71.95 million Illumina sequence reads of a radish taproot library, a collection of 11,928 genic-SSR loci were successfully identified in 11,311 unigene sequences. Trinucleotide repeats were the most abundant repeat units, as in many other plants, with a frequency of 52 %. Furthermore, a total of 5,503 genic-SSR primers were developed, from which 1,052 SSR primers were synthesized, and a subset of 823 (78.23 %) primers could generate stable bands. Moreover, 67 selected informative genic-SSR markers were used to determine the genetic diversity of 32 radish genotypes, in which the polymorphism information content values ranged from 0.49 to 0.89. For effective cultivar identification, a novel strategy called manual cultivar identification diagram was employed. Thirty-two radish accessions were clearly separated by six genic-SSR markers. Additionally, the cross-species/genera transferability of these SSRs was further validated in nine relatives in Brassicaceae. These results suggested that the novel genic-SSR markers, as a basis for future genetic linkage and gene tagging analysis, could be very valuable in facilitating genetic mapping, marker-assisted selection and comparative genome analysis.

A new strategy for identification of currant (Ribes nigrum L.) cultivars using RAPD markers

We developed a new approach using RAPD fingerprints to distinguish 37 currant cultivars from northeastern China based on optimization of RAPD by choosing 11 nucleotide primers and strict screening PCR annealing temperature. We found that the manual cultivar identification diagram (MCID) approach clearly developed fingerprints from 8 different primers that were useful for cultivar identification; a cultivar identification diagram (CID) was readily constructed. This CID allows efficient currant cultivar identification, providing information to separate all the currant cultivars from each other, based on the detail polymorphic bands from the corresponding primers, which were marked in the correct positions on the currant CID. According to the CID, 10 currant cultivars in 5 groups were randomly selected for the referable and workable identification of this strategy. The results proved the workability and efficiency of the MCID method, facilitating the identification of fruit cultivars with DNA markers. This MCID approach will be useful for early identification of seedlings in the nursery industry and protection of cultivar rights.

Methodology An efficient approach to identify Ginkgo biloba cultivars by using random amplified polymorphic DNA markers with a manual cultivar identification diagram strategy

Cultivar identification is a key step to avoid the formation of homonyms and synonyms of Ginkgo biloba. In this study, a new approach based on combinational utilization of polymorphic bands produced from 6 different random amplified polymorphic DNA (RAPD) primers was developed for identifying 42 Ginkgo cultivars, and a manual cultivar identification diagram that consisted of polymorphic bands produced from different RAPD primers was reported. To check the reliability and efficiency of the cultivar identification diagram, 5 randomly chosen cultivars were further tested, and the workability of the diagram was verified. This new approach will be very helpful for Ginkgo cultivar discrimination and protection, and will also be beneficial for the nursery industry for early identification of Ginkgo seedlings.

Analysis of genetic relationships and identification of flowering-mei cultivars using EST-SSR markers developed from apricot and fruiting-mei

Sixteen pairs of EST-SSR primers developed from apricot (Prunus armeniaca L) and 11 pairs from fruiting-mei (Prunus mume) were employed in PCR amplification for analysis of genetic relationships and identification of 54 flowering-mei accessions. The results show that 14 pairs of apricot and 11 pairs of fruiting-mei EST-SSR primers had clear amplification products in flowering-mei, among which 8 apricot and 9 fruiting-mei EST-SSR primer pairs could amplify polymorphic bands and a total of 65 alleles, were detected from all the 54 flowering-mei accessions. The ratio of polymorphic bands amplified by the 8 apricot and 9 fruiting-mei EST-SSR primers 59.1% and 72.1%, respectively, indicating that the levels of polymorphism from fruiting-mei EST-SSR primers were relatively high. Analysis of genetic relationships of all the 54 accessions by the polymorphic SSR markers showed that EST-SSR markers could clearly separate flowering-mei accessions and the clustering pattern of most cultivars was in accordance with their pedigree information. In addition, a manual cultivar identification diagram (MCID) of the 54 cultivars was constructed using polymorphic bands from the DNA fingerprints. To the best of our knowledge, this new strategy is by far the most efficient and workable in identifying plant varieties using EST-SSR markers. This MCID strategy could separate all the cultivars from each other, with the polymorphic bands employed for identifying the cultivars and the corresponding primers being marked in the correct position on the MCID. This flowering-mei MCID can provide the information needed to separate any of the 54 important cultivars studied here, which is of benefit to flowering-mei cultivar identification for cultivar-right-protection and the general flowering-mei industry in China.