Simple Sequence Repeat Molecular Markers Research Articles

Use of SSR Markers for the Exploration of Genetic Diversity and DNA Finger-Printing in Early-Maturing Upland Cotton (Gossypium hirsutum L.) for Future Breeding Program

DNA fingerprinting and genetic diversity analysis of 79 early-maturing upland cotton (Gossypium hirsutum L.) cultivars were performed using Simple Sequence Repeat (SSR) molecular markers. Out of 126 pairs of SSR primers, we selected 71 pairs that gave good polymorphisms and clear bands, had good stability, and showed even distribution on the cotton chromosomes, and 142 polymorphic genotypes were amplified. The average number of alleles amplified with the SSR primers was 2.01. The polymorphism information content (PIC) of the markers ranged from 0.1841 to 0.9043, with an average of 0.6494. The results of fingerprint analysis showed that nine varieties had characteristic bands, and at least six primer pairs could be used to completely distinguish all 79 cotton accessions. Using NTSYS-pc 2.11 cluster analysis, the genetic similarity coefficients between the cotton genotypes ranged from 0.3310 to 0.8705, with an average of 0.5861. All cotton accessions were grouped into five categories at a similarity coefficient of 0.57, which was consistent with the pedigree sources. At the same time, the average genetic similarity coefficients of early-maturing upland cotton varieties in China showed a low-high-low pattern of variation over time, revealing the development history of early-maturing upland cotton varieties from the 1980s to the present. This also indirectly reflects that in recent years, China’s cotton breeders have focused on innovation and have continuously broadened the genetic resources for early-maturing upland cotton.

Recovering triploid citrus hybrids from 2x × 2x sexual crosses with the aid of embryo rescue and flow cytometry in Turkey.

Turkey is one of the major exporters of mandarins in the Mediterranean region. Seedlessness in citrus, which is one of the most desired fruit quality traits, especially in fresh mandarin export markets, can be obtained via triploidy as in many fruit species. Triploid plants can be recovered by 2x × 2x hybridizations in citrus, as well as 2x × 4x and 4x × 2x crosses. Hence, we aimed to develop local triploid hybrids by using the embryo rescue technique in five crosses using eight different citrus varieties in the present study. Embryos isolated from abortive seeds derived by 135days after pollinations were cultured on modified Murashige and Tucker (MT) medium by adding different levels of GA3 to achieve a high germination rate. A population of 574 plants was developed as a result of embryo rescue. After screening the ploidy levels of this 574-plant population with the aid of flow cytometry, 4 triploids from 'Encore' × 'Murcott', 8 triploids from 'Fortune' ×'Willow leaf', 1 triploid from 'Kiyomi' × 'Murcott', and 1 triploid from 'Ortanique' × 'Murcott' hybridization were recovered. Triploid hybrid plants and related parents were analyzed with SSR markers heterozygotic for parental mandarin varieties. In addition, we evaluated stomatal characteristics of diploid and triploid hybrids obtained from different crosses. Stomatal traits of diploid and triploid hybrids in all crosses significantly differed except the stomata index. Genotyping of triploid plants confirmed using Simple Sequence Repeat (SSR) molecular markers and five SSRs were able to identify three alleles of triploid hybrids. Selected triploid mandarin hybrids have been grafted on several rootstocks for field trials and are in the process of yield and quality performances.

Development and Application of Fruit Color-Related Expressed Sequence Tag-Simple Sequence Repeat Markers in Abelmoschus esculentus on the Basis of Transcriptome Sequencing.

Abelmoschus esculentus is a medicinal and edible plant that contains large amounts of active ingredients, including anthocyanins, polysaccharides, flavonoids, and terpenoids. However, because of a relative lack of molecular research, there are few molecular markers applicable for this plant species. In this study, on the basis of A. esculentus fruit color-related transcriptome sequencing data, we analyzed the patterns of simple sequence repeats (SSRs) in differentially expressed genes (DEGs) and revealed the biological processes and metabolic pathways associated with the related genes. We also designed primers for SSR loci to develop SSR molecular markers. Primers were synthesized using a DEG associated with a protein–protein interaction network. Polymorphic SSR markers were screened for the subsequent examination of A. esculentus germplasm resources and fruit color association analysis. The results indicated that 24.98% of the unigenes contained SSR motifs. Single-base (mononucleotide) repeats were the main SSRs, followed by trinucleotide and dinucleotide repeats. We selected 47 expressed sequence tag (EST)-SSR primer pairs for the genotyping of 153 A. esculentus varieties/lines. We ultimately obtained 21 EST-SSR markers suitable for genotyping. A generalized linear model-based association analysis detected two EST-SSR markers significantly associated with A. esculentus fruit color. In conclusion, several EST-SSR and SSR molecular markers in A. esculentus were developed in this study. The fruit color-associated markers may be useful for the molecular marker-assisted breeding of new A. esculentus varieties.

Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content

Abstract. Jannah N, Pharmawati M, Uslan. 2022. Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content. Biodiversitas 23: 2690-2698. This study investigated the genetic diversity of Sterculia quadrifida R.Br.or faloak based on Inter Simple Sequence Repeats (ISSR) molecular markers and determined stomatal density and chlorophyll content from several localities in Kupang District, East Nusa Tenggara, Indonesia. For genetic diversity analysis, samples were collected from the Oelsonbai forest area of Maulafa Sub-district, Kupang City, whereas for stomatal density and chlorophyll content analysis, samples were collected from areas with various degrees of anthropogenic activity from Kupang city. DNA was isolated using Cetyl Trimethyl Ammonium Bromide (CTAB) method and amplified by three ISSR primers (UBC 811, UBC 812, and UBC 815), whereas stomatal density was observed using a microscope and chlorophyll content was determined using a spectrophotometer. Genetic data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA) method and Nei & Li coefficient similarity in the MVSP software. A total of 78 ISSR bands ranging from 320-3200 bp were obtained, where populations were divided into four main clusters and six sub-clusters, similarly having moderate heterozygosity (He=0.50) not clustered based on their geographical distribution. There was a difference in stomatal density and chlorophyll content, probably influenced by anthropogenic disturbances. A conservation action plan is required to ensure the S. quadrifida's survival in the future.

Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollen formation in a woody plant.

The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats(SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

Characterization of Selected Zambian Rice (Oryza sativa) Accessions using Simple Sequence Repeat (SSR) Markers

Background: Rice (Oryza sativa L.) is grown in various parts of Zambia. Response of available varieties towards biotic (bacterial and fungal pathogens) and abiotic factors such as water stress remains unavailable. A systematic rice seed system or breeding program appears to be non-existent. This study was undertaken to assess the genetic diversity among some selected Zambian rice accessions using simple sequence repeat (SSR) molecular markers. Methods: Thirty accessions were selected for genetic diversity analysis using ten SSR markers (RM5, RM168, RM7, RM13, RM225, RM452, RM211, RM205, RM413 and RM46) selected from various chromosomal loci of the rice genome. Rice genomic DNA was amplified by the polymerase chain reaction and products were analyzed by 1% agarose gel electrophoresis. Most products appeared as single alleles in form of single bands averaging between 79-200 base pairs (bp). Significant relatedness within and among accessions was observed while most of the primers showed distinct polymorphism. Cluster analysis via a dendrogram revealed that most of the accessions were closely related and clustered into two major groups designated A and B. Result: Only one accession (ZM8321) appeared to be distinct and distantly related to all the other 29 accessions Cluster B contained the rest of the 29 accessions which were subdivided into 8 sub-clusters. Of the 29, accessions ZM8295 from Samfya District and ZM8313 from Kaputa district showed 100% similarity meaning that they are the same accession.

Characterizing Tetraploid Populations of Actinidia chinensis for Kiwifruit Genetic Improvement.

Understanding genetic diversity and structure in natural populations and their suitable habitat response to environmental changes is critical for the protection and utilization of germplasm resources. We evaluated the genetic diversity and structure of 24 A. chinensis populations using simple sequence repeat (SSR) molecular markers. The potential suitable distribution of tetraploid A. chinensis estimated under the current climate and predicted for the future climate was generated with ecological niche modeling (ENM). The results indicated that the polyploid populations of A. chinensis have high levels of genetic diversity and that there are distinct eastern and western genetic clusters. The population structure of A. chinensis can be explained by an isolation-by-distance model. The results also revealed that potentially suitable areas of tetraploids will likely be gradually lost and the habitat will likely be increasingly fragmented in the future. This study provides an extensive overview of tetraploid A. chinensis across its distribution range, contributing to a better understanding of its germplasm resources. These results can also provide the scientific basis for the protection and sustainable utilization of kiwifruit wild resources.

Molecular diversity analysis of rice (Oryza sativa L.) genotypes using RAPD and SSR marker

Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) molecular markers were used for detecting the genetic variability of 20 rice genotypes using 79 SSRs and 30 RAPD primers. Among primers used, a set of 16 SSR and 14 RAPD markers showed polymorphism, and banding patterns were scored as 1 (present) or 0 (absent) in the datasheet which was further analyzed by using Jaccard’s similarity coefficient and SAHN clustering. The number of alleles, PIC value, and heterozygosity for individual 16 SSR and 14 RAPD markers were used to assess the degree of polymorphism among the rice genotypes. A total of 36 polymorphic loci were found in 16 polymorphic SSR markers and 77 polymorphic loci were observed in 14 polymorphic RAPD markers. The number of alleles produced per locus ranged from 2 (RM447, RM21, RM171, RM237, RM25, RM283, RM510, RM259, RM334, RM433, RM489, and RM212) to 3 (RM263, RM152, RM413, and RM3331) in 16 SSRs markers and 3 (OPR-05) to 9 (OPR-02 and OPC-06) in 14 RAPD. In SSRs, the PIC value diverse from 0.30 (RM447) to 0.58 (RM152) with an average of 0.38 per locus and an average value of heterozygosity (0.49). RAPD analysis showed an average PIC value of 0.78. Based on the information generated, the 20 rice genotypes were grouped into two main clusters in both the analysis of the marker. In SSR markers analysis, cluster I comprised 4 genotypes and cluster II comprised 16 genotypes and in RAPD cluster I was shown 2 genotypes and cluster II show 18 genotypes.

Efeitos de tratamentos com nanopartículas e estresse salino na estrutura genética e características fisiológicas de Lavandula angustifolia Mill.

Lavandula angustifolia Mill. is an aromatic herb of the Lamiaceae family, which has been widely used by humans for many centuries. In the current study, we treated L. angustifolia samples with various concentrations of ZnO and Fe2O3 nanoparticles in the presence/ absence of NaCl salinity stress to evaluate the composition of essential oils, genetic structure, glandular trichome density and cellular Zn2+ and Fe2+ contents. We used Inter Simple Sequence Repeat (ISSR) molecular markers to investigate the parameters of genetic diversity among the treated samples. Furthermore, the hydro-distilled essential oil from the aerial parts of the samples was subjected to GC and GC / MS analyses. SPSS ver. 15, PAST, PopGene, and GenAlex software were employed for statistical analyses. Intracellular concentrations of Fe2+ and Zn2+ differed under various concentrations of nanoparticles and salinity treatments, and a significant negative correlation was observed between these elements, however, nanoparticles treatment significantly increased intracellular concentrations of iron and zinc ions. We found four types of glandular trichomes on the surface of the leaf of the treated plants, and the ANOVA test revealed a significant variation for most of them. Meanwhile, the short-stalked capitate trichomes were the most frequent in most of the evaluated samples. The main and trace essential oil compounds were the same among the treated plants, meanwhile, their percentages varied among the samples. The percentages of 1,8- cineole and camphor decreased in treated plants, which affects the quality of essential oils. Parameters of genetic diversity differed among the treated samples. Furthermore, the AMOVA test demonstrated a significant genetic variation that its substantial part belonged to among treated samples. These findings revealed that the treatment of nanoparticles and salinity stress strongly influenced the genetic diversity, trichomes density, iron and zinc ions content in lavender plants.

Diversidade genética molecular e agronômica entre genótipos de cevada sob irrigação no Cerrado brasileiro

Abstract The objective of this work was to evaluate and compare the genetic diversity of 29 barley genotypes, on the basis of molecular markers and quantitative agronomic traits, under irrigation in the Brazilian Cerrado. The randomly amplified polymorphic DNA (RAPD), inter sequence simple repeats (ISSR), and simple sequence repeat (SSR) molecular markers were used. The quantitative agronomic traits were evaluated in two irrigated environments in the Brazilian Cerrado, for the following parameters: estimated grain yield, grain size, thousand seed weight, plant height, lodging degree, and days to heading. Marker polymorphisms were 91, 51.46, and 85% for RAPD, ISSR, and SSR, respectively. The RAPD, ISSR, and SSR markers are complementary for the identification of genetic variability among barley genotypes. The low correlations between the distances estimated on the basis of molecular markers and the distances estimated on the basis of agronomic traits emphasize the importance of using complementary analyses of molecular markers for more complete studies on genetic variability. The agronomic traits of the genotypes are different in the two environments. The selected genotypes can compose the working collection of irrigated barley in the Brazilian Cerrado because of their wide genetic variability.

Blend wheat AL-type hybrid and using SSRs to determine the purity of hybrid seeds

Heterosis is a promising approach to increase wheat yield from a limited planting area. In this study, a fine quality restorer line 99AR144-1 and three stable male sterile lines, AL18A, AL36A and AL20A, were assigned as male and female, respectively. Seeds of the wheat line 99AR144-1 and three male sterile lines were mixed according to different proportions and then planted at an experimental farm at the Xinjiang Academy of Agri- Reclamation Sciences from 2013 to 2016. When the mixed sowing ratios of combinations 2 (AL36A × 99AR144- 1) and 3 (AL20A × 99AR144-1) were 6 to 8%, the seed production yields were higher than the control; the yield of hybrid seed production increased by 98.8 and 19.9%, respectively. This increase was attributed to a rise in the outcrossing seed setting rate. Further, this study used the Xbarc-8 SSR (simple sequence repeat) molecular marker to identify the purity of blend hybrid seeds and establish a regression equation for hybrid seed purity testing. The coefficient of the regression equation were 0.9878 and 0.9689 respectively, which shows that the purity of hybrids can be accurately predicted by using this equation. This method can quickly and accurately identify the seed purity in mixed seed production.

Analysis of genetic diversity in selected sugarcane (Saccharum officinarum L.) accessions using inter simple sequence repeat (ISSR) markers

Genetic diversity information among a population is important in exploiting heterozygosity for the improvement of crop species through breeding programmes. This study was therefore, conducted to assess genetic diversity and establish molecular relationships among 20 selected exotic sugarcane accessions from the Unilorin Sugar Research Institute germplasm using Inter Simple Sequence Repeat (ISSR) molecular markers. Genomic DNA was extracted from the sugarcane leaf. Fragments amplification was then performed by polymerase chain reaction (PCR) with ISSR markers and the data obtained were analyzed using MEGA 4 software. Analysis of the electropherogram showed a total of 39 loci consisting of 369 bands, out of which 95.8% were polymorphic. The biplot analysis showed all the markers contributed to the observed diversity with the least achieved with ISSR6. The principal co-ordinate analysis grouped the accessions into four clusters, comprising mixtures of all the six collection sites. The polymorphism obtained in the present study showed that the ISSR markers are effective for assessment of genetic diversity of the sugarcane accessions as it reveals the genetic similarity or divergence of the accessions regardless their place of origin or cultivation.

Selection of a core collection of Prunus sibirica L. germplasm by a stepwise clustering method using simple sequence repeat markers.

Prunus sibirica is an economically important tree species that occurs in arid and semi-arid regions of northern China. For this species, creation of a core collection is critical for future ecological and evolutionary studies, efficient economic utilization, and development and management of the broader collection of its germplasm resources. In this study, we sampled 158 accessions of P. sibirica from Russia and China using 30 pair of simple sequence repeat molecular markers and 30 different schemes to identify candidate core collections. The 30 schemes were based on combinations of two different sampling strategies, three genetic distances, and five different sample sizes of the complete germplasm resource. We determined the optimal core collection from among the 30 results based on maximization of genetic diversity among groups according to Number of observed alleles (Na), Number of effective alleles (Ne), Shannon’s information index (I), Polymorphic information content (PIC), Nei gene diversity (H) and compared to the initial collection of 158 accessions. We found that the optimal core collection resulted from preferred sampling at 25% with Nei & Li genetic distance these ratios of Na, Ne, I, PIC and H to the complete 158 germplasm resources were 73.0%, 113%, 102%, 100% and 103%, respectively, indicating that the core collection comprised a robust representation of genetic diversity in P. sibirica. The proposed core collection will be valuable for future molecular breeding of this species and management of its germplasm resources.

Appropriate statistical methods for analysis of safflower genetic diversity using agglomerative hierarchical cluster analysis through combination of phenotypic traits and molecular markers

AbstractCombining phenotypic and genotypic germplasm characterization is a key to efficient and successful safflower (Carthamus tinctoriusL.) breeding program by identifying valuable and confirmed parents. This study aimed to investigate and use appropriate statistical methods for such a characterization, and to identify potential genetic pools in safflower germplasm that may be useful for breeding program implementation. The genetic diversity of 45 accessions from different countries, provided by the USDA‐ARS, was assessed during two cropping seasons, using agromorphological traits and inter simple sequence repeat (ISSR) molecular markers. Agglomerative hierarchical cluster analysis (AHCA) was used with appropriate similarity distances, and Ward and unweighted pair group method with arithmetic mean (UPGMA) linkages. Agreement between distance–linkage combinations was evaluated using cophenetic correlation, Mantel test, Fisher exact test, Cramer'sV, overall accuracy, and Cohen's κ. Both agromorphological phenotyping and molecular genotyping revealed significant genetic diversity. Ward linkage was better than UPGMA, using simple matching distance for molecular markers and Gower distance for phenotypic traits as well as for combined phenotypic traits and molecular markers. It delineated the studied accessions into four main clusters. Some accessions showed desirable profiles that can be used in future breeding programs. This is the first report of a series of appropriate statistical methods that can be used for assessing genetic diversity in safflower, combining phenotypic traits and molecular markers, and thus identifying relevant genetic pools for breeding program.

Genetic analysis of Lepidium spp. by SSR and ISSR molecular markers

Simple sequence repeat (SSR) and inter simple sequence repeat (ISSR) markers were employed to evaluate the genetic diversity and population structure of 22 accessions belonging to three Lepidium species. Results revealed a high level of genetic diversity in the Lepidium accessions. A total of 58 bands were amplified by 11 ISSR primers, of which all were polymorphic, while 11 SSR primers produced 30 bands, of which 27 (90.0%) were polymorphic. The average values of polymorphism information content (PIC), marker index (MI), effective multiplex ratio (EMR) and percentage of polymorphic loci (PPL) in the ISSR markers were higher than those in SSRs, whereas higher values of number of effective alleles (Ne) and Shannon's information index (I) were observed by SSR markers. Based on the number of observed alleles (Na), Ne, I, expected heterozygosity (He) and PPL, the highest genetic diversity was found in L.draba followed by L.sativum by both markers. The analysis of molecular variance showed that higher allelic diversity within species than among species. The 22 Lepidium accessions grouped into three main clusters based on neighbor-joining (NJ) clustering and principal coordinate analysis (PCoA), and three (ISSR) or four (SSR and pooled data) sub-populations based on STRUCTURE analysis independent of their taxonomy. This is the first report of genetic diversity and relationships among accessions of L.sativum, L.draba and L. latifolium using SSR and ISSR markers. Therefore, these results can be utilized to improve these Lepidium species for various desired traits through molecular and hybridization breeding techniques.

Inter Simple Sequence Repeat molecular markers to reveal the genetic diversity of superior durian of Gunungpati, Semarang, Indonesia

Abstract. Nasrika E, Retnonongsih A. 2021. Inter Simple Sequence Repeat molecular markers to reveal the genetic diversity of superior durian of Gunungpati, Semarang, Indonesia. Biodiversitas 22: 4054-4059. Kalimantan is the center of many tropical fruits, including durian. One of the edible durians and favorited by many is Durio zibethinus, which has many superior varieties. Almost all regions in Indonesia have superior durian varieties; for example, superior durian Gunungpati from Gunungpati Sub-district, the center for producing durian in Semarang City, Central Java, Indonesia. ISSR analysis was carried out to reveal the genetic diversity of the superior durian Gunungpati. DNA isolation of 16 superior durians used the modified CTAB method. The genomic DNA was amplified using 10 ISSR primers and then electrophoresed using 2% agarose gel. Data were analyzed using NTSYS PC version 2.02. The resulting allele has a relative size of 220 bp-1800 bp, with 87.9% are polymorphic. The similarity coefficient of 16 varieties was ranged between 0.54-0.88. All the superior durian varieties examined were different accessions so that each variety has the potential to be registered as a new variety of superior Indonesian durian. Specific alleles are found in G1, G3, G7, G8, and G13, which can be an identity of these varieties.

Low Outcrossing from an Apple Field Trial Protected with Nets

Regulatory compliance of experimental releases into the environment of not yet approved genetically modified plants often requires implementation of measures to reduce the dispersal of reproductive material. To study the impact of nets on pollen flow in an experimental field site in Switzerland, non-GM apple varieties ‘Ladina’ and ’Nicogreen’ were planted inside and outside a netted plot, respectively. Seeds harvested from mature fruits were germinated and the paternal variety of the seedlings was determined using simple sequence repeat (SSR) molecular markers. We demonstrate that pollination frequency from trees inside the netted plot to trees outside over a two-year (2018 and 2019) study of 4500 seedlings is 0.6% (26 seedlings). Moreover, these outcrossing events decreased with increasing distance from the pollen donor. Over the study period, we found on average 0.9%, 0.5%, 0.4% and 0.09% of the seedlings derived from apples of ‘Nicogreen’ trees at 8 m, 15 m, 72 m and 117 m being generated by fertilisations of ‘Ladina’ pollen, respectively. In comparison, 48.3% (2018 season) and 75.1% (2019 season) of examined ‘Ladina’ seedlings in the netted plot originated from ‘Nicogreen’ tree pollen outside the netted plot. The results suggest that insect netting is effective in minimizing egress of apple pollen from an experimental site and that the likelihood of outcrossing is reduced further (<0.1%) when there are no compatible apple trees within a radius of 100 m of the pollen donor. These data are important for biosafety research/regulation to aid understanding of pollen flow in experimental field sites.

Evaluation of the crossings between local and drought-tolerant rice varieties using simple sequence repeat (SSR) molecular marker

Two cultivars of local rice, namely Mentik Wangi and Mentik Susu, have been grown around west to south flank of Merapi volcano. They are highly valued for their good taste and cooking characteristics. An attempt to introduce drought-tolerant rice varieties has been conducted by crossing them with two sources, namely Kasalath and Bluebonnet. Molecular-assisted selection using a set of SSR markers was applied to check the F1 and F2 generation trueness and segregation. This research was conducted in the facilities of Department of Agronomy, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta. The research was a field experiment arranged in a completely randomized design, consisting of Mentik Wangi, Mentik Susu, Bluebonnet, and Kasalath, as well as their crossings’ F1 and F2 plants as treatments. The SSR markers used are RM72, RM228, RM518, and RM20(A). Polymorphism test of the parents showed that polymorphism exists between local and donor parents, thus these markers were considered eligible for the F1 and F2 tests. The heterozygous individuals of F1 were 75% for Bluebonnet × Mentik Wangi, 44.4% for Bluebonnet × Mentik Susu, and 46.7% for Mentik Wangi × Bluebonnet; all were consistent in every primer used. We could not confirm that the F2 populations showed segregation pattern that followed Mendelian segregation in some crosses due to too small size of the sample. Heterozygous individuals in F2 showed the differences pattern for each marker, indicating that the location of the SSR markers were far from each other in the genome.

Metabolic profiling, in vitro propagation, and genetic assessment of the endangered rare plant Anarrhinum pubescens

BackgroundAnarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. ResultsTwenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard’s similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. ConclusionsThis study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.

ISSR based analysis of genetic variability of plantlets culture of pineapple (Ananas comosus L.) from Sipahutar, North Sumatera, Indonesia

Sipahutar pineapple is very popular in North Sumatra, because of the distinctive sweet taste and normal water content. Furthermore, it is large in size and has a yellow fruit skin color with greenish tips. However, the problem with Sipahutar pineapple production is the limited amount of good quality seeds. The utilization of in vitro culture techniques on pineapples allows the emergence of somaclonal variations, especially in plantlets that have undergone the subculture stage. This somaclonal variation is one of the problems in commercial seedling production, therefore, it is necessary to conduct an initial examination using the inter simple sequence repeat (ISSR) molecular marker. This study aimed to determine the genetic and primary stability of ISSR, which can be used in pineapple plants native to Sipahutar. The methods used include sample preparation, DNA isolation, primer optimization, ISSR primer amplification by PCR method, and electrophoresis. Furthermore, a total of 15 samples were amplified with six ISSR primers, and the data were analyzed by cluster method using the NTSYS-PC software. The final result was visualized in a dendrogram and analysis of diversity was conducted using GenAlex. The results showed that the level of genetic variability of the Sipahutar pineapple, which has undergone in vitro culture using six ISSR molecular markers was 76-97%. Meanwhile, the genetic variability level of the native to Sipahutar pineapple can be influenced by the long culture period and the use of N6-benzyladenine. The primers can be used to observe the genetic variability, except for ISSR 25 with a PIC value of 0.000.