Induced genetic diversity through mutagenesis in wheat gene pool and significant use of SCoT markers to underpin key agronomic traits.

BACKGROUND: This research explores the efficacy of mutagenesis, specifically using sodium azide (SA) and hydrazine hydrate (HZ) treatments, to introduce genetic diversity and enhance traits in three wheat (Triticum aestivum L.) genotypes. The experiment entails subjecting the seeds to different doses of SA and HZ and cultivating them in the field for two consecutive generations: M1 (first generation) and M2 (second generation). We then employed selective breeding techniques with Start Codon Targeted (SCoT) markers to select traits within the wheat gene pool. Also, the correlation between SCoT markers and specific agronomic traits provides insights into the genetic mechanisms underlying mutagenesis-induced changes in wheat. RESULTS: In the study, eleven genotypes were derived from parent varieties Sids1, Sids12, and Giza 168, and eight mutant genotypes were selected from the M1 generation and further cultivated to establish the M2 generation. The results revealed that various morphological and agronomical characteristics, such as plant height, spikes per plant, spike length, spikelet per spike, grains per spikelet, and 100-grain weight, showed increases in different genotypes from M1 to M2. SCoT markers were employed to assess genetic diversity among the eleven genotypes. The bioinformatics analysis identified a correlation between SCoT markers and the transcription factors ABSCISIC ACID INSENSITIVE3 (ABI3) and VIVIPAROUS1 (VP1), crucial for plant development, growth, and stress adaptation. A comprehensive examination of genetic distance and the function identification of gene-associated SCoT markers may provide valuable insights into the mechanisms by which SA and HZ act as mutagens, enhancing wheat agronomic qualities. CONCLUSIONS: This study demonstrates the effective use of SA and HZ treatments to induce gene diversity through mutagenesis in the wheat gene pool, resulting in the enhancement of agronomic traits, as revealed by SCoT markers. The significant improvements in morphological and agronomical characteristics highlight the potential of mutagenesis techniques for crop improvement. These findings offer valuable information for breeders to develop effective breeding programs to enhance wheat quality and resilience through increased genetic diversity.

Centella asiatica (L.) Urb. and Hydrocotyle umbellata L. identification and quality assessment: A methodology comparison.

INTRODUCTION: Centella is an important genus in the Apiaceae family. It includes Centella asiatica, which has significant edible and medicinal values. However, this species is easily confused due to its similar morphological traits to Hydrocotyle umbellata, hindering its utilization in the consumer and pharmacological industries. OBJECTIVE: The study aims to differentiate these two closely related plant species using reliable methods of confirming the authenticity of natural herbal medicines. METHODS: Our work mainly focuses on the basic morphological characteristics, chemical markers, genetic fingerprints, and their biological responses. RESULTS: The plants can be clearly differentiated using their leaf shapes, stipules, petioles, inflorescences, and fruit structures. Although the phytochemical compositions of the C. asiatica extract were similar to that of H. umbellata which included flavonoids, tannins, and saponins important to the plant's ability to reduce inflammation and promote healing of wounds, the H. umbellata extract showed significantly higher toxicity than that of C. asiatica. High-performance liquid chromatography analysis was used to identify chemical fingerprints. The result revealed that C. asiatica had major triterpene glycoside constituents including asiaticoside, asiatic acid, madecassoside, and madecassic acid, which have a wide range of medicinal values. In contrast, triterpenoid saponins were not identified in H. umbellata. Furthermore, using SCoT1-6 primers was possible to effectively and sufficiently created a dendrogram which successfully identified the closeness of the plants and confirmed the differences between the two plant species. CONCLUSION: Therefore, differentiation can be achieved through the combination of morphometrics, molecular bioactivity, and chemical analysis.

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.

Landscape genetics reveals that adaptive genetic divergence in Pinus bungeana (Pinaceae) is driven by environmental variables relating to ecological habitats.

BACKGROUND: Understanding the genetic basis of local adaptation has long been the concern of biologists. Identifying these adaptive genetic variabilities is crucial not only to improve our knowledge of the genetic mechanism of local adaptation but also to explore the adaptation potential of species. RESULTS: Using 10 natural populations and 12 start codon targeted (SCoT) markers, a total of 430 unambiguous loci were yielded. The Bayesian analysis of population structure clearly demonstrated that the 10 populations of P. bungeana could be subdivided into three groups. Redundancy analysis showed that this genetic divergence was caused by divergence selection from environmental variables related to the ecological habitats of "avoidance of flooding" and "avoidance of high temperature and humidity." LFMM results indicated that Bio1, Bio5, Bio8, Bio12, Bio14, and Bio16, which are related to the ecological habitat of P. bungeana, were correlated with the highest numbers of environment-associated loci (EAL). CONCLUSIONS: The results of EAL characterization in P. bungeana clearly supported the hypothesis that environmental variations related to the ecological habitat of species are the key drivers of species adaptive divergence. Moreover, a method to calculate the species landscape adaptation index and quantify the adaptation potential of species was proposed and verified using ecological niche modeling. This model could estimate climatically suitable areas of species spatial distribution. Taking the results together, this study improves the current understanding on the genetic basis of local adaptation.

The study of inter-specific relationships of Bromus genus based on SCoT and ISSR molecular markers.

The genus of Bromus is one of the most important collection of rangeland plants, which are distributed in a wide range of natural areas of Iran. Interspecific relationships were evaluated in 90 accessions of 18 Bromus species based on 15 ISSR and 15 SCoT primers. SCoT markers separated the accessions better than ISSR marker. In addition, there was a high interspecific diversity between surveying germplasm. The sections of Bromus genus completely separated based on DNA molecular markers. SCoT markers could separate the accessions in each species. The primers of SC5 and SC35 from SCoT marker and UBC861, UBC857 and UBC844 primers from ISSR marker were identified as the best primers in revealing of genetic diversity between accessions. The sections of Ceratochloa, Genea, Pnigma and Bromus were monophyletic and were placed in one cluster. The section Bromus had a direct relationship with section Genea. In other words, section Ceratochloa has a direct relationship with Pnigma. B. tectorum and B. sericeus. B. sterilis had the most distance with other species in section Genea. B. squarrosus and B. japonicus had the most similarity and B. briziformis with B. danthoniae and B. scoparius with B. rechingeri had a moderate relationship in section Bromus. B. tomentosus and B. persicus had the highest similarity and B. riparius with B. biebersteinii and B. tomentellus with B. inermis had a moderate similarity in section Pnigma.

Analysis of Genetic Diversity and Development of a SCAR Marker for Green Tea (Camellia sinensis) Cultivars in Zhejiang Province: The Most Famous Green Tea-Producing Area in China.

Camellia sinensis (L.) O. Kuntze is one of the most important non-alcoholic beverage crops in Asian and African countries. In recent years, many green tea cultivars have been released and played an important role in improving the production and quality of tea trees. The objectives of this study were to assess the genetic diversity of the eighteen main green tea cultivars in Zhejiang Province-the most famous green tea-producing area of China-using start codon-targeted (SCoT) markers and to develop a specific sequence-characterized amplified region (SCAR) marker for application in cultivar diagnosis. Thirty-one SCoT primers produced 264 loci, 226 of which were polymorphic. The genetic similarity coefficients among these green tea cultivars ranged from 0.587 to 0.814, indicating that a high level of genetic diversity was present. Both a UPGMA dendrogram and a PCoA plot grouped the tea cultivars into three groups. The partitioning of groups in the UPGMA and PCoA was similar, and much of the clustering was highly consistent with the classification of tea cultivars according to their genetic backgrounds. A unique SCoT band, SCoT4-1649, specific to the tea cultivar 'Yingshuang,' was transformed into a SCAR marker. This SCAR marker is highly useful for the identification and germplasm conservation of green tea cultivars.

Molecular insights of genetic variation in milk thistle (Silybum marianum [L.] Gaertn.) populations collected from southwest Iran.

Milk thistle (Silybum marianum) is among the world's popular medicinal plants. Start Codon Targeted (SCoT) marker system was utilized to investigate the genetic variability of 80 S. marianum genotypes from eight populations in Iran. SCoT marker produced 255 amplicons and 84.03% polymorphism was generated. The SCoT marker system's polymorphism information content value was 0.43. The primers' resolving power values were between 4.18 and 7.84. The percentage of polymorphic bands was between 33.3 and 100%. The Nei's gene diversity (h) was 0.19-1.30 with an average 0.72. The Shannon's index (I) ranged from 0.29 to 1.38 with an average value of 0.83. The average gene flow (0.37) demonstrated a high genetic variation among the studied populations. The variation of 42% was displayed by the molecular variance analysis among the populations while a recorded variation of 58% was made within the populations. Current investigation suggested that SCoT marker system could effectively evaluate milk thistle genotypes genetic diversity.

Development of SCoT-Based SCAR Marker for Rapid Authentication of Taxus Media.

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530 bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.

Landscape genomics reveal that ecological character determines adaptation: a case study in smoke tree (Cotinus coggygria Scop.).

BACKGROUND: The adaptive evolution of species response to environment are the key issues in molecular ecology and evolutionary biology. The direction of adaptive differentiation of species in regions lacking strong selection pressure is usually diverse. However, the driving mechanism of the diverse adaptive differentiation for regional species is still undetermined to date. In this study, we used landscape genomics modelling to infer the adaptive evolution of Cotinus coggygria in China's warm-temperate zone. RESULTS: Using fifteen natural populations and nine start codon targeted (SCoT) markers, a total of 1131 unambiguous loci were yielded. Our results showed two genetic groups existed in the fifteen natural populations of C. coggygria, which is due to the divergent selection driven by six environmental factors. Environmental association analyses revealed the environmental variables related to precipitation were associated with high numbers of environment-associated loci. CONCLUSIONS: Our results indicated that the ecological characters of C. coggygria, i.e. avoiding wetness and tolerating drought, determine its adaptive evolution. This study provides a reference that ecological character determines the adaptive evolution of species in regions lacking strong selection pressure.

Optimizing Sample Size to Assess the Genetic Diversity in Common Vetch (Vicia sativa L.) Populations Using Start Codon Targeted (SCoT) Markers.

Common vetch (Vicia sativa subsp. sativa L.) is a self-pollinating annual forage legume with worldwide importance. Here, we investigate the optimal number of individuals that may represent the genetic diversity of a single population, using Start Codon Targeted (SCoT) markers. Two cultivated varieties and two wild accessions were evaluated using five SCoT primers, also testing different sampling sizes: 1, 2, 3, 5, 8, 10, 20, 30, 40, 50, and 60 individuals. The results showed that the number of alleles and the Polymorphism Information Content (PIC) were different among the four accessions. Cluster analysis by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) and STRUCTURE placed the 240 individuals into four distinct clusters. The Expected Heterozygosity (HE) and PIC increased along with an increase in sampling size from 1 to 10 plants but did not change significantly when the sample sizes exceeded 10 individuals. At least 90% of the genetic variation in the four germplasms was represented when the sample size was 10. Finally, we concluded that 10 individuals could effectively represent the genetic diversity of one vetch population based on the SCoT markers. This study provides theoretical support for genetic diversity, cultivar identification, evolution, and marker-assisted selection breeding in common vetch.

Evaluation of Morpho-Physiological and Yield-Associated Traits of Rice (Oryza sativa L.) Landraces Combined with Marker-Assisted Selection under High-Temperature Stress and Elevated Atmospheric CO2 Levels.

Rice (Oryza sativa L.) is an important cereal crop worldwide due to its long domestication history. North-Eastern India (NEI) is one of the origins of indica rice and contains various native landraces that can withstand climatic changes. The present study compared NEI rice landraces to a check variety for phenological, morpho-physiological, and yield-associated traits under high temperatures (HTs) and elevated CO2 (eCO2) levels using molecular markers. The first experiment tested 75 rice landraces for HT tolerance. Seven better-performing landraces and the check variety (N22) were evaluated for the above traits in bioreactors for two years (2019 and 2020) under control (T1) and two stress treatments [mild stress or T2 (eCO2 550 ppm + 4 °C more than ambient temperature) and severe stress or T3 (eCO2 750 ppm + 6 °C more than ambient temperature)]. The findings showed that moderate stress (T2) improved plant height (PH), leaf number (LN), leaf area (LA), spikelets panicle-1 (S/P), thousand-grain weight (TGW), harvest index (HI), and grain production. HT and eCO2 in T3 significantly decreased all genotypes' metrics, including grain yield (GY). Pollen traits are strongly and positively associated with spikelet fertility at maturity and GY under stress conditions. Shoot biomass positively affected yield-associated traits including S/P, TGW, HI, and GY. This study recorded an average reduction of 8.09% GY across two seasons in response to the conditions simulated in T3. Overall, two landraces-Kohima special and Lisem-were found to be more responsive compared to other the landraces as well as N22 under stress conditions, with a higher yield and biomass increment. SCoT-marker-assisted genotyping amplified 77 alleles, 55 of which were polymorphic, with polymorphism information content (PIC) values from 0.22 to 0.67. The study reveals genetic variation among the rice lines and supports Kohima Special and Lisem's close relationship. These two better-performing rice landraces are useful pre-breeding resources for future rice-breeding programs to increase stress tolerance, especially to HT and high eCO2 levels under changing climatic situations.

Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro.

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Natural Biostimulant Attenuates Salinity Stress Effects in Chili Pepper by Remodeling Antioxidant, Ion, and Phytohormone Balances, and Augments Gene Expression.

A biostimulant is any microorganism or substance used to enhance the efficiency of nutrition, tolerance to abiotic stress and/or quality traits of crops, depending on its contents from nutrients. Plant biostimulants like honey bee (HB) and silymarin (Sm) are a strategic trend for managing stressed crops by promoting nutritional and hormonal balance, regulating osmotic protectors, antioxidants, and genetic potential, reflecting plant growth and productivity. We applied diluted honey bee (HB) and silymarin-enriched honey bee (HB- Sm) as foliar nourishment to investigate their improving influences on growth, yield, nutritional and hormonal balance, various osmoprotectant levels, different components of antioxidant system, and genetic potential of chili pepper plants grown under NaCl-salinity stress (10 dS m‒1). HB significantly promoted the examined attributes and HB-Sm conferred optimal values, including growth, productivity, K+/Na+ ratio, capsaicin, and Sm contents. The antioxidative defense components were significantly better than those obtained with HB alone. Conversely, levels of oxidative stress markers (superoxide ions and hydrogen peroxide) and parameters related to membrane damage (malondialdehyde level, stability index, ionic leakage, Na+, and Cl- contents) were significantly reduced. HB-Sm significantly affects inactive gene expression, as a natural biostimulator silencing active gene expression. SCoT primers were used as proof in salt-treated or untreated chili pepper plants. There were 41 cDNA amplicons selected by SCoT-primers. Twenty of them were EcDNA amplicons (cDNA-amplicons that enhanced their genes by one or more treatments) representing 49% of all cDNA amplicons, whereas 7 amplicons for ScDNA (whose genes were silenced in one or more treatments) represented 17%, and 14 McDNA (monomorphic cDNA-amplicons with control) amplicons were represented by 34% from all cDNA amplicons. This indicates the high effect of BH-Sm treatments in expression enhancement of some inactive genes and their silenced effect for expression of some active genes, also confirming that cDNA-SCoT markers succeeded in detection of variable gene expression patterns between the untreated and treated plants. In conclusion, HB-Sm as a natural multi-biostimulator can attenuate salt stress effects in chili pepper plants by remodeling the antioxidant defense system and ameliorating plant productivity.

Genetic diversity and genetic structure of Acer monspessulanum L. across Zagros forests of Iran using molecular markers.

Acer monspessulanum L. is an important tree species found in the temperate Zagros forests of Iran. Despite its importance, the long-term persistence of its small and fragmented populations is jeopardised by genetic erosion and hence, monitoring its genetic resource and variability is practically required for providing conservation measures of the species germplasm in Zagros woodland ecosystem. The present study aimed to provide the first data on genetic diversity and genetic differentiation pattern of 19 natural populations comprising 240 individuals of A. monspessulanum across its growing area in Zagros forests using three molecular tools including inter-simple sequence repeats (ISSR), start codon targeted (SCoT), and simple sequence repeat (SSR). In total, ISSR and SCoT primers generated a total of 141 and 121 clear and scorable bands for analysis with the polymorphism rate of 90.50 and 90.02% and a mean of 10.85 and 11 fragments per marker, respectively. In addition, 73 alleles were achieved using 10 polymorphic SSR loci from the studied accessions with 100% polymorphism, ranging between 5 and 10 alleles per locus. Average percentage of polymorphic alleles per population for ISSR, SCoT, and SSR data was 84.02%, 83%, and 100%, respectively, and generally, Nei's gene diversity (H) and Shannon's index of diversity (I) values for all populations demonstrated moderate to high levels of genetic diversity (H = 0.267-0.707; I = 0.38-1.38). The AMOVA results of the three marker systems attributed higher genetic variation to individuals within in each population than among populations. Furthermore, overall GST value for all populations detected the moderate to high levels of genetic differentiation, indicating a limited gene flow occurrence among the populations. STRUCTURE analysis (K = 5) clustered the populations into four to five distinct groups, in accordance with geographical distances. These results could represent an important contribution for effective germplasm characterization and could be eventually used in in situ or ex situ conservation of A. monspessulanum genetic resources.

Relationship analysis for Ludisia discolor germplasm resources based on SCoT molecular marker / 中草药

Objective: To make a distinction between Ludisia discolor and its relatives genus in molecular level, SCoT markers were employed to assess the genetic relationship and construct the DNA fingerprint. Methods: Orthogonal design method were carried out to optimize the suitable SCoT-PCR reaction system based on five factors. The optimum annealing temperature and SCoT primers were also screened. The 12 germplasm resources were used as materials, the screened primers were selected to analyze the genetic relationship of 12 materials. POPGENE was used to calculate the genetic diversity, NTSYS was performed to analyze cluster, and DNA map was constructed. Results: The optimized SCoT-PCR reaction system was constructed and a total of 12 rich bands were screened out as the primers of SCoT molecular marker with polymorphism ratio of 98.98%. According to Nei's genetic similarity coefficient, a total of 12 materials were divided into three cluster when coefficient was 0.45. Goodyera schlechtendaliana was in category I with seven L. discolor lines, indicating that these samples had close relationship. In category II, there were three samples came from Anoectochilus roxburghii. Moreover, a green L. discolor sample was alone clustered into category III. The DNA fingerprint map by the SC8 primer could identify the 12 materials. Conclusion: There are rich genetic diversities in 12 samples of L. discolor and its relatives genus, and the construction of DNA fingerprint map provides a theoretical basis for the identification of L. discolor and its relatives genus, which were tested in this study.