Distinctness, Uniformity And Stability Testing Research Articles

Comparative Analysis of Traditional Scented Rice (Oryza sativa L.) Varieties under Organic Cultivation Based on DUS Testing and Yield Assessment

An agricultural field study was conducted during the kharif season of 2020 at the G.B. Pant University of Agriculture and Technology in Uttarakhand, India, to investigate the qualitative and quantitative attributes of traditionally scented rice cultivated through organic methods. The experiment, a randomized block design with three replications, evaluated ten rice varieties, labeled as V1 to V10 (Kubir Mamhani, Kudrat-5, etc). The morphological variations among the varieties (registered under PPV&FR Act 2001) are based on DUS (Distinctness, Uniformity, and Stability) is required for protecting the uniqueness of traditional scented varieties of rice. It can also be the indicative of the yield. The grain, straw and harvest index were found the highest with variety Kudrat-5 followed by Kudrat-1.

Evaluation of SNP fingerprinting for variety identification of tomato by DUS testing

Variety identification is crucial for PBR (plant breeders’ rights) protection and PVR (plant variety registration). DUS (Distinctness, Uniformity and Stability) testing, utilizing field-based morphological inspection and DNA fingerprinting with molecular markers in the laboratory are commonly employed methods for variety identification. However, the limited number of molecular markers used in DNA fingerprinting often lacks close linkage to DUS traits. In this study, 116 tomato varieties were well identified both by SNP fingerprinting and DUS testing. PCA (Principal Component Analysis) and population classification demonstrated a highly consistent outcome between SNP fingerprinting and DUS testing, resulting in the division of 116 varieties into three groups: big fruit, cherry, and processing tomatoes. Furthermore, we selected a new set of 16 core SNPs and 18 core DUS traits, which exhibited higher efficiency in variety identification due to their convenient and easy processing. Moreover, the observed variations in SNP markers among each pair of tomato varieties were linearly correlated with those comparison of all different DUS traits (R2 ​= ​0.85), and the linear correlation was also obtained based on the comparison of different core SNP fingerprints with those of the core DUS traits (R2 ​= ​0.86). In conclusion, we evaluated SNP fingerprinting for variety identification in comparison to DUS testing, and found these two methods had consistent result. This study also highlights the potential of limited core DUS traits and core SNP fingerprints for effective identification and discrimination of tomato varieties.

Genetic diversity for morphophysiological character studies in rainfed sorghum (Sorghum bicolor[L.] Moench) parental lines

The experimental trial was conducted at Sorghum Research Centre, Marathwada Agricultural University, Parbhani during kharif 2021 in randomized design with 3 replications. Eighteen parental (B & R) lines were evaluated to study genetic diversities and variability for fifteen yield contributing characters. Among all the eighteen genotypes of parental lines significant differences were observed for yield and its yield contributing traits except total chlorophyll content. Coefficient of variance at phenotypic level showed higher values than at genotypic level among all traits. Coefficient of variance for the traits like chlorophyll stability index (G=23.035, P=23.714), leaf area (G=21.064, P=21.673), 1000 grain weight (G=28.845, P=29.929), fodder yield (G=21.858, P=22.458), grain number per panicle (G=29.564, P=30.158), leaf dry weight (G=29.044, P=29.903) was observed high at both level. Traits like plant height (H=90, GAM=21.041), grain yield (H=86, GAM=29.074), leaf dry weight (H=94, GAM=23.664), leaf area (H=94, GAM=42.17), chlorophyll stability index (H=94, GAM=46.093), grain number per panicle (H=96, GAM=59.703), fodder yield (H=94, GAM=43.823),1000 grain weight (H=92, GAM=52.270) and harvest index (H=84, GAM=32.321) were recorded high heritability with high genetic advancement. Genotype AKR 504, NR 39-15, KR 218, KR 219, PMS 100B, AKMS 90 B and INDORE 12 showed the better performance for all characters thus, should be used for development of hybrids and inbreds in breeding programmes by DUS (Distinctness, Uniformity and Stability) testing.

Genetic Analysis of Potato Breeding Collection Using Single-Nucleotide Polymorphism (SNP) Markers.

The autotetraploid potato (Solanum tuberosum L.) is an important crop in China, and it is widely cultivated from Northeast China to South China. Thousands of varieties are bred by breeding institutions or companies, and distinguishing the different varieties based on morphological characteristics is difficult. Using DNA fingerprints is an efficient method to identify varieties that plays an increasingly important role in germplasm identification and property rights protection. In this study, the genetic diversity and population structure of 135 autotetraploid potatoes were evaluated using specific-locus amplified fragment sequencing (SLAF-seq) methods. A total of 3,397,137 high-quality single-nucleotide polymorphisms (SNPs), which were distributed across 12 chromosomes, were obtained. Principal component analysis (PCA), neighbour-joining genetic trees, and model-based structure analysis showed that these autotetraploid potato subpopulations, classified by their SNPs, were not consistent with their geographical origins. On the basis of the obtained 3,397,137 SNPs, 160 perfect SNPs were selected, and 71 SNPs were successfully converted to penta-primer amplification refractory mutation (PARMS-SNP) markers. Additionally, 190 autotetraploid potato varieties were analysed using these 71 PARMS-SNP markers. The PCA results show that the accessions were not completely classified on the basis of their geographical origins. The SNP DNA fingerprints of the 190 autotetraploid potato varieties were also constructed. The SNP fingerprint results show that both synonyms and homonyms were present amongst the 190 autotetraploid potatoes. Above all, these novel SNP markers can lay a good foundation for the analysis of potato genetic diversity, DUS (distinctness, uniformity, and stability) testing, and plant variety protection.

MORPHOLOGICAL CHARACTERISTICS OF SIXTEEN SESAMUM VARIETIES FOR DISTINCTNESS, UNIFORMITY AND STABILITY (DUS) TEST

Sesamum indicum is an important food oil crop of India. It is useful in many aspects like seeds for oil, seed cake (after oil extraction) as fodder, Seeds as one of the richest sources of calcium carrying many nutrients, seeds having therapeutic properties along with many cosmetic uses. For releasing any new variety of crop DUS (Distinctness, Uniformity and Stability) Test is done. For conducting DUS Test many morphological characteristics are observed for several generations and uniformity stability of characteristics are noted down. We observed morphological characteristics of 16 Sesamum crop varieties from different regions of India for DUS Testing. Result shown that even geographically different Sesamum crop can be grouped into one. So, along with morphological characteristics, Biochemical and Molecular markers are also suggested to be included for DUS Testing.

​Morphological Characterization and Morphological Traits based Genetic Diversity Analysis of Farmer’s Pea (Pisum sativum L.) Varieties of Uttar Pradesh using DUS Descriptors, as per PPV and FRA, 2001

Background: The conditions of distinctness, uniformity and stability (DUS) must be met in order for a new variety to be registered or protected. DUS testing ensures that a novel variety is unique from other existing varieties and that the farmers will produce a variety with consistent traits that will remain intact in subsequent generations to follow. However, DUS testing does not provide information about genetic diversity as a result, cluster analysis was performed. Methods: Twenty-two farmers’ varieties (FVs) of pea (Pisum sativum L.) were collected from Vindhyan zone of eastern Uttar Pradesh and were grown in randomized block design with three replications during rabi 2019-20 and 2020-2021. FVs were characterized according to DUS guidelines of the PPV and FRA, 2001 and were subjected for Agglomerative cluster analysis by using Ward’s method. Result: All the characters were found uniform and stable in their expression in consecutive two seasons. PKKK-227 and PMKK-232 were similar to each other, while rest FVs were found distinct. The varieties were grouped into 5 clusters, among which maximum genotypes (8) were in cluster II and minimum genotypes (2) were in cluster V. Cluster I, III and IV had 4, 3 and 5 farmers’ varieties respectively. FVs in cluster II are more or less similar to each other; hence they have a common ancestor. Ramaipur had maximum diversity among the villages of Vindhyan zone.

Overcoming barriers to the registration of new plant varieties under the DUS system

Distinctness, Uniformity and Stability (DUS) is an intellectual property system introduced in 1961 by the International Union for the Protection of New Varieties of Plants (UPOV) for safeguarding the investment and rewarding innovation in developing new plant varieties. Despite the rapid advancement in our understanding of crop biology over the past 60 years, the DUS system has changed little and is still largely dependent upon a set of morphological traits for testing candidate varieties. As the demand for more plant varieties increases, the barriers to registration of new varieties become more acute and thus require urgent review to the system. To highlight the challenges and remedies in the current system, we evaluated a comprehensive panel of 805 UK barley varieties that span the entire history of DUS testing. Our findings reveal the system deficiencies such as inconsistencies in DUS traits across environments, limitations in DUS trait combinatorial space, and inadequacies in currently available DUS markers. We advocate the concept of genomic DUS and provide evidence for a shift towards a robust genomics-enabled registration system for new crop varieties.

Is plant variety registration keeping pace with speed breeding techniques?

Recent innovations in breeding technologies have reduced the timeframe to develop improved plant varieties compared to conventional breeding processes. Technologies like speed breeding, or rapid generation advancement, may also accelerate the process of statutory variety registration. Within this procedure, improved varieties are required to satisfy distinctness, uniformity and stability (DUS) criteria to establish the unique identity of a given submission during the variety registration process. The DUS standard also provides a solid basis for seed certification, plant breeders’ rights, as well as variety maintenance throughout commercial lifespan of varieties. Currently, the overall timeline of variety registration may vary from 2 to 4 years, depending on crop type and country. In this article, we propose the concept of ‘speed DUS testing’: a rapid phenotype-based method, which could be integrated with approaches that take advantage of DNA markers. We compare methods and discuss how DUS testing could be modernized to fast-track variety registration.

Development of model web-server for crop variety identification using throughput SNP genotyping data

Crop varieties or genotypes of a given species are pivotal for agricultural production and ownership, management and improvement of their germplasm is a great challenge. Its morphological identification requires time, cost and descriptors are often compromised statistically due to phenotypic plasticity. Development of DNA based signature of varieties can overcome these limitations. There is a global need to implement world trade organization (WTO) and intellectual property rights (IPR) guidelines of Plant Breeders Rights (PBR) where DUS (distinctness, uniformity and stability) testing can be supplemented by DNA profile. Universalization and minimization of SNP number without compromising identification accuracy is the major challenge in development of varietal profile by rapid genotype assay. Besides this, there is no server-based approach reducing computational skill with global accessibility of referral phenotypic and genotypic data. We report world’s first model web server for crop variety identification using >350 Indian wheat varieties and Axiom 35 K SNP chip data. Standard filtering and linkage disequilibrium approach were used to develop varietal signature in Linux using HTML, Java, PHP and MySQL with provision of QR code generator to facilitate bar-coding. Phylogenetic tree constructed by selected SNPs confirms six major trait based clusters of varieties and their pedigree. Our user friendly server based tool, VISTa (Variety Identification System of Triticum aestivum) (http://webtom.cabgrid.res.in/vista) can be used in DUS testing having dispute resolution of sovereignty and access benefit sharing (ABS) issues. This model approach can be used in other crops with pan-global level management of crop germplasm in endeavour of crop productivity.

Development of DUS Test Guidelines for new varieties of Viburnum L.

Distinctness, uniformity and stability (DUS) Test Guidelines for new varieties of Viburnum L. were developed according to the principles and requirements of the International Union for the Protection of New Varieties of Plants (UPOV) and China. The development processes, including selection and determination of testing characteristics, determination of expression states of characteristics, selection of example varieties, determination of the order of characteristics, and assessment of distinctness, uniformity and stability, are described in this paper. There were 54 testing characteristics: 24 qualitative characteristics (QL), 15 quantitative characteristics (QN), 15 pseudo-qualitative characteristics (PQ) and 34 example varieties in the DUS Test Guidelines. Among the 54 testing characteristics, 20 were asterisked and 34 were non-asterisked. Seven were selected for variety groupings and 18 were adopted in the Technical Questionnaire. The development of these DUS Test Guidelines will be useful for the application and protection of new Viburnum varieties. The Guidelines will provide technical support for DUS testing of Viburnum and serve as a reference for the development of new varieties in other countries.

Genetic variability of wheat germplasm represented in the south Pannonian region

In this study, genetic variability was investigated among 50 winter wheat varieties (Triticum aestivum L.) which are grown in parts of Croatia, Hungary, Serbia and Slovenia according to 22 morphological characteristics used for DUS (distinctness, uniformity and stability) testing. The average Dice similarity coefficient was 0.371. The determined similarity coefficient was in range 0.083 - 0.776. A significant variability of 6.21% in the breeding programs according to period was determined as well as significant variability of 3.10% between breeding programs. The UPGMA clustering divided investigated varieties into four main clusters. Based on data analysis, most distant varieties with best morphological characteristics were found which will provide valuable resource of new parent's combinations in future breeding programs. This paper also provided valuable assessment of morphological characteristics to define distinctness criteria in the DUS examination of wheat.

Protecting vegetable cultivars in Brazil: a chili pepper case-study research

ABSTRACT: In all countries, members of the UPOV (Union Internationale pour la Protection des Obtentions Végétales), of which Brazil is a signatory, to protect a new plant variety is necessary to carry out DUS (Distinctness, Uniformity and Stability) tests. DUS testing forCapsicum spp. is based on 48 descriptors, which involve qualitative and quantitative traits, observed from germination to fruit harvest. This paper describes the performance of DUS tests on lines of chili peppers, which are candidates for protection; we discuss the main aspects related to operational difficulties, the relevance of some descriptors for the protection process and highlight the importance of protecting new plant varieties as national intellectual property. Four C. annuum var.annuum recombinant inbred lines were tested. The Capsicum Breeding Program of the Universidade Estadual do Norte Fluminense Darcy Ribeiro developed the lines, which are resistant to bacterial spot. They were tested under greenhouse conditions from June to November 2013; and from January to July 2014 in Campos dos Goytacazes, Rio de Janeiro State, Brazil. The 'Jalapeño M' commercial genotype was used as control and treatments were arranged in a randomized block design with seven plants per plot with five replications. Besides the 48 descriptors, stipulated by legislation for Capsicum DUS testing, we included a descriptor for bacterial spot resistance. The descriptors that enabled distinction varied with each line. Although being distinctive for some descriptors, L1 and L2 lines were neither homogeneous nor stable. L6 and L8 recombined lines were characterized by homogeneity and stability. Fruit shape, capsaicin presence, number of days for flowering and bacterial spot resistance were descriptors that allowed for differentiation between treatments. In conclusion, the L6 and L8 recombined lines met the DUS test requirements; therefore, they may be subjected to the protection process with the Brazilian Ministry of Agriculture, Livestock and Food Supply.

The dependence of DUS (distinctness, uniformity and stability) decisions concerning white mustard and oilseed rape varieties on the number of measurements

Summary An analysis is made of the possibility of reducing numbers of measurements in DUS (distinctness, uniformity and stability) trials on varieties of white mustard and oilseed rape, based on the results of DUS trials performed in the years 2005–2009 by the Research Centre for Cultivar Testing. It is shown that, when decisions are taken after three years of testing, it is possible to decrease the number of measurements by 50%, with the decisions practically unchanged for both considered species. For so-called early decisions (after two years of testing) the distinctness decisions are again practically the same for full and reduced data, although the decisions concerning uniformity differ significantly. This means that the number of measurements can be reduced only in cases where DUS testing is continued for three years.

Molecular Profiling of Major Indian Rice Cultivars Using a Set of Eight Hypervariable Microsatellite Markers

India bred high yielding rice varieties have enriched to a great extent the global rice germplasm since the mid-sixties. Systematic research efforts for development of cultivar-specific DNA fingerprints of major Indian rice cultivars, however, have not received due attention. The present investigation was aimed at development of DNA fingerprints for 90 high yielding rice varieties using hypervariable microsatellite (hvRM) markers. A panel of eight markers, viz. RM11313, RM13584, RM15004, RM5844, RM22250, RM22565, RM24260 and RM8207 was chosen from 52 polymorphic markers based on their highly polymorphic nature, SSR repeat type and number and ability to distinguish genotypes, in order to develop DNA fingerprints of 90 varieties. The remaining high polymorphic hvRM markers could be of immense value in future to distinguish new cultivars, in case they could not be distinguished by the 8 marker panel. Four of the 8 markers, viz. RM22250, RM13584, RM24260 and RM5844 were located in expressed genes and could be of value in DUS (Distinctness, Uniformity and Stability) testing. Thus we suggested, that this set of 8 loci could be used as standard for DNA fingerprinting of Indian rice cultivars.

Comparison of uniformity decisions in DUS testing for full and reduced numbers of measurements

Summary It is investigated how a reduction in the number of measurements influences uniformity decisions in distinctness, uniformity and stability (DUS) testing. Using real data from DUS trials performed in Poland for several species, it is shown that when final decisions are taken after three years of testing, a reduction in the number of measurements by 50% of the numbers indicated in the Guidelines has very limited impact on decisions (rejection or approval of candidate variety). Decisions taken after one year (or two years) are more dependent on the numbers of measurements.

Microsatellite-based DNA fingerprinting and genetic diversity of bottle gourd genotypes

In India, the registration and protection of new and notified/extant plant varieties are based on the criteria of distinctness, uniformity and stability (DUS) of morphological characteristics. However, these morphological traits have not been helpful in resolving closely related genotypes. The molecular markers can very well support the DUS testing in such cases. Therefore, in the present study, 20 accessions of bottle gourd were fingerprinted using 20 simple sequence repeat (SSR) primers. Of these, ten primers exhibited polymorphic profiles, while nine exhibited monomorphic patterns and one revealed a null allele. The number of alleles ranged from 2 to 4 with an average of 2.6 alleles per locus. Unique DNA profiles of all the accessions could be created using a set of five polymorphic primers. Therefore, SSR markers used in the present study could precisely distinguish all the 20 accessions from each other, and these SSR markers can be further used to differentiate the future genotypes from the existing ones. The dendrogram depicting the genetic relationships as revealed by NTSYS-pc 2.02 and the tree diagram generated using the DARwin 5.0 program classified the accessions into two main clusters. There is no strong association between the clustering pattern and geographical origin of these accessions. This SSR marker-based diversity would facilitate the implementation of marker-assisted breeding schemes for efficient introduction of the desired traits into bottle gourd.

Evaluation of the use of high-density SNP genotyping to implement UPOV Model 2 for DUS testing in barley

Developments in high-throughput genotyping provide an opportunity to explore the application of marker technology in distinctness, uniformity and stability (DUS) testing of new varieties. We have used a large set of molecular markers to assess the feasibility of a UPOV Model 2 approach: "Calibration of threshold levels for molecular characteristics against the minimum distance in traditional characteristics". We have examined 431 winter and spring barley varieties, with data from UK DUS trials comprising 28 characteristics, together with genotype data from 3072 SNP markers. Inter varietal distances were calculated and we found higher correlations between molecular and morphological distances than have been previously reported. When varieties were grouped by kinship, phenotypic and genotypic distances of these groups correlated well. We estimated the minimum marker numbers required and showed there was a ceiling after which the correlations do not improve. To investigate the possibility of breaking through this ceiling, we attempted genomic prediction of phenotypes from genotypes and higher correlations were achieved. We tested distinctness decisions made using either morphological or genotypic distances and found poor correspondence between each method.

MANAGEMENT OF PEACH TREE REFERENCE COLLECTIONS: ONGOING RESEARCH & DEVELOPMENT PROGRAM RELEVANT TO THE COMMUNITY PLANT VARIETY RIGHTS PROTECTION SYSTEM

Peach/nectarine is an important fruit tree crop in Europe and indeed world-wide. The registration of new cultivars, either for Plant Breeders' Rights or National Listing (PBR/NL) purposes in the European Union (EU), requires the completion of a distinctness, uniformity and stability (DUS) test in one of the EU Member States. The number of candidate cultivars and potentially required example cultivars entered for DUS testing is steadily increasing annually. Moreover, DUS testing of this type of tree crops requires the maintenance of large orchards, particularly with the aim of having a complete reference collection. In France, Hungary, Spain and Italy, peach tree breeding activity is characterized by an important number of cultivars available and a short turn-over of the cultivars. This situation will extend to new EU members states in the coming years, with the development of the European trade. However, although the diversification increases the number of segments, for each segment, new released cultivars are genetically closer and closer. The consequence is that it is now difficult in some cases to distinguish between cultivars. Clearly, developing means of managing reference collections is highly desirable in order to be able to compare candidate cultivars with the closest cultivars of common knowledge in the reference collections prior to planting them, and so reduce the number of cultivars that need to be grown, without eroding the strength of PBR and the relevance of DUS tests. Effective means of such a management include the definition of a standardised way to compare phenotypic data and to use molecular markers to eliminate the reference cultivars which do not need to be compared to the candidate cultivars. In peach tree, various molecular markers such as DNA microsatellites (SSR) have been developed and evaluated, but they have not yet been used for the characterization of large collections. Therefore, this project will generate a database compiling both phenotypic data, including standardised morphological descriptions and digital pictures, and a large data set of DNA profiles for 522 peach tree cultivars selected among the EU granted and listed cultivars.

Comparison of image analysis and direct measurement of UPOV taxonomic characteristics for variety discrimination as determined over five growing seasons, using industrial chicory as a model crop

Tests for distinctness, uniformity and stability (DUS) are an essential component of variety registration and granting plant breeders’ rights. This study, performed on industrial chicory (Cichorium intybus L.), included (1) an evaluation of the discriminating power of the UPOV characteristics and (2) a comparison of the root shape between the UPOV-based characteristics and characteristics determined via image analysis. Five consecutive growing periods were studied to assess the long-term stability of the parameters. Lack of discriminating power of characteristics for examining distinctness may impede breeding progress by failing to discriminate novel candidate varieties. However, a balance needs to be set so that newly introduced characteristics or new evaluation methodologies are not too powerful as to damage the protection of registered varieties. The results showed great differences in the discriminating power of the UPOV characteristics. The elimination of some of these low-discriminating characteristics would probably result in savings, but would have no consequences on the decisions taken by DUS testing authorities. Image analysis-derived characteristics could either replace currently-used parameters or provide additional characteristics with good discriminating power, determined in an objective and standardized way. Here we discuss possible implications for variety registration.

Use of DNA markers for cereal line uniformity assessment

Use of DNA markers for cereal line uniformity assessment Prior to the registration of a new variety, it is required to undergo Distinctness, Uniformity and Stability (DUS) testing. Preparing a newly developed variety to meet the requirements of DUS testing is a lengthy process, particularly regarding aspects of uniformity and stability. Field testing of a large number of lines is time and resource intensive. In addition, the expression of certain traits may be influenced by environmental conditions. The use of DNA markers may allow rapid assessment of the level of genetic diversity within a particular line or variety, and to remove individuals that are genetically differentiated, thus accelerating the homogenisation of a newly developed variety. In this study, we utilised AFLP and the iPBS marker techniques to assess genetic variation within advanced breeding lines of several cereal species (triticale, wheat, barley). The combined use of molecular and morphological selection over three years of analysis and selection resulted in the reduction of genetic diversity within breeding lines.