Browsing by Author "Yussif, Toufiq Soale"
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- Identification of Chestnut Hybrids Using SSR Markers and Bioinformatic ToolsPublication . Yussif, Toufiq Soale; Choupina, Altino; Gouveia, Maria EugéniaChestnut trees serve as a vital resource for food and timber production, yet invasive pests and diseases increasingly threaten their survival and productivity. In Europe, hybridization programs have been launched to develop resilient rootstocks, such as the ink disease-resistant CA90 hybrid (Castanea sativa × Castanea crenata), to combat pathogens like Phytophthora cinnamomi. However, distinguishing these hybrids from conventional varieties remains challenging, as current methods rely heavily on field observations and morphological traits. To address this gap, this study identified molecular markers using Simple Sequence Repeats (SSR) microsatellite markers and bioinformatics to differentiate CA90 hybrids from other chestnut varieties. We analyzed 35 chestnut samples, including three CA90 controls, hybrids, and non-hybrid plants from Portugal, to establish genetic profiles based on SSR band patterns and motif variations. From 43 existing SSR markers, nine primers with null allelic features and low observed heterozygosity (Ho) were selected and modified for this study. Polymerase Chain Reaction (PCR) amplification and agarose gel electrophoresis were employed to visualize Deoxyribonucleic Acid. (DNA) bands, followed by Sanger sequencing of 27 amplified products to confirm genetic variations. This approach identified 31 SSRs across 22 sequences, with trinucleotide repeats dominating (67.74%), followed by dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%) motifs. Across the nine loci, 18 alleles were detected, ranging from one to three alleles per locus among the samples. Crucially, the CP4 locus emerged as a novel, hybrid-specific marker, exclusively present in CA90 samples, and should be combined with primer loci CP2, CP6, CP9, and CP10 during identification on gel for cost-effectiveness. These SSR-based markers offer a reliable, cost-efficient solution to identify disease-resistant CA90 chestnut hybrids, surpassing traditional morphological methods. By enabling precise selection of resilient rootstocks, they enhance sustainable cultivation and pathogen management. These tools can optimize breeding programs, boost orchard productivity, and protect genetic diversity, proving essential for mitigating pest and disease threats while ensuring the long-term health of chestnut ecosystems.
- A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSRPublication . Yussif, Toufiq Soale; Cruz, Nadine Evora da; Coelho, Valentim; Gouveia, Maria Eugénia; Choupina, AltinoChestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and Morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.
- A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSRPublication . Yussif, Toufiq Soale; Cruz, Nadine Evora da; Coelho, Valentim; Gouveia, Maria Eugénia; Choupina, Altino BrancoChestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.