Comparative transcriptome analysis of vegetable soybean grain discloses genes essential for grain quality.

BACKGROUND: Vegetable soybean is an important vegetable crop in world. Seed size and soluble sugar content are considered crucial indicators of quality in vegetable soybean, and there is a lack of clarity on the molecular basis of grain quality in vegetable soybean. RESULTS: In this context, we performed a comprehensive comparative transcriptome analysis of seeds between a high-sucrose content and large-grain variety (Zhenong 6, ZN6) and a low-sucrose content and small-grain variety (Williams 82, W82) at three developmental stages, i.e. stage R5 (Beginning Seed), stage R6 (Full Seed), and stage R7 (Beginning Maturity). The transcriptome analysis showed that 17,107 and 13,571 differentially expressed genes (DEGs) were identified in ZN6 at R6 (vs. R5) and R7 (vs. R6), respectively, whereas 16,203 and 16,032 were detected in W82. Gene expression pattern and DEGs functional enrichment proposed genotype-specific biological processes during seed development. The genes participating in soluble sugar biosynthesis such as FKGP were overexpressed in ZN6, whereas those responsible for lipid and protein metabolism such as ALDH3 were more enhanced in W82, exhibiting different dry material accumulation between two genotypes. Furthermore, hormone-associated transcriptional factors involved in seed size regulation such as BEH4 were overrepresented in ZN6, exhibiting different seed size regulation processes between two genotypes. CONCLUSIONS: Herein, we not only discovered the differential expression of genes encoding metabolic enzymes involved in seed composition, but also identified a type of hormone-associated transcriptional factors overexpressed in ZN6, which may regulate seed size and soluble content. This study provides new insights into the underlying causes of differences in the soybean metabolites and appearance, and suggests that genetic data can be used to improve its appearance and textural quality.

ResNet incorporating the fusion data of RGB & hyperspectral images improves classification accuracy of vegetable soybean freshness.

The freshness of vegetable soybean (VS) is an important indicator for quality evaluation. Currently, deep learning-based image recognition technology provides a fast, efficient, and low-cost method for analyzing the freshness of food. The RGB (red, green, and blue) image recognition technology is widely used in the study of food appearance evaluation. In addition, the hyperspectral image has outstanding performance in predicting the nutrient content of samples. However, there are few reports on the research of classification models based on the fusion data of these two sources of images. We collected RGB and hyperspectral images at four different storage times of VS. The ENVI software was adopted to extract the hyperspectral information, and the RGB images were reconstructed based on the downsampling technology. Then, the one-dimensional hyperspectral data was transformed into a two-dimensional space, which allows it to be overlaid and concatenated with the RGB image data in the channel direction, thereby generating fused data. Compared with four commonly used machine learning models, the deep learning model ResNet18 has higher classification accuracy and computational efficiency. Based on the above results, a novel classification model named ResNet-R &H, which is based on the residual networks (ResNet) structure and incorporates the fusion data of RGB and hyperspectral images, was proposed. The ResNet-R &H can achieve a testing accuracy of 97.6%, which demonstrates a significant enhancement of 4.0% and 7.2% compared to the distinct utilization of hyperspectral data and RGB data, respectively. Overall, this research is significant in providing a unique, efficient, and more accurate classification approach in evaluating the freshness of vegetable soybean. The method proposed in this study can provide a theoretical reference for classifying the freshness of fruits and vegetables to improve classification accuracy and reduce human error and variability.

Genome and Transcriptome Analysis of Ascochyta pisi Provides Insights into the Pathogenesis of Ascochyta Blight of Pea.

Ascochyta blight caused by Ascochyta pisi is a major constraint to pea (Pisum sativum L.) production worldwide. Deciphering the pathogenic mechanism of A. pisi on peas will help in breeding resistant pea varieties and developing effective approaches for disease management. However, little is known about the genomic features and pathogenic factors of A. pisi. In this study, we first report that A. pisi is one of the causal agents of ascochyta blight disease of pea in China. The genome of the representative isolate A. pisi HNA23 was sequenced using PacBio and Illumina sequencing technologies. The HNA23 genome assembly is almost 41.5 Mb in size and harbors 10,796 putative protein-encoding genes. We predicted 555 carbohydrate-active enzymes (CAZymes), 1,008 secreted proteins, 74 small secreted cysteine-rich proteins (SSCPs), and 26 secondary metabolite biosynthetic gene clusters (SMGCs). A comparison of A. pisi genome features with the features of 6 other available genomes of Ascochyta species showed that CAZymes, the secretome, and SMGCs of this genus are considerably conserved. Importantly, the transcriptomes of HNA23 during infection of peas at three stages were further analyzed. We found that 245 CAZymes and 29 SSCPs were upregulated at all three tested infection stages. SMGCs were also trigged, but most of them were induced at only one stage of infection. Together, our results provide important genomic information on Ascochyta spp. and offer insights into the pathogenesis of A. pisi. IMPORTANCE Ascochyta blight is a major disease of legumes worldwide. Ascochyta pisi and other Ascochyta species have been identified as pathogens of ascochyta blight. Here, we first report that A. pisi causes ascochyta blight of pea in China, and we report the high-quality, fully annotated genome of A. pisi. Comparative genome analysis was performed to elucidate the differences and similarities among 7 Ascochyta species. We predict abundant CAZymes (569 per species), secreted proteins (851 per species), and prolific secondary metabolite gene clusters (29 per species) in these species. We identified a set of genes that may be responsible for fungal virulence based on transcriptomes in planta, including CAZymes, SSCPs, and secondary metabolites. The findings from the comparative genome analysis highlight the genetic diversity and help in understanding the evolutionary relationship of Ascochyta species. In planta transcriptome analysis provides reliable information for further investigation of the mechanism of the interaction between Ascochyta spp. and legumes.

Promoter of Vegetable Soybean GmTIP1;6 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

Tonoplast intrinsic proteins (TIPs), a sub-family of aquaporins (AQPs), are known to play important roles in plant abiotic stress responses. However, evidence for the promoters of TIPs involvement in abiotic stress processes remains scarce. In this study, the promoter of the vegetable soybean GmTIP1;6 gene, which had the highest similarity to TIP1-type AQPs from other plants, was cloned. Expression pattern analyses indicated that the GmTIP1;6 gene was dramatically induced by drought, salt, abscisic acid (ABA), and methyl jasmonate (MeJA) stimuli. Promoter analyses revealed that the GmTIP1;6 promoter contained drought, ABA, and MeJA cis-acting elements. Histochemical staining of the GmTIP1;6 promoter in transgenic Arabidopsis corroborated that it was strongly expressed in the vascular bundles of leaves, stems, and roots. Beta-glucuronidase (GUS) activity assays showed that the activities of the GmTIP1;6 promoter were enhanced by different concentrations of polyethylene glycol 6000 (PEG 6000), NaCl, ABA, and MEJA treatments. Integrating these results revealed that the GmTIP1;6 promoter could be applied for improving the tolerance to abiotic stresses of the transgenic plants by promoting the expression of vegetable soybean AQPs.

Genome sequencing and population resequencing provide insights into the genetic basis of domestication and diversity of vegetable soybean.

Vegetable soybean is one of the most important vegetables in China, and the demand for this vegetable has markedly increased worldwide over the past two decades. Here, we present a high-quality de novo genome assembly of the vegetable soybean cultivar Zhenong 6 (ZN6), which is one of the most popular cultivars in China. The 20 pseudochromosomes cover 94.57% of the total 1.01 Gb assembly size, with contig N50 of 3.84 Mb and scaffold N50 of 48.41 Mb. A total of 55 517 protein-coding genes were annotated. Approximately 54.85% of the assembled genome was annotated as repetitive sequences, with the most abundant long terminal repeat transposable elements. Comparative genomic and phylogenetic analyses with grain soybean Williams 82, six other Fabaceae species and Arabidopsis thaliana genomes highlight the difference of ZN6 with other species. Furthermore, we resequenced 60 vegetable soybean accessions. Alongside 103 previously resequenced wild soybean and 155 previously resequenced grain soybean accessions, we performed analyses of population structure and selective sweep of vegetable, grain, and wild soybean. They were clearly divided into three clades. We found 1112 and 1047 genes under selection in the vegetable soybean and grain soybean populations compared with the wild soybean population, respectively. Among them, we identified 134 selected genes shared between vegetable soybean and grain soybean populations. Additionally, we report four sucrose synthase genes, one sucrose-phosphate synthase gene, and four sugar transport genes as candidate genes related to important traits such as seed sweetness and seed size in vegetable soybean. This study provides essential genomic resources to promote evolutionary and functional genomics studies and genomically informed breeding for vegetable soybean.

Investigation of the AQP Family in Soybean and the Promoter Activity of TIP2;6 in Heat Stress and Hormone Responses.

Aquaporins (AQPs) are one diverse family of membrane channel proteins that play crucial regulatory roles in plant stress physiology. However, the heat stress responsiveness of AQP genes in soybean remains poorly understood. In this study, 75 non-redundant AQP encoding genes were identified in soybean. Multiple sequence alignments showed that all GmAQP proteins possessed the conserved regions, which contained 6 trans-membrane domains (TM1 to TM6). Different GmAQP members consisted of distinct Asn-Pro-Ala (NPA) motifs, aromatic/arginine (ar/R) selectivity filters and Froger's positions (FPs). Phylogenetic analyses distinguished five sub-families within these GmAQPs: 24 GmPIPs, 24 GmTIPs, 17 GmNIPs, 8 GmSIPs, and 2 GmXIPs. Promoter cis-acting elements analyses revealed that distinct number and composition of heat stress and hormone responsive elements existed in different promoter regions of GmAQPs. QRT-PCR assays demonstrated that 12 candidate GmAQPs with relatively extensive expression in various tissues or high expression levels in root or leaf exhibited different expression changes under heat stress and hormone cues (abscisic acid (ABA), l-aminocyclopropane-l-carboxylic acid (ACC), salicylic acid (SA) and methyl jasmonate (MeJA)). Furthermore, the promoter activity of one previously functionally unknown AQP gene-GmTIP2;6 was investigated in transgenic Arabidopsis plants. The beta-glucuronidase (GUS) activity driven by the promoter of GmTIP2;6 was strongly induced in the heat- and ACC-treated transgenic plants and tended to be accumulated in the hypocotyls, vascular bundles, and leaf trichomes. These results will contribute to uncovering the potential functions and molecular mechanisms of soybean GmAQPs in mediating heat stress and hormone signal responses.

QTL Mapping Combined With Bulked Segregant Analysis Identify SNP Markers Linked to Leaf Shape Traits in Pisum sativum Using SLAF Sequencing.

Leaf shape is an important trait that influences the utilization rate of light, and affects quality and yield of pea (Pisum sativum). In the present study, a joint method of high-density genetic mapping using specific locus amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) was applied to rapidly detect loci with leaf shape traits. A total of 7,146 polymorphic SLAFs containing 12,213 SNP markers were employed to construct a high-density genetic map for pea. We conducted quantitative trait locus (QTL) mapping on an F2 population to identify QTLs associated with leaf shape traits. Moreover, SLAF-BSA was conducted on the same F2 population to identify the single nucleotide polymorphism (SNP) markers linked to leaf shape in pea. Two QTLs (qLeaf_or-1, qLeaf_or-2) were mapped on linkage group 7 (LG7) for pea leaf shape. Through alignment of SLAF markers with Cicer arietinum, Medicago truncatula, and Glycine max, the pea LGs were assigned to their corresponding homologous chromosomal groups. The comparative genetic analysis showed that pea is more closely related to M. truncatula. Based on the sequencing results of two pools with different leaf shape, 179 associated markers were obtained after association analysis. The joint analysis of SLAF-seq and BSA showed that the QTLs obtained from mapping on a high-density genetic map are convincing due to the closely associated map region with the BSA results, which provided more potential markers related to leaf shape. Thus, the identified QTLs could be used in marker-assisted selection for pea breeding in the future. Our study revealed that joint analysis of QTL mapping on a high-density genetic map and BSA-seq is a cost-effective and accurate method to reveal genetic architecture of target traits in plant species without a reference genome.

Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness.

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, a family of plant-specific proteins, play crucial roles in plant growth and development and stress response. However, systematical information is unknown regarding the TCP gene family in soybean. In the present study, a total of 54 GmTCPs were identified in soybean, which were grouped into 11 groups with the typical TCP conserved domains. Phylogenetic relationship, protein motif and gene structure analyses distinguished the GmTCPs into two homology classes: Class I and Class II. Class II was then differentiated into two subclasses: CIN and CYC/TB1. Unique cis-element number and composition existed in the promoter regions which might be involved in the gene transcriptional regulation of different GmTCPs. Tissue expression analysis demonstrated the diverse spatiotemporal expression profiles of GmTCPs. Furthermore, the interaction protein of one previously functionally unknown TCP protein-GmTCP8 was investigated. Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10). QRT-PCR assays indicated the distinct expression profiles of GmTCPs in response to abiotic stresses (heat, drought and salt) and stress-related signals (abscisic acid, brassinolide, salicylicacid and methyl jasmonate). These results will facilitate to uncover the possible roles of GmTCPs under abiotic stress and hormone signal responses in soybean.

Identification of the AQP members involved in abiotic stress responses from Arabidopsis.

Aquaporins (AQPs) constitute a highly diverse family of water channel proteins that play crucial biological functions in plant growth and development and stress physiology. In Arabidopsis, 35 AQPs are classified into four subfamilies (PIPs, TIPs, NIPs and SIPs). However, knowledge about the roles of different subfamily AQPs remains limited. Here, we explored the chromosomal location, gene structure and expression patterns of all AQPs in different tissues or under different abiotic stresses based on available microarray data. Tissue expression analysis showed that different AQPs had various expression patterns in tissues (root, leaf, flower and seed). Expression profiles under stress conditions revealed that most AQPs were responsive to osmotic, salt and drought stresses. Phenotypic and physiological identification showed that Tip2;2 loss-of-function mutant exhibited less sensitive to abiotic stresses (mannitol, NaCl and PEG) compared with wild-type, as evident by analysis of germination rate, root growth, survival rate, ion leakage, malondialdehyde (MDA) and proline contents. Mutant of TIP2;2 modulated the transcript levels of SOS1, SOS2, SOS3, DREB1A, DREB2A and P5CS1, under abiotic stress conditions. This study provides a basis for further functional identification of stress-related candidate AQPs in Arabidopsis.

Studies on the Control of Ascochyta Blight in Field Peas (Pisum sativum L.) Caused by Ascochyta pinodes in Zhejiang Province, China.

Ascochyta blight, an infection caused by a complex of Ascochyta pinodes, Ascochyta pinodella, Ascochyta pisi, and/or Phoma koolunga, is a destructive disease in many field peas (Pisum sativum L.)-growing regions, and it causes significant losses in grain yield. To understand the composition of fungi associated with this disease in Zhejiang Province, China, a total of 65 single-pycnidiospore fungal isolates were obtained from diseased pea samples collected from 5 locations in this region. These isolates were identified as Ascochyta pinodes by molecular techniques and their morphological and physiological characteristics. The mycelia of ZJ-1 could penetrate pea leaves across the stomas, and formed specific penetration structures and directly pierced leaves. The resistance level of 23 available pea cultivars was tested against their representative isolate A. pinodes ZJ-1 using the excised leaf-assay technique. The ZJ-1 mycelia could penetrate the leaves of all tested cultivars, and they developed typical symptoms, which suggested that all tested cultivars were susceptible to the fungus. Chemical fungicides and biological control agents were screened for management of this disease, and their efficacies were further determined. Most of the tested fungicides (11 out of 14) showed high activity toward ZJ-1 with EC50 < 5 µg/mL. Moreover, fungicides, including tebuconazole, boscalid, iprodione, carbendazim, and fludioxonil, displayed more than 80% disease control efficacy under the recorded conditions. Three biocontrol strains of Bacillus sp. and one of Pantoea agglomerans were isolated from pea-related niches and significantly reduced the severity of disease under greenhouse and field conditions. To our knowledge, this is the first study on ascochyta blight in field peas, and results presented here will be useful for controlling the disease in this area.

Identification of chilling-responsive microRNAs and their targets in vegetable soybean (Glycine max L.).

Chilling stress is a major factor limiting the yield and quality of vegetable soybean (Glycine max L.) on a global scale. In the present study, systematic identification and functional analysis of miRNAs under chilling stress were carried out to clarify the molecular mechanism of chilling resistance. Two independent small RNA libraries from leaves of soybean were constructed and sequenced with the high-throughput Illumina Solexa system. A total of 434 known miRNAs and 3 novel miRNAs were identified. Thirty-five miRNAs were verified by qRT-PCR analysis. Furthermore, their gene targets were identified via high-throughput degradome sequencing. A total of 898 transcripts were targeted by 54 miRNA families attributed to five categories. More importantly, we identified 51 miRNAs differentially expressed between chilling stress and control conditions. The targets of these miRNAs were enriched in oxidation-reduction, signal transduction, and metabolic process functional categories. Our qRT-PCR analysis confirmed a negative relationship among the miRNAs and their targets under chilling stress. Our work thus provides comprehensive molecular evidence supporting the involvement of miRNAs in chilling-stress responses in vegetable soybean.

Comparative Transcriptomic Analyses of Vegetable and Grain Pea (Pisum sativum L.) Seed Development.

Understanding the molecular mechanisms regulating pea seed developmental process is extremely important for pea breeding. In this study, we used high-throughput RNA-Seq and bioinformatics analyses to examine the changes in gene expression during seed development in vegetable pea and grain pea, and compare the gene expression profiles of these two pea types. RNA-Seq generated 18.7 G of raw data, which were then de novo assembled into 77,273 unigenes with a mean length of 930 bp. Our results illustrate that transcriptional control during pea seed development is a highly coordinated process. There were 459 and 801 genes differentially expressed at early and late seed maturation stages between vegetable pea and grain pea, respectively. Soluble sugar and starch metabolism related genes were significantly activated during the development of pea seeds coinciding with the onset of accumulation of sugar and starch in the seeds. A comparative analysis of genes involved in sugar and starch biosynthesis in vegetable pea (high seed soluble sugar and low starch) and grain pea (high seed starch and low soluble sugar) revealed that differential expression of related genes at late development stages results in a negative correlation between soluble sugar and starch biosynthetic flux in vegetable and grain pea seeds. RNA-Seq data was validated by using real-time quantitative RT-PCR analysis for 30 randomly selected genes. To our knowledge, this work represents the first report of seed development transcriptomics in pea. The obtained results provide a foundation to support future efforts to unravel the underlying mechanisms that control the developmental biology of pea seeds, and serve as a valuable resource for improving pea breeding.

Foxtail Millet NF-Y Families: Genome-Wide Survey and Evolution Analyses Identified Two Functional Genes Important in Abiotic Stresses.

It was reported that Nuclear Factor Y (NF-Y) genes were involved in abiotic stress in plants. Foxtail millet (Setaria italica), an elite stress tolerant crop, provided an impetus for the investigation of the NF-Y families in abiotic responses. In the present study, a total of 39 NF-Y genes were identified in foxtail millet. Synteny analyses suggested that foxtail millet NF-Y genes had experienced rapid expansion and strong purifying selection during the process of plant evolution. De novo transcriptome assembly of foxtail millet revealed 11 drought up-regulated NF-Y genes. SiNF-YA1 and SiNF-YB8 were highly activated in leaves and/or roots by drought and salt stresses. Abscisic acid (ABA) and H2O2 played positive roles in the induction of SiNF-YA1 and SiNF-YB8 under stress treatments. Transient luciferase (LUC) expression assays revealed that SiNF-YA1 and SiNF-YB8 could activate the LUC gene driven by the tobacco (Nicotiana tobacam) NtERD10, NtLEA5, NtCAT, NtSOD, or NtPOD promoter under normal or stress conditions. Overexpression of SiNF-YA1 enhanced drought and salt tolerance by activating stress-related genes NtERD10 and NtCAT1 and by maintaining relatively stable relative water content (RWC) and contents of chlorophyll, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in transgenic lines under stresses. SiNF-YB8 regulated expression of NtSOD, NtPOD, NtLEA5, and NtERD10 and conferred relatively high RWC and chlorophyll contents and low MDA content, resulting in drought and osmotic tolerance in transgenic lines under stresses. Therefore, SiNF-YA1 and SiNF-YB8 could activate stress-related genes and improve physiological traits, resulting in tolerance to abiotic stresses in plants. All these results will facilitate functional characterization of foxtail millet NF-Ys in future studies.

Determination of the genetic diversity of vegetable soybean [Glycine max (L.) Merr.] using EST-SSR markers.

The development of expressed sequence tag-derived simple sequence repeats (EST-SSRs) provided a useful tool for investigating plant genetic diversity. In the present study, 22 polymorphic EST-SSRs from grain soybean were identified and used to assess the genetic diversity in 48 vegetable soybean accessions. Among the 22 EST-SSR loci, tri-nucleotides were the most abundant repeats, accounting for 50.00% of the total motifs. GAA was the most common motif among tri-nucleotide repeats, with a frequency of 18.18%. Polymorphic analysis identified a total of 71 alleles, with an average of 3.23 per locus. The polymorphism information content (PIC) values ranged from 0.144 to 0.630, with a mean of 0.386. Observed heterozygosity (Ho) values varied from 0.0196 to 1.0000, with an average of 0.6092, while the expected heterozygosity (He) values ranged from 0.1502 to 0.6840, with a mean value of 0.4616. Principal coordinate analysis and phylogenetic tree analysis indicated that the accessions could be assigned to different groups based to a large extent on their geographic distribution, and most accessions from China were clustered into the same groups. These results suggest that Chinese vegetable soybean accessions have a narrow genetic base. The results of this study indicate that EST-SSRs from grain soybean have high transferability to vegetable soybean, and that these new markers would be helpful in taxonomy, molecular breeding, and comparative mapping studies of vegetable soybean in the future.

Development and characterization of 41 novel EST-SSR markers for Pisum sativum (Leguminosae).

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers were developed in Pisum sativum for further use in genetic studies and breeding programs. METHODS AND RESULTS: Forty-one novel EST-SSR primers were developed and characterized for size polymorphism in 32 Pisum sativum individuals from four populations from China. In each population, the number of alleles per locus ranged from one to seven, with observed heterozygosity and expected heterozygosity ranging from 0 to 0.8889 and 0 to 0.8400, respectively. Furthermore, 53.7% of these markers could be transferred to the related species, Vicia faba. CONCLUSIONS: The developed markers have potential for application in the study of genetic diversity, germplasm appraisal, and marker-assisted breeding in pea and other legume species.

Developing new SSR markers from ESTs of pea (Pisum sativum L.).

The development of expressed sequence tags (ESTs) from pea has provided a useful source for mining novel simple sequence repeat (SSR) markers. In the present research, in order to find EST-derived SSR markers, 18 552 pea ESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and assembled into 10 086 unigenes. A total of 586 microsatellites in 530 unigenes were identified, indicating that merely 5.25% of sequences contained SSRs. The most abundant SSRs within pea were tri-nucleotide repeat motifs, and among all the tri-nucleotide repeats, the motif GAA was the most abundant type. In total, 49 SSRs were used for primer design. EST-SSR loci were subsequently screened on 10 widely adapted varieties in China. Of these, nine loci showed polymorphic profiles that revealed two to three alleles per locus. The polymorphism information content value ranged from 0.18 to 0.58 with an average of 0.41. Furthermore, transferable analysis revealed that some of these loci showed transferability to faba bean. Because of their polymorphism and transferability, these nine novel EST-SSRs will be valuable tools for marker-assisted breeding and comparative mapping of pea in the future.

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. • METHODS AND RESULTS: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. • CONCLUSIONS: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.