SUnSeT: spectral unmixing of hyperspectral images for phenotyping soybean seed traits.

KEY MESSAGE: Hyperspectral features enable accurate classification of soybean seeds using linear discriminant analysis and GWAS for novel seed trait genes. Evaluating crop seed traits such as size, shape, and color is crucial for assessing seed quality and improving agricultural productivity. The introduction of the SUnSet toolbox, which employs hyperspectral sensor-derived image analysis, addresses this necessity. In a validation test involving 420 seed accessions from the Korean Soybean Core Collections, the pixel purity index algorithm identified seed- specific hyperspectral endmembers to facilitate segmentation. Various metrics extracted from ventral and lateral side images facilitated the categorization of seeds into three size groups and four shape groups. Additionally, quantitative RGB triplets representing seven seed coat colors, averaged reflectance spectra, and pigment indices were acquired. Machine learning models, trained on a dataset comprising 420 accession seeds and 199 predictors encompassing seed size, shape, and reflectance spectra, achieved accuracy rates of 95.8% for linear discriminant analysis model. Furthermore, a genome-wide association study utilizing hyperspectral features uncovered associations between seed traits and genes governing seed pigmentation and shapes. This comprehensive approach underscores the effectiveness of SUnSet in advancing precision agriculture through meticulous seed trait analysis.

QTL mapping reveals key factors related to the isoflavone contents and agronomic traits of soybean (Glycine max).

BACKGROUND: Soybean is a valuable source of edible protein and oil, as well as secondary metabolites that can be used in food products, cosmetics, and medicines. However, because soybean isoflavone content is a quantitative trait influenced by polygenes and environmental interactions, its genetic basis remains unclear. RESULTS: This study was conducted to identify causal quantitative trait loci (QTLs) associated with soybean isoflavone contents. A mutant-based F2 population (190 individuals) was created by crossing the Korean cultivar Hwanggeum with low isoflavone contents (1,558 µg g-1) and the soybean mutant DB-088 with high isoflavone contents (6,393 µg g-1). A linkage map (3,049 cM) with an average chromosome length of 152 cM was constructed using the 180K AXIOM® SoyaSNP array. Thirteen QTLs related to agronomic traits were mapped to chromosomes 2, 3, 11, 13, 19, and 20, whereas 29 QTLs associated with isoflavone contents were mapped to chromosomes 1, 3, 8, 11, 14, 15, and 17. Notably, the qMGLI11, qMGNI11, qADZI11, and qTI11, which located Gm11_9877690 to Gm11_9955924 interval on chromosome 11, contributed to the high isoflavone contents and explained 11.9% to 20.1% of the phenotypic variation. This QTL region included four candidate genes, encoding ß-glucosidases 13, 14, 17-1, and 17-2. We observed significant differences in the expression levels of these genes at various seed developmental stages. Candidate genes within the causal QTLs were functionally characterized based on enriched GO terms and KEGG pathways, as well as the results of a co-expression network analysis. A correlation analysis indicated that certain agronomic traits (e.g., days to flowering, days to maturity, and plant height) are positively correlated with isoflavone content. CONCLUSIONS: Herein, we reported that the major QTL associated with isoflavone contents was located in the interval from Gm11_9877690 to Gm11_9955924 (78 kb) on chromosome 11. Four ß-glucosidase genes were identified that may be involved in high isoflavone contents of soybean DB-088. Thus, the mutant alleles from soybean DB-088 may be useful for marker-assisted selection in developing soybean lines with high isoflavone contents and superior agronomic traits.

QTL Mapping of Tiller Number in Korean Japonica Rice Varieties.

Tiller number is an important trait associated with yield in rice. Tiller number in Korean japonica rice was analyzed under greenhouse conditions in 160 recombinant inbred lines (RILs) derived from a cross between the temperate japonica varieties Odae and Unbong40 to identify quantitative trait loci (QTLs). A genetic map comprising 239 kompetitive allele-specific PCR (KASP) and 57 cleaved amplified polymorphic sequence markers was constructed. qTN3, a major QTL for tiller number, was identified at 132.4 cm on chromosome 3. This QTL was also detected under field conditions in a backcross population; thus, qTN3 was stable across generations and environments. qTN3 co-located with QTLs associated with panicle number per plant and culm diameter, indicating it had pleiotropic effects. The qTN3 regions of Odae and Unbong40 differed in a known functional variant (4 bp TGTG insertion/deletion) in the 5' UTR of OsTB1, a gene underlying variation in tiller number and culm strength. Investigation of variation in genotype and tiller number revealed that varieties with the insertion genotype had lower tiller numbers than those with the reference genotype. A high-resolution melting marker was developed to enable efficient marker-assisted selection. The QTL qTN3 will therefore be useful in breeding programs developing japonica varieties with optimal tiller numbers for increased yield.

Genome wide association study to detect genetic regions related to isoflavone content in a mutant soybean population derived from radiation breeding.

Isoflavones are major secondary metabolites that are exclusively produced by legumes, including soybean. Soy isoflavones play important roles in human health as well as in the plant defense system. The isoflavone content is influenced by minor-effect quantitative trait loci, which interact with polygenetic and environmental factors. It has been difficult to clarify the regulation of isoflavone biosynthesis because of its complex heritability and the influence of external factors. Here, using a genotype-by-sequencing-based genome-wide association mapping study, 189 mutant soybean genotypes (the mutant diversity pool, MDP) were genotyped on the basis of 25,646 high-quality single nucleotide polymorphisms (SNPs) with minor allele frequency of >0.01 except for missing data. All the accessions were phenotyped by determining the contents of 12 isoflavones in the soybean seeds in two consecutive years (2020 and 2021). Then, quantitative trait nucleotides (QTNs) related to isoflavone contents were identified and validated using multi-locus GWAS models. A total of 112 and 46 QTNs related to isoflavone contents were detected by multiple MLM-based models in 2020 and 2021, respectively. Of these, 12 and 5 QTNs were related to more than two types of isoflavones in 2020 and 2021, respectively. Forty-four QTNs were detected within the 441-Kb physical interval surrounding Gm05:38940662. Of them, four QTNs (Gm05:38936166, Gm05:38936167, Gm05:38940662, and Gm05:38940717) were located at Glyma.05g206900 and Glyma.05g207000, which encode glutathione S-transferase THETA 1 (GmGSTT1), as determined from previous quantitative trait loci annotations and the literature. We detected substantial differences in the transcript levels of GmGSTT1 and two other core genes (IFS1 and IFS2) in the isoflavone biosynthetic pathway between the original cultivar and its mutant. The results of this study provide new information about the factors affecting isoflavone contents in soybean seeds and will be useful for breeding soybean lines with high and stable concentrations of isoflavones.

Identification of Loci Governing Agronomic Traits and Mutation Hotspots via a GBS-Based Genome-Wide Association Study in a Soybean Mutant Diversity Pool.

In this study, we performed a genotyping-by-sequencing analysis and a genome-wide association study of a soybean mutant diversity pool previously constructed by gamma irradiation. A GWAS was conducted to detect significant associations between 37,249 SNPs, 11 agronomic traits, and 6 phytochemical traits. In the merged data set, 66 SNPs on 13 chromosomes were highly associated (FDR p < 0.05) with the following 4 agronomic traits: days of flowering (33 SNPs), flower color (16 SNPs), node number (6 SNPs), and seed coat color (11 SNPs). These results are consistent with the findings of earlier studies on other genetic features (e.g., natural accessions and recombinant inbred lines). Therefore, our observations suggest that the genomic changes in the mutants generated by gamma irradiation occurred at the same loci as the mutations in the natural soybean population. These findings are indicative of the existence of mutation hotspots, or the acceleration of genome evolution in response to high doses of radiation. Moreover, this study demonstrated that the integration of GBS and GWAS to investigate a mutant population derived from gamma irradiation is suitable for dissecting the molecular basis of complex traits in soybeans.

Comparative Study on Phenolic Compounds and Antioxidant Activities of Hop (Humulus lupulus L.) Strobile Extracts.

In this study, we investigated the phenolic compounds in hop strobile extracts and evaluated their antioxidant property using DPPH and ABTS assay. The total phenolic compound (TPC) and total flavonoid compound (TFC) estimated in two different solvent extracts considerably varied depending on the extraction solvent. The most abundant phenolic compound in hop strobile was humulones (α-acid) with levels ranging from 50.44 to 193.25 µg/g. El Dorado accession revealed higher antioxidant activity in ethanol extracts (DPPH: IC50 124.3 µg/mL; ABTS: IC50 95.4 µg/mL) when compared with that of the other accessions. Correlations between DPPH (IC50) scavenging TFC in ethanol extract (TFC_E, -0.941), and TPC_E (-0.901), and between ABTS (IC50) scavenging TFC_E (-0.853), and TPC_E (-0.826), were statistically significant at p < 0.01 level, whereas no significant correlation was observed between antioxidant activities, TPC and TFC in water extract. This study is the first to report that variations in the level of phenolic contents and antioxidant activity of various hop cultivars depended on the type of extraction solvent used and the cultivation regions. These results could provide valuable information on developing hop products.

Unraveling the complexity of faba bean (Vicia faba L.) transcriptome to reveal cold-stress-responsive genes using long-read isoform sequencing technology.

Faba bean (Vicia faba L.), a globally important grain legume providing a stable source of dietary protein, was one of the earliest plant cytogenetic models. However, the lack of draft genome annotations and unclear structural information on mRNA transcripts have impeded its genetic improvement. To address this, we sequenced faba bean leaf transcriptome using the PacBio single-molecule long-read isoform sequencing platform. We identified 28,569 nonredundant unigenes, ranging from 108 to 9669 bp, with a total length of 94.5 Mb. Many unigenes (3597, 12.5%) had 2-20 isoforms, indicating a highly complex transcriptome. Approximately 96.5% of the unigenes matched sequences in public databases. The predicted proteins and transcription factors included NB-ARC, Myb_domain, C3H, bHLH, and heat shock proteins, implying that this genome has an abundance of stress resistance genes. To validate our results, we selected WCOR413-15785, DHN2-12403, DHN2-14197, DHN2-14797, COR15-14478, and HVA22-15 unigenes from the ICE-CBF-COR pathway to analyze their expression patterns in cold-treated samples via qRT-PCR. The expression of dehydrin-related genes was induced by cold stress. The assembled data provide the first insights into the deep sequencing of full-length RNA from faba bean at the single-molecule level. This study provides an important foundation to improve gene modeling and protein prediction.

Analysis of genetic diversity and relationships of Perilla frutescens using novel EST-SSR markers derived from transcriptome between wild-type and mutant Perilla.

BACKGROUND: Perilla frutescens (Lamiaceae) is distributed in East Asia and is classified into var. frutescens and crispa. P. frutescens is multipurpose crop for human health because of a variety of secondary metabolites such as phenolic compound and essential oil. However, a lack of genetic information has hindered the development and utilization of Perilla genotypes. METHODS AND RESULTS: This study was performed to develop expressed sequence tag-simple sequence repeat (EST-SSR) markers from P. frutescens var. crispa (wild type) and Antisperill (a mutant cultivar) and used them to assess the genetic diversity of, and relationships among, 94 P. frutescens genotypes. We obtained 65 Gb of sequence data comprising 632,970 transcripts by de novo RNA-sequencing. Of the 14,780 common SSRs, 102 polymorphic EST-SSRs were selected using in silico polymerase chain reaction (PCR). Overall, successful amplification from 58 EST-SSRs markers revealed remarkable genetic diversity and relationships among 94 P. frutescens genotypes. In total, 268 alleles were identified, with an average of 4.62 alleles per locus (range 2-11 alleles/locus). The average polymorphism information content (PIC) value was 0.50 (range 0.04-0.86). In phylogenetic and population structure analyses, the genotypes formed two major groups: Group I (var. crispa) and Group II (var. frutescens). CONCLUSION: This results suggest that 58 novel EST-SSR markers derived from wild-type cultivar (var. crispa) and its mutant cultivar (Antisperill) have potential uses for population genetics and recombinant inbred line mapping analyses, which will provide comprehensive insights into the genetic diversity and relationship of P. frutescens.

Differential Gene Expression Associated with Altered Isoflavone and Fatty Acid Contents in Soybean Mutant Diversity Pool.

Soybean seeds are consumed worldwide owing to their nutritional value and health benefits. In this study we investigated the metabolic properties of 208 soybean mutant diversity pool (MDP) lines by measuring the isoflavone and fatty acid contents of the seed. The total isoflavone content (TIC) ranged from 0.88 mg/g to 7.12 mg/g and averaged 3.08 mg/g. The proportion of oleic acid among total fatty acids (TFA) ranged from 0.38% to 24.66% and averaged 11.02%. Based on the TIC and TFA among the 208 MDP lines, we selected six lines with altered isoflavone content and six lines with altered oleic acid content compared with those of the corresponding wild-types for measuring gene expression. Each of twelve genes from the isoflavone and fatty acid biosynthesis pathways were analyzed at three different seed developmental stages. Isoflavone biosynthetic genes, including CHI1A, IFS1, and IFS2, showed differences in stages and expression patterns among individuals and wild-types, whereas MaT7 showed consistently higher expression levels in three mutants with increased isoflavone content at stage 1. Expression patterns of the 12 fatty acid biosynthetic genes were classifiable into two groups that reflected the developmental stages of the seeds. The results will be useful for functional analysis of the regulatory genes involved in the isoflavone and fatty acid biosynthetic pathways in soybean.

Measurement of Genetic Mobility Using a Transposon-Based Marker System in Sorghum.

Transposable elements (TEs) are ubiquitous repetitive components of eukaryotic organisms that show mobility in the genome against diverse stresses. TEs contribute considerably to the size, structure, and plasticity of genomes and also play an active role in genome evolution by helping their hosts adapt to novel conditions by conferring useful characteristics. We developed a simple and rapid method for investigation of genetic mobility and diversity among TEs in combination with a target region amplification polymorphism (TE-TRAP) marker system in gamma-irradiated sorghum mutants. The TE-TRAP marker system reveals a high level of genetic diversity, which provides a useful marker resource for genetic mobility research.

Detecting Genetic Mobility Using a Transposon-Based Marker System in Gamma-Ray Irradiated Soybean Mutants.

Transposable elements (TEs)-major components of eukaryotic genomes-have the ability to change location within a genome. Because of their mobility, TEs are important for genome diversification and evolution. Here, a simple rapid method, using the consensus terminal inverted repeat sequences of PONG, miniature inverted-repeat transposable element (MITE)-Tourist (M-t) and MITE-Stowaway (M-s) as target region amplification polymorphism (TE-TRAP) markers, was employed to investigate the mobility of TEs in a gamma-irradiated soybean mutant pool. Among the different TE-TRAP primer combinations, the average polymorphism level and polymorphism information content value were 57.98% and 0.14, respectively. Only the PONG sequence separated the mutant population into three major groups. The inter-mutant population variance, determined using the PONG marker (3.151 and 29%) was greater than that of the M-t (2.209 and 20%) and M-s (2.766 and 18%) markers, whereas the reverse was true for the intra-mutant population variations, with M-t and M-s values, being 15.151 (82%) and 8.895 (80%), respectively, compared with the PONG marker (7.646 and 71%). Thus, the MITE markers revealed more dynamic and active mobility levels than the PONG marker in gamma-ray irradiated soybean mutant lines. The TE-TRAP technique associated with sensitive MITEs is useful for investigating genetic diversity and TE mobilization, providing tools for mutant selection in soybean mutation breeding.

Effects of UV-A radiation on organ-specific accumulation and gene expression of isoflavones and flavonols in soybean sprout.

Organ-specific flavonoid destination in soybean sprouts following UV irradiation is still unclear although the metabolic pathway of flavonoid synthesis and UV responded flavonoid accumulation have been well investigated. We report the identification of organ-specific localization and specific gene expression of isoflavones and kaempferol glycosides in the soybean sprouts responded to UV-A irradiation. UV-A irradiation stimulated only root isoflavones, especially increase of genistein types. The daidzein types predominated in non-UV-A treated roots. Kaempferol glycosides were not increased in roots by UV-A, but distinctly increased in aerial organs, especially in the cotyledons. These results demonstrate that UV-A upregulates the naringenin pathway synthesizing genistin and kaempferol rather than the liquiritigenin pathway synthesizing daidzin and glycitin. High GmUGT9 and other gene expression related to isoflavone synthesis in roots clearly demonstrate the UV-A-induced isoflavone accumulation. Aerial organ specific increase of GmF3H, GmFLS1, and GmDFR1 expression by UV-A distinctly demonstrates the flavonol increase in aerial organs.

Complete chloroplast genome sequence of an orchid hybrid Cymbidium sinense (♀) × C. goeringii (♂).

The complete chloroplast genome sequence of the Cymbidium hybrid, C. sinense (♀) × C. goeringii (♂) was assembled in this study. The circular genome was 150,149 bp in length with an overall GC content of 37.1% and consisted of a pair of 25,691 bp inverted repeats, and two single-copy regions that were 84,987 bp and 13,780 bp, respectively. Gene annotation analysis identified 109 genes including 75 protein-coding genes, 30 transfer RNA, and 4 ribosomal RNA genes. Phylogenetic analysis showed its closest relationship to Cymbidium sinense, reflecting a maternal inheritance of chloroplasts.

Comparative Analysis of Volatile Compounds of Gamma-Irradiated Mutants of Rose (Rosa hybrida).

Roses are one of the most important floricultural crops, and their essential oils have long been used for cosmetics and aromatherapy. We investigated the volatile compound compositions of 12 flower-color mutant variants and their original cultivars. Twelve rose mutant genotypes were developed by treatment with 70 Gy of 60Co gamma irradiation of six commercial rose cultivars. Essential oils from the flowers of the 18 genotypes were analyzed by gas chromatography-mass spectrometry. Seventy-seven volatile compounds were detected, which were categorized into six classes: Aliphatic hydrocarbons, aliphatic alcohols, aliphatic ester, aromatic compounds, terpene alcohols, and others. Aliphatic (hydrocarbons, alcohols, and esters) compounds were abundant categories in all rose flowers. The CR-S2 mutant had the highest terpene alcohols and oil content. Three (CR-S1, CR-S3, and CR-S4) mutant genotypes showed higher ester contents than their original cultivar. Nonacosane, 2-methylhexacosane, and 2-methyltricosane were major volatile compounds among all genotypes. Hierarchical cluster analysis (HCA) of the rose genotypes gave four groups according to grouping among the 77 volatile compounds. In addition, the principal component analysis (PCA) model was successfully applied to distinguish most attractive rose lines. These findings will be useful for the selection of rose genotypes with improved volatile compounds.

Phenolic Compounds in Extracts of Hibiscus acetosella (Cranberry Hibiscus) and Their Antioxidant and Antibacterial Properties.

Hibiscus species are rich in phenolic compounds and have been traditionally used for improving human health through their bioactive activities. The present study investigated the phenolic compounds of leaf extracts from 18 different H. acetosella accessions and evaluated their biofunctional properties, focusing on antioxidant and antibacterial activity. The most abundant phenolic compound in H. acetosella was caffeic acid, with levels ranging from 14.95 to 42.93 mg/100 g. The antioxidant activity measured by the ABTS assay allowed the accessions to be classified into two groups: a high activity group with red leaf varieties (74.71-84.02%) and a relatively low activity group with green leaf varieties (57.47-65.94%). The antioxidant activity was significantly correlated with TAC (0.933), Dp3-Sam (0.932), Dp3-Glu (0.924), and Cy3-Sam (0.913) contents (p < 0.001). The H. acetosella phenolic extracts exhibited antibacterial activity against two bacteria, with zones of inhibition between 12.00 and 13.67 mm (Staphylococcus aureus), and 10.67 and 13.33 mm (Pseudomonas aeruginosa). All accessions exhibited a basal antibacterial activity level (12 mm) against the Gram-positive S. aureus, with PI500758 and PI500764 exhibiting increased antibacterial activity (13.67 mm), but they exhibited a more dynamic antibacterial activity level against the Gram-negative P. aeruginosa.

Characterization of Gene Isoforms Related to Cellulose and Lignin Biosynthesis in Kenaf (Hibiscus cannabinus L.) Mutant.

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier flowering than the wild type. We sequenced and analyzed the transcriptome of apical leaf and stem using Pacific Biosciences single-molecule long-read isoform sequencing platform. De novo assembly yielded 26,822 full-length transcripts with a total length of 59 Mbp. Sequence similarity against protein sequence allowed the functional annotation of 11,370 unigenes. Among them, 10,100 unigenes were assigned gene ontology terms, the majority of which were associated with the metabolic and cellular process. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 8875 of the annotated unigenes to 149 metabolic pathways. We also identified the majority of putative genes involved in cellulose and lignin-biosynthesis. We further evaluated the expression pattern in eight gene families involved in lignin-biosynthesis at different growth stages. In this study, appropriate biotechnological approaches using the information obtained for these putative genes will help to modify the desirable content traits in mutants. The transcriptome data can be used as a reference dataset and provide a resource for molecular genetic studies in kenaf.

ATM suppresses leaf senescence triggered by DNA double-strand break through epigenetic control of senescence-associated genes in Arabidopsis.

All living organisms are unavoidably exposed to various endogenous and environmental stresses that trigger potentially fatal DNA damage, including double-strand breaks (DSBs). Although a growing body of evidence indicates that DNA damage is one of the prime drivers of aging in animals, little is known regarding the importance of DNA damage and its repair on lifespan control in plants. We found that the level of DSBs increases but DNA repair efficiency decreases as Arabidopsis leaves age. Generation of DSBs by inducible expression of I-PpoI leads to premature senescence phenotypes. We examined the senescence phenotypes in the loss-of-function mutants for 13 key components of the DNA repair pathway and found that deficiency in ATAXIA TELANGIECTASIA MUTATED (ATM), the chief transducer of the DSB signal, results in premature senescence in Arabidopsis. ATM represses DSB-induced expression of senescence-associated genes, including the genes encoding the WRKY and NAC transcription factors, central components of the leaf senescence process, via modulation of histone lysine methylation. Our work highlights the significance of ATM in the control of leaf senescence and has significant implications for the conservation of aging mechanisms in animals and plants.

Transcriptome analysis to identify candidate genes associated with the yellow-leaf phenotype of a Cymbidium mutant generated by γ-irradiation.

Leaf color is an important agronomic trait in flowering plants, including orchids. However, factors underlying leaf phenotypes in plants remain largely unclear. A mutant displaying yellow leaves was obtained by the γ-ray-based mutagenesis of a Cymbidium orchid and characterized using RNA sequencing. A total of 144,918 unigenes obtained from over 25 million reads were assigned to 22 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes database. In addition, gene ontology was used to classify the predicted functions of transcripts into 73 functional groups. The RNA sequencing analysis identified 2,267 differentially expressed genes between wild-type and mutant Cymbidium sp. Genes involved in the chlorophyll biosynthesis and degradation, as well as ion transport, were identified and assayed for their expression levels in wild-type and mutant plants using quantitative real-time profiling. No critical expression changes were detected in genes involved in chlorophyll biosynthesis. In contrast, seven genes involved in ion transport, including two metal ion transporters, were down-regulated, and chlorophyllase 2, associated with chlorophyll degradation, was up-regulated. Together, these results suggest that alterations in chlorophyll metabolism and/or ion transport might contribute to leaf color in Cymbidium orchids.

Selection of mutants with high linolenic acid contents and characterization of fatty acid desaturase 2 and 3 genes during seed development in soybean (Glycine max).

BACKGROUND: Soybean seeds contain 18-24% lipids, which are made up of 85% polyunsaturated fatty acids. Two of these (linoleic and linolenic acids) comprise essential fatty acids that are not synthesized in humans and animals. Linolenic acid plays a vital role in the maintenance of brain function and is a source of docosahexaenoic acid for retinal and nerve tissue, with its physiological functions being a focus of attention. RESULTS: We developed mutant soybean populations via gamma irradiation of Korean cultivars Danbaek and Daepung and evaluated the linolenic acid content of 78 and 154 M9 mutant progenies. We selected the four mutant lines with the highest linolenic acid contents based on 2 years of investigation of fatty acids. The selected mutant lines had linolenic acid contents that were 33.9% to 67.7% higher than those of the original cultivars and exhibited increased fatty acid desaturase (FAD) gene expression levels during seed development. We also identified nucleotide polymorphisms of FAD genes in the four mutant lines. CONCLUSION: The present study found that linolenic acid content is related to significantly increased expression levels of the FAD3C and FAD3D genes in the endoplasmic reticulum, which was uncovered by radiation mutation breeding of soybean. © 2019 Society of Chemical Industry.

Utility of TRAP markers to determine indel mutation frequencies induced by gamma-ray irradiation of faba bean (Vicia faba L.) seeds.

Purpose: The aims were to determine the optimal gamma ray dose for faba bean (Vicia faba) and to identify genetic variation and indel mutation frequency among mutants using target region amplification polymorphism (TRAP) markers. Materials and methods: Seeds from 10 elite lines were irradiated with gamma rays (50-700 Gy), and germination, survival rate, and representative morphological traits were measured. The extent of DNA damage was investigated using comet assays, and TRAP markers were used to evaluate genetic variation, genetic diversity, and mutation frequencies. Results: Germination percentages significantly decreased at doses >100 Gy. Survival percentages and morphological traits decreased with elevation in dose. The comet assays revealed that high irradiation doses decreased head DNA levels. Phylogenetic and principal component analysis of 555 individual faba bean plants resolved eight major groups. Genetic variation between controls and mutants was limited to within groups. Mutation frequencies were associated with gamma dose in each mutant line. Conclusions: The optimal gamma dose was 100-150 Gy on the basis of survival percentage and morphological response analysis. The TRAP markers distinguished mutant lines and showed association between mutation frequency and gamma doses. This study will be useful for faba bean mutation breeding and may be applicable to other crops.