SCoT Markers Research Articles

Limited-scale field trial confirmed differences in growth and agarose characteristics in life-cycle stages of industrially important marine red alga Gracilaria dura (Gracilariales, Rhodophyta)

The red alga Gracilaria dura is economically important due to its high-quality agarose. Previous studies with wild populations reported the existence of specific differences in functional traits as well as agar characteristics among life cycle stages. In farmed populations, such differences can be exploited for commercial gains. For that, the variation among stages still needs to be well established under farming scenarios. Here, we compared the life cycle stages of G. dura regarding morphological and anatomical structures, growth performance under preliminary field trials, characteristics of agarose of cultivated biomass with biochemical (NMR) and molecular profiling (SCoT). The male gametophyte was found to have a significantly higher growth rate of 6.23 ± 0.59% day−1 than the tetrasporophyte (5.10 ± 0.14% day−1) and cystocarpic female gametophyte (2.67 ± 0.32% day−1). A maximum agarose yield of 28.6 ± 1.53% was obtained from the tetrasporophyte, significantly higher than 27.4 ± 0.60% in cystocarpic female gametophyte and 25.2 ± 0.36% in male gametophyte. The gel strength of agarose from male gametophytes was 2384 ± 124.13 g cm−2, which was significantly higher than the 1900 ± 50 g cm−2 and 2122 ± 124.03 g cm−2 recorded from tetrasporophytes and cystocarpic female gametophytes, respectively. A metabolomic study by NMR spectroscopy showed critical differences in alanine, lactate and isethionic acid among stages. The genetic correlation studied with the SCoT marker showed an average polymorphism of 47.02%. The average heterozygosity and Shannon-Wiener index were 0.63 and 1.06 respectively. This study of characterising and differentiating isomorphic life phases of G. dura by a decisive biomarker could be a valuable reference point to select an appropriate cultivar for commercial farming and breeding programmes.

Cross infection and molecular characterization of Colletotrichum spp. infecting coffee and black pepper

Anthracnose caused by Colletotrichum species is an economically important fungal disease in both coffee and black pepper leading to substantial crop losses. In India, coffee is cultivated under natural shade conditions and black pepper is grown as an inter-crop. Since Colletotrichum species can infect a broad range of host plants, there is an apprehension of the spread of anthracnose from coffee to black pepper and vice versa. Because of this, it is essential to identify the Colletotrichum strains infecting coffee and black pepper as an aid in developing effective control measure strategies. Cross-infection studies using the strain isolated from coffee and black pepper failed to infect each other. However, the ITS sequence of Colletotrichum spp. isolated from coffee and black pepper shared a similarity of 96.45% and placed both the strains under Colletotrichum gloeosporioides. The genetic variability between both strains was studied using 20 SRAP and 15 SCoT polymorphic molecular markers. The average polymorphism information content (PIC) and resolving power (Rp) of the 20 SRAP primer combinations were 0.52 and 14.65 and for SCoT primers it was 0.39 and 12.27 respectively. The UPGMA dendrogram and principal coordinate analysis divided the Colletotrichum species into two clusters and placed them in two different coordinates. Based on the Jacquard coefficient index, the similarity between the two strains was 31.0% using SRAP and 60.0% using SCoT markers whereas using SRAP and SCoT combined assay it was 43.0%. The study showed that the two Colletotrichum strains infecting coffee and black pepper are genetically distinct.

Molecular Characterization of Ten Cicer arietinum L. Genotypes Using SCOT Marker

The up-to-date technique of start codon targeted (SCoT) polymorphism (sites around start codon) is efficient in genetic diversity analysis and fingerprinting. In the current study, 94 amplified fragments were obtained using 10 SCoT Primers with 10 genotypes of Cicer arietinum L with 35% polymorphism, where the polymorphic bands were 33 and 61 bands were monomorphic. The SCoT data analysis showed genetic similarity ranging from 84 to 98%. A dendrogram separated the studied ten chickpea genotypes into two major clusters, divided into two main sub-clusters. The difference in Principal Coordinate Analysis (PCA) established genetic variance between the investigated samples. In four quadrants, the PCA variable demonstrated clustering for the 10 genotypes of chickpea that were examined.

Molecular characterization of four Mullet species based on SCoT and ISSR markers

Molecular markers were used as indicators in assessing the genetic diversity of a fish, they allow for direct observation of genetic information and estimation of genetic relationships between the population and species. In the present study, eleven SCoT primers and ten ISSR primers were used to estimate the genetic diversity among the four mullet species namely Mugil cephalus, Liza ramada, Liza grana, and Valamugil seheli collected from four different Egypt governorates Alexandria, Ismailia, Port Said and Damietta. The two markers SCoT and ISSR successfully amplified amplicons with a total number of 176 and 132 of which 153 and 111 were polymorphic, representing a percentage of polymorphism of 86.9% and 84.1% polymorphic amplicons/ primer, respectively. The similarity indices ranged from 0.47 to 0.84, 054 to 0.92 and 0.52 to 0.86 for SCoT, ISSR and combined, respectively. Cluster analysis based on similarity matrices of SCoT, ISSR and combined dendrograms revealed some similarities; for example, the grouping of the Mugile cephallus (Alexandria, Ismailia and Port Said) together and the grouping Valamugil seheli (Ismailia and Damietta) based on the three dendrograms. Also, Liza ramada, Liza garana (Damietta) and Mugile cephallus ( Damietta) were clustered together based on the three dendrograms. From gene sequencing and NCBI Blast analysis identified of gene location in fish genomes. The NCBI blast tool was used to align our gene with the NCBI database and it revealed that this sequence is highly similar to a sequence located on fish species called Dicentrarchus labrax (the European bass). Also, the sequence is located on Chr1 near a gene called “Type II keratin E3”.

Gene flow and population structure in Allochrusa (caryophylloideae, caryophyllaceae) with the use of molecular markers

Allochrusa Bunge is a genus of subfamily Caryophylloideae Rabeler & Bittrich contains about 8 species distributed in Turkey, Central Asia, Afghanistan, Caucasus, Transcaucasus and Iran. Three species of Allochrusa versicolor, A. bungei and A. persica occur in Iran show some degree of morphological overlaps that make the species delimitation difficult. Till present time, there has been no detailed information available on morphological and genetic structure of these species in the country. The aims of the present study are: 1) to find the diagnostic value of SCoT markers in delimitation of Allochrusa species, 2) to find the genetic structure of these taxa in Iran, and 3) to investigate the species inter-relationship. For this study, 97 randomly collected plants from 7 geographical populations in three Allochrusa species were used. We encountered extensive within species genetic and morphological diversity. SCoT molecular markers could delimit the studied species. AMOVA and STRUCTURE analysis revealed that the species of Allochrusa are genetically differentiated. The Mantel test showed correlation between genetic distance and geographical distance of the populations studied. Genetic affinity of the studied species has been discussed.

Genetic diversity analysis of the medicinal plant Heracleum persicum desf. ex fischer

Persian hogweed (Heracleum persicum Desf. ex Fischer) is native to Iran but was introduced to Europe as an invasive plant. It has medicinal and edible values. Its fruits have been used to relieve flatulence, stomach aches as well as a flavoring as a digestive and an antiseptic. This medicinal plant forms many geographical populations in the country, particularly in mountainous regions; however, we have no data on the genetic structure and genetic diversity of this plant species. Such information can be used in gene pool identification and future genetic conservation and breeding programs. Therefore, the present study was performed with the following aims: 1- Provide data on genetic diversity of geographical populations, 2- Identify the potential gene pools for future genetic conservation and breeding programs. We used both Inter-simple sequence repeats (ISSR) and the Start Codon Targeted (SCoT) molecular markers as these are suitable molecular markers for genetic diversity investigations. There are highly polymorphic, reproducible, and relatively low cost markers. The present study revealed that both ISSR and SCoT markers produce data on the genetic variability and genetic affinity of the local populations. Both molecular markers revealed a good level of genetic variability within and among Heracleum persicum populations. Analysis of molecular variance (AMOVA) produced a significant differences between geographical populations for both markers. We could identify few ISSR as well as SCoT bands which can differentiate the studied populations. The moderate to a good level of genetic diversity we observed within each H. persicum may be due to cross pollination. In conclusion, we suggest using combination of ISSR and SCoT molecular markers to study population genetic variability in H. persicum geographical populations for future genetic conservation and germplasm collection of this medicinal plant.

The using of gene-oriented SCoT markers in genotyping of the Apple genus (Malus Mill.)

This paper discusses the aspects of optimization of the SCoT genotyping method for representatives of the genus Apple (Malus Mill.), including the orchard apple (Malus domestica). Special attention is paid to the methods of total DNA isolation from apple plant tissue, which is due to the sensitivity of multilocus marker systems to the quality of the nucleic acid preparation. On a sample of total DNA isolated from the leaves of the Golden Delicious cultivar, 18 SCoT markers were tested, from which 4 Malus Mill cultivars and species most promising for genotyping were selected.

Study on genetic diversity between Malva l. (Malvaceae): A high value medicinal plant using SCoT molecular markers

The Malva L. genus, popularly known as mallow, grows spontaneously in almost all of Europe and the Mediterranean region. The Malva genus has 25-40 species and it can be considered as an annual and/or biannual herb. This genus contains species that reveal therapeutic properties and are mostly important in medicine and the functional food industry. Therefore, due to the importance of these plant species, we performed a combination of morphological and molecular data for this species. For this study, we used 97 randomly collected plants from seven species in 7 provinces. According to the SCoT markers analysis, Malva sylvestris and Malva aegyptia had the lowest similarity and the species of Malva neglecta and Malva vericillata had the highest similarity. The aims of present study are: 1) can SCoT markers identify Malva species, 2) what is the genetic structure of these taxa in Iran, and 3) to investigate the species inter-relationship? The present study revealed that combination of morphological and SCoT data can identify the species.

GENETIC DIVERSITY OF NATURALLY DISTRIBUTED Rhododendron moulmainense Hook. f. POPULATIONS IN LAM VIEN PLATEAU, VIETNAM REVEALED BY ISSR AND SCoT MARKERS

Genetic diversity and variation of three naturally distributed Rhododendron moulmainense populations in the Lam Vien Plateau of Vietnam were analyzed using ISSR and SCoT markers separately and data from the two types of markers were then combined to show the overall results. Based on combined data, the genetic diversity parameters of the Tuyen Lam population were percentage of polymorphic bands (PPB) = 28.79%; genetic heterozygosity (He) = 0.1041; Shannon index (I) = 0.1552. The corresponding parameters were PPB = 37.88%; He = 0.1434; I = 0.2131 in the Hon Nga population, and PPB = 46.21%; He = 0.1857; I = 0.2709 in the Bidoup population. At the species level in Lam Dong province, the corresponding parameters were PPB = 62.12%; He = 0.1747; I = 0.2756. The average of pairwise genetic similarity coefficients among investigated individuals of Tuyen Lam population was 0.973, of Hon Nga population was 0.911, and of Bidoup population was 0.895. The genetic differentiation and the number of migrants per generation among investigated populations were GSTS = 0.1735 and Nm = 2.3824 individuals, respectively. Genetic distance between Tuyen Lam and Hon Nga populations was 0.0399, between Tuyen Lam and Bidoup populations were 0.0621, and Bidoup and Hon Nga populations were 0.0670. Data generated by both marker types and the combined revealed similar trends of genetic diversity levels among the studied populations. The SCoT data showed higher values of genetic diversity and population gene differentiation than did the ISSR data, but the latter showed higher gene differentiation among sampled individuals in the study populations. Comparing the values of Polymorphism Information Content (PIC) for discriminatory ability; Effective Multiplex Ratio (EMR) for several revealed loci and/or efficiency of the primer-marker system; Marker Index (MI) for the efficiency of used method; and Resolving Power (Rp) between used techniques indicated that discriminatory ability, efficiency, and ability of the ISSR primer combinations were higher but using SCoT marker system was more efficient than ISSR marker system for evaluating genetic diversity.

Genetic diversity studies on seven Egyptian wheat (Triticum aestivum) cultivars using Scot and ISSR polymorphism markers.

Assessment of genetic diversity of crops by using informative molecular markers is important for their genetic improvement and conservation. Genetic diversity and relationships among seven Egyptian cultivars of wheat (Triticum aestivum L) (Masr1, Swiss2, Swiss4, Giza7, Giza9, Giza10 and Sakha94)were analyzed using SCoT and ISSR markers. A total of 7 SCoT and 7 ISSR primers were used to estimate genetic polymorphism among seven wheat cultivars (Masr1, Swiss2, Swiss4, Giza7, Gisa9, Giza10 and Sakha94) . DNA extraction was done using Thermo Kit according to its manufacturer’s instructions followed by amplifications with ISSR and SCoT and agarose gel electrophoresis. Based on UPGMA both ISSR and SCoT markers resolved cultivars into dendrogram with three major clusters .The reproducible bands were scored for analyses polymorphic information content (PIC), Resolving power (RP) and Marker index(MI). Comparatively, two markers were effective. The average polymorphic information content (PIC), Resolving power (RP)and Marker index (MI) of SCoT were reflected relatively higher than those of ISSR. According to the present results, SCoT markers proved more informative in studying genetic diversity among seven wheat cultivars. The results demonstrated that SCoT and ISSR markers are useful for genetic diversity analysis of wheat cultivars. This information is useful for utilization in plant breeding programs.

Assessment of the Suitability of Molecular SCoT Markers for Genetic Analysis of Coffee Species

AbstractStart codon targeted polymorphism (SCoT), a novel and gene-targeted marker, has recently become the marker of choice in genetic diversity studies. In the present study, 31 SCoT primers were tested for their suitability in the genetic analysis of 21 coffee genotypes representing 18 species. A total of 647 distinct PCR amplified fragments were produced with a mean of 20.9 fragments per primer and 80.80% of which were polymorphic. The polymorphic information content of SCoT primers ranged from 0.16 to 0.86, with a mean value of 0.63. Resolving power ranged from 6.19 to 28.29, with a mean value of 20.2. Species-specific unique PCR amplified fragments were identified for 16 species, which could be used as genetic fingerprints. The genetic similarity among various coffee species calculated using the Dice similarity coefficient ranged between 0.60 and 0.89. The dendrogram constructed using the unweighted pair group of arithmetic means (UPGMA) clustered the 21 coffee genotypes into two major groups. The study revealed that Coffea jenkinsii, an indigenous species from India, showed the highest similarity with C. arabica, which is of Ethiopian origin. The results proved the suitability of SCoT markers in genetic analysis of coffee genotypes.

DNA barcoding and genetic fidelity assessment of micropropagated Aenhenrya rotundifolia (Blatt.) C.S. Kumar and F.N. Rasm.: a critically endangered jewel orchid.

Aenhenrya rotundifolia is a critically endangered terrestrial jewel orchid. It is monotypic and endemic to evergreen forests of southern western ghats of India. In the present study, identification of this plant species is validated with DNA barcoding using matK and rbcL chloroplast markers. Further, germ-free juvenile axillary bud explants were cultured on Mitra medium supplemented with different kinds of cytokinins like 6-benzyladenine, 6-furfurylaminopurine, N6-(Δ2-isopentyl) adenine, thidiazuron, zeatinandmeta-topolin as well as auxins such as α-naphthaleneacetic acid, indole-3-acetic acid and indole-3-butyric acid at different concentrations and combinations for successful proliferation and establishment in vitro. After 12weeks of culture, axillary bud explants produced an average of 30.12 ± 0.71 shoots per explant, 3.87 ± 0.06cm shoot length, 1671 ± 2.82mg fresh mass of proliferated shoots with a proliferation frequency of 100% on Mitra medium supplemented with 6.20µM meta-topolin and 2.25µM thidiazuron. No root formation was observed in in vitro proliferated microshoots. However, tiny hair like projections were observed in some elongated shoots on Mitra medium pertaining to5.37µM NAA. The tiny hair like structure bearing plantlets were hardened and acclimatized with 100% survival rate in the polytunnel chamber. After 8-10months of establishment ex vitro, flowering was observed. Additionally, the genetic fidelity of in vitro derived plants was tested with ISSR and SCoT marker profiling. The test results revealed that the plants derived from the protocol has 99% genetic similarity to that of the donor mother plant. This study can be applied in forensic interventions of this species, describes the maintenance of germplasm in vitro and establishment of new viable population in its original habitats by restoring existing sites of this critically endangered jewel orchid.

Genetic fidelity assessment of long term in vitro shoot cultures and regenerated plants in Japanese plum cvs Santa Rosa and Frontier through RAPD, ISSR and SCoT markers

In vitro propagation can be explored to overcome the constraint of limited planting material in plum. Although it ensures the production of plants in large number, but there are chances of somaclonal variations using this technology. Thus, clonal fidelity of in vitro raised plants should be checked to obtain true to type planting material prior to transplantation in the field. Genetic fidelity of in vitro cultures of plum (Prunus salicina) cvs. Santa Rosa and Frontier multiplied for 5 years (60 passages) through enhanced axillary bud proliferation was tested and compared with 2 year old in vitro raised and mother plants of respective cultivars using RAPD, ISSR and SCoT markers. Out of twenty-eight RAPD primers, eighteen produced 29 and 27 distinct bands in Santa Rosa and Frontier whereas, all of the fifteen ISSR primers screened generated clear reproducible bands in both the cultivars. In SCoT assay, eight primers out of twenty-six generated reproducible bands in both the cultivars. Homogenous amplification was observed in all the samples thereby confirming the genetic fidelity of tissue culture raised plants, thus suggesting that in vitro propagation using axillary buds is the safest mode for the production of clonal planting material in plum.

Highly efficient rapid micropropagation and assessment of genetic fidelity of regenerants by ISSR and SCoT markers of Solanum khasianum Clarke

An efficient method of rapid micropropagation of Solanum khasianum Clarke was successfully established from the leaf, petiole, and nodal explants. The morphogenetic response of different concentrations of TDZ and BAP individually or in combination with auxins (IAA/IBA/2,4-D) was tested. Friable callus was obtained on different concentrations of BAP alone or in combination with IAA/IBA/2,4-D. Rapid multiple shoot induction was achieved from friable callus on MS medium supplemented with varying concentrations of TDZ and IBA. The leaf explants exhibited a high frequency of multiple shoots than petiole and nodal explants with an optimal percentage of response (92.73%), mean shoot number (53.5 ± 0.47), and shoot length (11.2 ± 0.53 cm) on MS medium augmented with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1). Maximum rooting efficiency was achieved on MS medium with 1.5 mg l−1 IBA with 12.8 ± 0.36 mean number of roots. The in vitro rooted plants were acclimatized with a survival rate of 80%. The genetic fidelity of the regenerants assayed by the ISSR and the SCoT markers showed no genetic variation. The study examined the micropropagation responses of S. khasianum in the presence of various growth regulators and provided a simple and more suitable protocol adapted for the mass propagation of clones in this species. We have established a highly efficient micropropagation system for large scale production in Solanum khasianum. Evaluation of clonal fidelity by using ISSR and SCoT markers detected no somaclonal variations. The present study helps to the enhancement of potential alkaloids (solasodine) with the help of biotechnological tools.

A New Aspect for In vitro Propagation of Jerusalem Artichoke and Molecular Assessment Using RAPD, ISSR and SCoT Marker Techniques

JERUSALEM artichoke is an important crop for a wide range of agricultural, medicinal and industrial purposes. Its tubers are rich in inulin and it is cultivated for food and animal feed. The current study aimed to develop an applicable protocol for micropropagation of three cultivars of Jerusalem artichoke (i.e., Balady, Fuza and Alba) using stem node explants. These explants were cultured on ½ MS medium augmented with different concentrations of kanamycin or cefotaxime. The lowest contamination correlated with highest survival percentages were recorded for ½ MS supplemented with 62.5mg L-1 cefotaxime. For shoot multiplication, the maximum number of shoots (11) were obtained for Alba cultivar cultured on MS fortified with 1mg L-1 BA + 0.1mg L-1 NAA + 50mg L-1 nano selenium. For rooting in vitro, the maximum values of rooting percent, root numbers/plantlet and length of roots were observed with ½ MS + 2mg L-1 IBA + 0.1mg L-1 NAA + 0.5mg L-1 KIN. Resulted in vitro regenerated plantlets, were acclimatized on perlite and peat moss mixture (1:1), which gave the highest percent of survival (100%) for Alba followed by Fuza and Balady (80 and 60%, respectively). Moreover, the molecular characterization based on RAPD, ISSR and SCoT techniques was carried out. The polymorphic percentages among in vivo shoots of the three cultivars recorded 38.09, 42.3 and 34.61%, respectively; 61.53, 67.8 and 50% between in vivo shoots, in vitro regenerates and stem derived calli of the three cultivars. The dendrogram analysis of combined techniques showed high similarity between Balady and Fuza followed by Alba.

Enhancement of Chilling Tolerance and Efficiency Using Start Codon Targeted (Scot) Technique for Molecular Assessment in some Tomato Genotypes. تحسین تحمل البرودة وکفاءة استخدام تکنیک SCOT فى التقییم الجزیئى فی بعض التراکیب الوراثیة للطماطم.

Significant reduction in tomato yield and its fruit quality occurs annually in Egypt during winter and early summer seasons as a result of chilling stress. In this study, a set of tomato lines and cultivars were estimated against their response to treatment by ATP and glycine betaine, in order to select the most promising genotypes for further breeding programs. The Field experiment was carried out at Genetics Department farm, Faculty of Agriculture, Mansoura University during the winter season of 2017/2018. A Randomized Complete Blocks Design in three replicates was adopted in current investigation. The Data were recorded on different vegetative, fruit qualityand yield component traits. The results showed that the Edkawy cultivar had the most useful yield components parameters affected by the exogenous treatment followed by S. Marmande and the line KL-45. This cultivar was characterized by a large number of fruits per inflorescence and average fruit weight around 90 grams. The genetic diversity using SCoT marker technique between 11 tomato genotypes was conducted for selecting the promising genotypes for further breeding programs. The SCoT primers produced a total of 94 bands with an average 11.75 bands per primer ranged from seven (SCoT-6 and SCoT-9) to 22 (SCoT-1) per primer. Among them 84 bands were polymorphic. The highest molecular distance was among KL-41 and KL-03 (0.482). While the lowest MD was between Floradade and Edkawy (0.215). The 11 genotypes showed 14 marker loci, these markers were spread over these genotypes variously differentiating each genotype from the other.

Population structure and genome-wide association analysis for salinity tolerance in wheat using SNP, SSR and SCoT marker assays.

Wheat is an essential staple food in the developing world, where demand is projected to grow exponentially in the future; simultaneously, climate changes are projected to reduce supply in the near future. One of the main consequences of climate change is salinity, which negatively impacts the world's cultivated area and therefore affects the global wheat production. Our objectives are to study the population structure of several Egyptian and international wheat accessions and to identify the genetic factors controlling the salinity stress response of b wheat. In addition, we have attempt to identify genes that control some important agronomic parameters of wheat under salinity stress were identified. The wheat germplasm panel consisted of 70 accessions obtained from Egypt, Syria and Iran. The assessment of salinity tolerance was conducted over the years of 2018 and 2019 in the field and in the greenhouse. The genome association analysis (GWAS) and population structure analysis was conducted using six SCoT, five SSR and 93 SNP markers. Analysis of the population structure using allele frequency and phylogenetic analysis indicated that the studied wheat accessions were belong to four population groups. Where, for the most portion, Egyptian, Syrian and Iranian accessions are clustered depending on their country of origin. The GWAS analysis revealed 13 SNP markers that were significantly associated with morpho-agronomic wheat traits during salinity stress. These markers were closely related to genes that are known to have a direct link to wheat response to salinity stress such as CYP709B2, MDIS2, STAY-GREEN, PIP5K9, and MSSP2 genes. This study revealed the genetic structure of adapted and imported wheat accessions, which could be used to select potential wheat accessions for local breeding programs. In addition, the SNP genotyping assay is a very potential technology that could be efficiently applied to detect genes that control bread wheat response to salinity stress.

Genetic diversity analysis of Hippeastrum rutilum cultivars based on SCoT markers

Objective The objective of this study is to further analyze the genetic diversity, genetic relationship and parental identification of Hippeastrum rutilum cultivars at molecular level. Method The SCoT marker system of H. rutilum was screened by orthogonal design method, and genetic diversity and genetic relationship of 41 cultivars were analyzed. Result (1) The optimum reaction system of SCoT markers for H. rutilum (20 μL) included DNA 40 ng, primer 0.1 μmol·L−1, MgCl2 2.0 mmol·L−1, dNTPs 0.4 mmol·L−1 and rTaq DNA polymerase 0.75 U (1 U=16.67 nkat). (2) 77 polymorphic bands were obtained from 41 H. rutilum cultivars by 12 SCoT primers, and the average polymorphic band ratio was up to 86.52%. The genetic similarity coefficient between H. rutilum cultivars was 0.292 3−0.834 3, indicating that the 41 cultivars had high genetic diversity and wide genetic range. (3)UPGMA(unweighted pair-group method with arithmetic means) cluster analysis showed that the SCoT marker clustering of H. rutilum had significant correlation with flower color, but not with the petal type. At genetic similarity coefficient of 0.420 0, the 41 cultivars were divided into two groups. The first group had both double and single petal cultivars. The first group was divided into four subgroups, among which those with similar flower colors were clustered into one group. In subgroupⅠd, the single-petal, white ‘Hydrangea’ (No. 20) and red ‘Miracle’ (No. 22) were the possible parents of double-petal, orange-red and white multicolor flowers (No. 21 ‘Yingchun’). The second group was mostly multicolor flowers. Conclusion SCoT marker technique can be effectively applied to genetic diversity analysis and identification of possible parents of H. rutilum cultivars. [Ch, 6 fig. 4 tab. 21 ref.]

Genetic relationships and diversity among populations of Paris polyphylla assessed using SCoT and SRAP markers.

The genetic diversity of 33 Paris polyphylla samples collected from the Dabie Mountains was analyzed using SCoT and SRAP molecular markers, revealing the genetic relationships among Paris polyphylla resources in the Dabie Mountains at the molecular level and providing a theoretical basis for genetic improvement and conservation. As a result, a total of 134 bands were amplified with 9 SCoT primers, the percentage of polymorphic bands was 100%, the average number of primers amplified was 14.89, the PIC value was 94.83% and the genetic similarity coefficient ranged from 0.463 to 0.896. Ten pairs of SRAP primer combinations amplified 135 bands, including 129 polymorphic bands, and the percentage of polymorphic bands was 95.56%. The average number of polymorphic bands obtained with each pair of SRAP primer combinations was 12.9, the PIC value was 93.91%, and the genetic similarity coefficient ranged from 0.533 to 0.904. This study showed that both SCoT and SRAP markers were suitable for the genetic diversity analysis of P. polyphylla, which belongs to a genus in which SRAP marker technology has not previously been applied, despite its application in a variety of other plants.

Droplet-vitrification of Aranda Broga Blue orchid: Role of ascorbic acid on the antioxidant system and genetic fidelity assessments via RAPD and SCoT markers

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.