RAPD Markers Research Articles

M-brigde- and elicitor-assisted enhanced post-storage germination of Rauvolfia serpentina synthetic seeds, their genetic fidelity assessment and reserpine estimation

The present study reports an amelioration of synthetic seed germination efficiency with a simple intervention of filter paper (M-bridge) drenched in ½Murashige and Skoog liquid (½MSL) medium in Rauvolfia serpentina. Synthetic seeds were developed from in vitro shoot tips using 3% (w/v) sodium alginate and 75 mM calcium chloride. Media for synthetic seed gemination were tested in cotton bed drenched with sterile water (SW), cotton bed drenched with ½MSL medium, M-bridge drenched with ½MSL medium, and ½MS semisolid (½MSS) medium alone. Synthetic seeds inoculated on ½MSL or SW or ½MSS media, exhibited slower germination and inferior performance; however, with the involvement of M-bridge, fastest germination (5.6 ± 0.4 days) and maximum proliferation (4.8 ± 0.7 shoots with 42.8 ± 2.4 mm length, and 19.8 ± 2.7 leaves/synthetic seed after 42 days of inoculation) were recorded. Among the tested elicitors (silver nitrate, salicylic acid and sodium nitroprusside) at their different concentrations supplemented in ½MSL medium (with M-bridge), 50 µM silver nitrate resulted in 100% germination frequency and resulted in germination within 5 days. Further, the synthetic seeds were stored for 42 days at − 20 °C, 8 °C, and 25 °C. Maximum germination efficiency was obtained in case of seeds stored at 8 °C with respect to earliness in germination (6.9 ± 0.3 days), germination percentage (71.8 ± 3.4%) and subsequent proliferation (4.9 ± 0.6 shoots with 18.9 ± 0.9 mm length, and 8.9 ± 0.7 leaves/synthetic seed). Clonal fidelity analysis of the in vitro regenerants performed using RAPD markers exhibited monomorphic banding pattern. The reserpine content of the regenerants was found to be maximum following 42 days-storage at 8 °C (126.9 µg/g), followed by a substantial decline in the synthetic seed-derived regenerants following 42-days-storage at 25 °C (120.7 µg/g) or − 20 °C (121.8 µg/g).

Genetic variation in Iranian populations of citrus cottony scale (Pulvinaria aurantii Cockerell) based on RAPD, mitochondrial DNA and RFLP markers

In the present study, we used RAPD marker, COI gene sequence and RFLP analysis to investigate the genetic variation of 11 Pulvinaria aurantii Cockerell populations from different regions in northern Iran. The twenty-one RAPD primers generated 834 amplification products, of which 702 (84.17%) bands were polymorphic. The genotypic similarity of the populations ranged from 0.46 to 0.85 based on the Dice similarity coefficient. UPGMA dendrogram indicated that there is no clear relationship between genetic distances and geographical distances. An 850 bp fragment of COI gene was sequenced for 33 samples, resulting in five haplotypes. COI sequencing indicated that 63.63% of the samples (seven populations) had the same nucleotide sequence as outgroup (Japan). Enzymatic digestion of P. aurantii COI gene (656 bp fragment) resulted in 315 restriction fragments with 11 different sizes. The results indicated genetically differences of the populations but the level of variations was independent of geographical distances. Successful amplification of the COI barcode region of Pulvinaria aurantii Twenty-one RAPD primers generated 834 bands with 84% polymorphism Identify five different haplotypes based on the COI barcode region Detection of genetic diversity among some populations based on RAPD, COI and RFLP No clear correlation was found between geographical distances and genetic distances

Genetic diversity and population structure of Indian willow (Salix tetrasperma Roxb.) with dominant molecular markers along its distribution range in Himalayan region

Indian willow (Salix tetrasperma) is an agriculturally useful tree which occurs over a wide geographic area across South Asia and bears importance. So far, this species has never been studied for molecular genetic diversity. The present study was, therefore, carried out to assess the genetic diversity and population structure analysis using RAPD and ISSR molecular markers in diverse genotypes from five populations covering North India. The mean number of effective alleles, Shannon information index and gene diversity i.e., 1.38 ± 0.013, 0.35 ± 0.010 and 0.23 ± 0.007, respectively were obtained with RAPD + ISSR markers. The analysis of molecular variance generated by RAPD + ISSR revealed a higher genetic variation (87%) within population as compared to that of among population (13%). Nei genetic distance was maximum (0.185) between Jammu and Kashmir and Punjab populations. Significant Mantel correlation (r=0.551,) was obtained between RAPD and ISSR markers. Bayesian clustering pattern obtained through STRUCTURE software showed four gene pools. Based on the genetic information obtained with regards to Indian willow by combining the RAPD and ISSR marker systems, it is proposed that an individual tree be selected within populations rather than among populations for the improvement of economic traits of Indian willow alongwith conservation of entire ecological populations.

Use of the RAPD marker to determine the genetic diversity of various Dalbergia sissoo Roxb. (Shisham) genotypes and their evolutionary relationship in Indian subcontinents

Use of the RAPD marker to determine the genetic diversity of various Dalbergia sissoo Roxb. (Shisham) genotypes and their evolutionary relationship in Indian subcontinents

Callus induction and multiple shoot proliferation from nodal explants of Mansonia altissima: confirmation of genetic stability using ISSR and RAPD markers

Callus induction and multiple shoot proliferation from nodal explants of Mansonia altissima: confirmation of genetic stability using ISSR and RAPD markers

Gold nanoparticles and electromagnetic irradiation in tissue culture systems of bleeding heart: biochemical, physiological, and (cyto)genetic effects

This study aimed to analyze the effect of various mutagens on the in vitro development, physiological activity, acclimatization efficiency, and genetic integrity of Lamprocapnos spectabilis ‘Valentine’. Gold nanoparticles (AuNPs), microwaves, and X-rays were used at different doses. The profiles of primary and secondary metabolites and the enzymatic activity in the produced plants were studied. The usefulness of various genetic markers in the detection of mutations in the species was compared. The genome size of L. spectabilis was estimated for the first time. It was found that the addition of AuNPs into the culture medium had a positive impact on the in vitro development and multiplication of plants. All of the shoots regenerated adventitious roots, but plants subjected to the longest microwave irradiation (3 × 9 s) and the non-treated control had the lowest acclimatization efficiency. Application of mutagens significantly affected the activity and profile of most enzymes and phytochemicals studied, however, the final effect depended on the agent type and dose. Mutations were detected by DAMD, RAPD, and SCoT markers in 7.5% of plants, but not by ISSRs. Phenotype variation in leaf shape was found in four plants. The genome size of L. spectabilis was found to be very small; about 1281 Mbp.

Structure and genetic diversity of two populations of Garcinia kola Heckel (Clusiaceae) revealed by RAPD markers

This study aims to characterize the genetic diversity of two populations of Garcinia kola using five RAPD markers in order to contribute to the development of sustainable management and conservation strategy for this species. Genomic Deoxyribonucleic acid (DNA) extracted from leaf fragments of 94 trees from two agroecological (south and west) zones according to Cethyl Trimethyl Ammonium Bromide (CTAB) protocol was analyzed. The mean number of alleles was ranged from 14.2 to 21.2 and the effective number of alleles ranged from 8.62 to 9.44. The average Shannon diversity index was ranged from 2.08 to 2.59 and the average genetic diversity was estimated to be 0.80. A total of 58 polymorphic bands were identified with a polymorphism rate of 100%. According to analysis of molecular variance the most important component of the genetic variation was obtained within population (94%). The analysis of the genetic diversity of all the trees revealed the existence of two groups composed of the trees from the two populations. In addition, genetic differentiation between the two populations was relatively moderate (Fst = 0.061). Sustainable management of G. kola trees should be achieved through in situ conservation. But for ex situ conservation it would be more practical to take as many individuals as possible within populations.

Phylogenetic Relationship among Indigenous Cattle Breeds of Pakistan Based on RAPD Markers

Phylogenetic Relationship among Indigenous Cattle Breeds of Pakistan Based on RAPD Markers

Genetic variability of pepper mutants (Piper nigrum l.) based on morphological and RAPD markers

The area expansion and volume of Indonesian palm oil production is not in line with its productivity. The productivity of Indonesian oil palm is inseparable from the plants age, most of which have exceeded their productive age. There are also significant indications of the use of illegitimate seeds,, causing a gap between yield and potential productivity. Efforts to increase the productivity and sustainability of oil palm plantations have been carried out by forming a particular agency that collects and manages oil palm plantation funds. One of the programs to increase productivity and sustainability is the “Replanting of Smallholder Palm Oil (PSR)” with a grant scheme for participating farmers. The progress of replanting realization is plodding. The target of 180,000 hectares per year cannot be realized. Accelerated breakthroughs without neglecting the primary mission of increasing productivity and sustainability are very urgent. This paper is a review on the importance of supporting the PSR seed institutional innovation in accelerating the productivity and sustainability of Indonesian oil palm.

Rapd marker to assess genetic diversity and exchange genes in Alcea (Malvaceae)

In order to comprehend the plant resources' protection and management in every setting, genetic variousness research is required. Because of the minimal number of distinguishing morphological features, for example, leaf sequence and carpel structure, Alcea includes a problematic taxonomy.34 Alcea species are reported in Iran among them, 15 species are endemic. There are no specifics. Alcea genetic variousness was studied using RAPD (Random Amplified Polymorphic DNA). As a result, we gathered and evaluated six species from five Iranian provinces. Seventy plant samples were gathered in total. Our objectives were to 1) determine the genetic variousness of Alcea species and 2) determine the genetic variousness of Alcea species. 2) Is there a link between a species's genetic makeup and geographical location? 3) Population and taxon genetic configuration. In-plant species, we found considerable variances in quantitative morphological features. Alcea species were separated into two categories using NJ clusters and principal component analysis. The unbiased anticipated heterozygozygosity (UHe) of Alcea rhyticarpa existed in the scope of 0.12. In Alcea aucheri, knowledge of Shannon was increased (0.39). The lowest value, 0.20, was found in Alcea rhyticarpa. In the Alcea species, gene flow (Nm) was quite modest (0.33). The current findings demonstrated RAPD markers and morphometry approaches for studying genetic variousness in Alcea species.

Genetic Fidelity Assessment of in Vitro Regenerated Plants of Ceropegia Thwaitesii Through Rapd and Scot Markers

Genetic Fidelity Assessment of in Vitro Regenerated Plants of Ceropegia Thwaitesii Through Rapd and Scot Markers

Assessment of phylogenetic diversity and relationships among <i>Grewia tenax</i> population in Sudan using RAPD and ISSR molecular markers

<abstract> <p>This research is conducted to study the genetic diversity of the genus <italic>Grewia tenax</italic> in Sudan. This plant has a common vernacular Sudanese Arabic name: “Gudeim”. It is mainly cultivated in North Darfur and Western Sudan. Common uses of <italic>Grewia</italic> species were overviewed in areas of nutrition, folk medicine and famine food. The genetic variation and genetic relationships among <italic>Grewia spp</italic> populations from different regions were efficiently determined using RAPD and ISSR markers.</p> <p>The results of genetic analysis were statistically analyzed by STATISTCA and GenALEx 6.5 software. The results of molecular variance revealed that 74% of total genetic diversity was due to within populations variations as opposed to 26% due to variations between populations unweighted pair- group method with arithmetic average (UPGMA) were constructed for and RAPD + ISSR. The UPGMA results showed variability of <italic>Grewia</italic> genotypes.</p> <p>It was concluded that both the marker systems RAPD and ISSR combination can be effectively used in determination of genetic relationships among <italic>Grewia tenax</italic> genotypes.</p> </abstract>

Detecting the effects of Wi-Fi waves on phenotypic and molecular markers of Vicia faba L.

The study objective was to evaluate the effects of exposure Vicia faba to Wi-Fi waves on growth and molecular levels. The seeds of the Vicia faba plant were soaked for 48 hours, then the seeds were divided into five groups, four for treatments and one for the control group, phenotypic characteristics were recorded, while the molecular markers were evaluated using RAPD-PCR markers to detect the effect of waves on DNA sequence. Morphological study, the highest plant height recorded in treatment B that was 123.9 followed by C, D and E. As for the bean pod number, the treatment E was 13.6 that showed the highest number that followed by D, C and B. For the seed number, the highest was E 34.4 seed that followed by D, B and C treatments. The number of leaves in the plants, the highest was in treatment B that was 251 followed by C, D and E treatments respectively. The leaf measurements recorded, the highest leaf area was in the treatment E that was 3.2-6.1 followed by D, C and B treatments respectively. Most of the treatments were distinguished by unique bands and absent of all mutant bands (51), unique bands (33) and absent bands (18), the treatment E (60) day got a higher number of unique bands that were (12), and the treatment B (15) day have got (4) bands, while the absent bands the treatment D (45) day got a higher number of the absent bands that were (6), and the treatment B (15) day got (4) bands. There was a difference in the results between the morphological and molecular markers because of the difference in mutation frequency on the genome of their coding and noncoding DNA. The Wi-Fi waves induced mutations in the genome of Vicia faba L. and the mutations increased with the period of exposure of the plant to the Wi-Fi waves. The efficiency of the RAPD markers was high in detecting the producing mutation in the genome of Vicia faba L

Determination of DNA Cytosine Methylation Polymorphism Using RAPD Markers in Some Nigella sativa L. Genotypes

Epigenetik, DNA baz değişimi olmaksızın gen ifadesi ve fonksiyonundaki kalıtsal değişimler olarak tanımlanmaktadır. DNA'da metilasyon ve histon proteinlerindeki kimyasal modifikasyonlar bitkilerde en çok çalışılan iki epigenetik mekanizmadır. Epigenetik araştırmalarda düşük işlem hacimli ve yüksek işlem hacimli DNA metilasyon saptama teknikleri vardır. Düşük işlem hacimli tekniklerden bir tanesi de enzim tabanlı DNA sitozin metilasyonunun varlığına ya da yokluğuna dayanan bir yaklaşımdır. Bu yaklaşım kullanılarak biyolojik aktiviteleri ve terapötik potansiyeli olan Nigella sativa L. genotipleri için rastgele genlerde DNA sitozin metilasyonu araştırılmıştır. Bu çalışmada, beş farklı Nigella sativa L. türüne ait genotipler (Çameli, Eskişehir, Konya, Suriye, Şanlıurfa) arasındaki sitozin metilasyon farklılıklarını araştırmak için touch-down polimeraz zincir reaksiyonları metilasyon duyarlı-rastgele arttırılmış polimorfik DNA (TD-MS RAPD) tekniği kullanılmıştır. Bu genotipler fidelerinden izole edilen genomik DNA örnekleri touch-down polimeraz zincir reaksiyonlarından önce metilasyona duyarsız olan MspI restriksiyon enzimi ve metilasyona duyarlı HpaII restriksiyon enzimi ile muamele edilmiştir. Kullanılan 8 oligonükleotid primerinden bir primer (OPB-12), beş genotip arasında metilasyon polimorfizmleri ile sonuçlanmıştır. TD-MS-RAPD-PZR yöntemi basit ve temel cihazları gerektiren uygun maliyetli yöntemdir. Bu yöntem normal bir DNA termal döngü cihazı ve DNA jel elektroforez sistemi kullanılarak kolayca uygulanabilmektedir. Ancak bu yöntemle saptanan metilasyon polimorfizmlerinin düzeyi çörek otunda çok düşüktür. Beş farklı çörek otu genomu arasında düşük düzeyde polimorfizm olduğu sonucuna varılmış olup bu durum çörek otu genomunun 5’-CCGG-3’ içeriklerinin düşük olmasından kaynaklandığı düşünülmektedir.

Determination of Genetic Variation in Some Teff Genotypes Using RAPD Markers

Günümüzde tüketici bilincinin artması sonucu sağlıklı gıda ürünlerine talep her geçen gün artmaktadır. Kimyasal bileşimi ve beslenme özelliklere sahip olan Tef bitkisi, insan beslenmesinde geçmişten beri kullanılan bir bitki olmakla beraber, ülkemizde de alternatif olarak yetiştirilebilecek kaynaklardan biridir. Sağlıklı yaşam açısından son derece önemli bir tahıl olan tef, glutensiz olması nedeniyle de özellikle çölyak hastalarının beslenmesinde önemli yer tutar. Bunun yanında yetişme koşullarının zor olmaması ve hasat süresinin kısa olmasıyla beraber gelecekte kuraklığa kolayca uyum sağlayabileceği düşüncesi tef tahılının değerini her geçen gün arttırmaktadır. Çalışma kapsamında Et 2016 tef bitkisi kullanılarak 19 RAPD markırı ile taranmıştır. İyi amplikon veren 10 tane RAPD primeri ile Tef bitkisinde genetik varyasyon araştırması yapılmıştır. Kullanılan 10 tane RAPD primeri ile PZR sonrası agaroz jel elektroforez yöntemi ile amplikonların ayrıştırılması sağlanarak varlık (1) ve yokluk (0) skorlaması gerçekleştirilmiştir. Elde edilen verilere ile Jaccard benzerlik indeksi kullanılarak temel koordinat analizi ve Bayesian istatistiği ile genotipler arasında dendogram oluşturulmuştur. Yapılan analizler sonucunda genotipler arasındaki genetik varyasyonun düşük olduğu saptanmıştır. Zobel Basic ve Dega Basic genotiplerinde çok az düzeyde farklılık tespit edilmiş olup, daha çok markır ile teyit edilmesi gerekmektedir. Sonuç olarak bu genotipler arasındaki varyasyonun artırılması için farklı yaklaşımlar kullanılarak (mutajenler vb.) varyasyonun artırılması önerilmektedir.

KERAGAMAN GENETIK SEMAI TIMOHO (Kleinhovia hospitaLinn.) BERDASARKAN PENANDA RANDOM AMPLIFIED POLYMORPHISM DNA (RAPD)

Timoho (Kleinhovia hospita Linn.) has high economic value for medicine and construction. However, information on genetic diversity and conservation genetic has been limited by the lack of genetic data. This study aimed to assess genetic diversity of timoho seedling using RAPD markers. Leaf samples of seedlings were collected from a nursery at CFBTI in Yogyakarta; the seedlings originated from plantation in Gunungkidul (GK) and arboretum in Faculty of Forestry, GMU (FK). Seven out of 22 screened RAPD markers were stable in amplification and consisted of 61 polymorphic loci; then these markers were used to analyse genetic diversity and population structure of 48 seedlings from GK and FK. The genetic diversity and structure parameters were calculated by GenAlex software. A moderate level of genetic diversity (mean uHE=0,326) and great genetic distance between the two populations (DA=0.119) were found in this species. PCoA analysis showed that the 48 individuals were divided into two clusters according to their originated population. Appropriate conservation strategies were proposed to protect genetic diversity of this species

Analysis of genetic diversity of bacteria Xanthomonas oryzae pv. oryzae causes leaf blight in North Sumatra

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight on rice plants, which is responsible for crop failure, resulting in economic losses. Control of Xoo bacteria can be accomplished through the use of Xoo-resistant rice varieties. Due to the shifting genetic structure and environmental conditions of Xoo bacteria, it is difficult to identify them according to their pathotype. This study aims to determine the genetic diversity of Xoo bacteria using the polymerase chain reaction (PCR) method and the IS1112-based RAPD marker, which is known to contain repetitive sequences. DNA was isolated from 52 isolates collected from 15 districts and cities in North Sumatra province and then used to amplify the IS1112 sequence. Dendrogram analysis revealed that cluster analysis of PCR findings classified isolates into 15 groups with a 90% similarity value. Genetic variation among Xoo isolates from North Sumatra ranged between 51% and 100%. North Sumatra Xoo isolates exhibited a high degree of genetic diversity. This findings can be used as a resource for future management of the Xoo bacteria by expediting disease-resistant rice breeding projects in various rice producing countries.

Androgenesis—Technology for Obtaining Genetically Stable Breeding Material of Capsicum annuum L.

Androgenesis in vitro is a basic method of obtaining haploid plants and DH (doubled haploid) lines of major crops such as potato, rapeseed, tomato, pepper, wheat, maize, and barley, and also many different minor crops and species with lower agricultural impact. Diploid plants appearing among androgenic regenerants are the effect of spontaneous doubling of the chromosome number in haploid cells during an embryo’s early developmental stages and are valuable fully homozygous breeding material. The subject of the presented research is spontaneous diploidization occurring in the development of androgenic, haploid pepper regenerants. In the presented experiment, the formation of diploid seeds was observed in the progeny of an androgenic, haploid plant derived in an anther culture of a hybrid (Capsicum annuum L. ATZ × Capsicum annuum L. ‘Corno di toro’)F2. Agromorphological and molecular analyses concerned eight diploid plants being progeny of the anther-derived haploid regenerant. Five of the plants constituted a phenotypically balanced group with valuable agromorphological features. Their genetic homogeneity was confirmed using 10 RAPD markers and 16 ISSR markers. Based on the results, it was concluded that anther-derived haploid plants of Capsicum can be the source of diploid, apomictic seeds, and the obtained offspring may constitute genetically stable, valuable breeding material.

Genetic profiles of three Cinchona species in Junghuhn Natural Reserve, Indonesia

Cinchona species were widely used as ancient medicines for different diseases because they contain the active component quinine and its derivatives. However, studies on the molecular aspects of cinchona, including its genetic diversity, have not been reported because most previous works focused on the administration of the antimalarial cinchona alkaloid. Quinine is also being tested as alternative compound for the treatment of Covid-19. The Junghuhn Natural Reserve in Indonesia contains three different types of cinchona plants, namely, Cinchona calisaya, Cinchona pubescens, and Cinchona sp. Given that the genetic diversity and kinship of these species have never been studied, collecting data on the cinchona gene pool has become imperative. This study analyzed the genetic diversity of the cinchona species in the Junghuhn Natural Reserve, Indonesia, by using eight RAPD markers, i.e., OPA-2, OPA-9, OPB-02, OPB-03, OPB-04, OPB-05, OPB-7, and OPJ-07, during 2020 at the University of Jenderal Soedirman, Purwokerto-Indonesia. Polymorphic band data were obtained. Then, phenogram analysis was conducted by using UPGMA and maximum parsimony with MEGA7. The RAPD profiles of Cinchona species (C. calisaya, C. pubescent, and Cinchona sp.) revealed polymorphism with different markers, i.e., OPA-2 (90%), OPB-2 (75%), OPB-5 (75%), OPB-3 (66.66%), OPB-4 (66.66%), OPB-7 (66.66%), OPJ-7 (66.66%), and OPA-9 (58.33%) sequentially with total polymorphism (70.62%). C. calisaya was identified as the most distinctive species. UPGMA yielded a coefficient of 0.200 and two distinctive groups: Group I, which comprised C. pubescens and Cinchona sp. with the p-distance value of 0.333, and Group II, which contained C. calisaya. Ixora sp. was treated as an outgroup plant. The topology of the dendrogram was consistent with that of the UPGMA dendrogram. Results may be used for the further exploration of the genetic diversity of cinchona species.

STUDY OF GENETIC DIVERSITY AND ANALYSIS OF THE DEGREE OF SIMILARITY IN SOME APPLE AND PLUM VARIETIES USING RAPD MARKERS

Molecular evaluation of germplasm is an important step in breeding programs, and the application of molecular biological techniques has led to important results in terms of both within- and between-species variability of traits. The RAPD technique has been successfully used to reveal allelic polymorphism as well as to measure genetic similarity. In this study, the genetic diversity of 25 genotypes and cultivars for apple species and 26 genotypes and cultivars for plum species was assessed with six RAPD markers. All these cultivars belong to the ex situ collection of apple and the ex situ collection of plum at the Research Institute for Fruit Growing Pitesti. The average number of amplified bands was 19.2 for apple and 17.66 for plum. Statistical analysis of intraspecific allelic polymorphism was expressed using the PIC (Polymorphic Information Content) index, which takes into account the allelic frequency. Two statistical indices were used to quantify genetic diversity: the Shannon index and the Simpson index. The degree of similarity between varieties was analyzed using the NTSYSpc version 2.1. Following RAPD analyses, the allele sizes of the analyzed varieties were within the range quoted in the literature, the genetic profiles of the studied varieties suggesting a medium to high genetic diversity, except for markers OPBC-04 and OPBB-05 for plum species, which expressed a high genetic diversity. Genetic distances calculated based on polymorphism of migrated bands in agarose gel confirmed the known genealogies of the apple and plum varieties studied. Thus, the smallest genetic distance for apple species was found between 'Jonagold' and 'Golden Delicious', 'Pionier' and 'Rustic', 'Jonathan' and 'Idared', 'Wagener Premiat' and 'Granny Smith', 'Remar' and 'Aura', 'Romus 3' and 'Rome Beauty', and the largest between Malus floribunda and the other genotypes studied. In plum, the smallest genetic distance was found between 'Dani' and 'Tita', 'Roman' and 'Tuleu gras', 'Dara' and 'Haganta', 'Romanța' and 'Stanley', 'Anna Spath' and 'Renclod Violet', and the largest between 'Lama', 'Black Diamond' and the other genotypes studied.