Anthracnose is a devastating fungal disease of common bean (Phaseolus vulgaris L.), the causal ag... more Anthracnose is a devastating fungal disease of common bean (Phaseolus vulgaris L.), the causal agent of which is Colletotrichum lindemuthianum. MSU 7-1 is a common bean breeding line that contains the Co-52 and Co-7 anthracnose resistance genes. In this study, phenotypic analyses were conducted in an F2 population derived from the Mexico 222 (S) x MSU 7-1 (R) cross that was inoculated with race 64 of C. lindemuthianum. Segregation analysis revealed a 3R:1S ratio, indicating that only Co-52 confers resistance to race 64 once the Co-7 gene has been defeated by this race. The molecular marker g12333250, in linkage group Pv07, was linked in coupling phase to Co-52 at a distance of 1.2 cM. The presence or absence of this marker was also determined in the G 2333, TU, H1 and PI 207262 cultivars. It was found that the g12333250 molecular marker was present in G 2333 and absent in the other cultivars. Because the allele and marker are physically linked in a cis configuration, the Co-52 resis...
Work flow used for assemblies allowing incorporation of reads from all 5 tissue libraries while r... more Work flow used for assemblies allowing incorporation of reads from all 5 tissue libraries while removing redundancy. Figure S2. Distributions of the number of isoforms for each transcript. Figure S3. SNP discovery pipeline with reads aligned to P. axillaris reference transcriptome assembly. Figure S4. Gene Ontology (GO) categories of the unigenes and unigenes with SNPs. Figure S5. Gene Ontology (GO) categories of the unigenes with more than ten SNPs. Distribution of the GO categories assigned to the unigenes were annotated in three categories: cellular components, molecular functions, and biological processes. Table S1. Primer sequences used for SNP validation by Sanger sequencing. Table S2. Primer sequences for CAPS markers developed from plastochron-related genes. (DOCX 782Â kb)
Background Petunia (Petuniaâ Ă â hybrida), derived from a hybrid between P. axillaris and P. inte... more Background Petunia (Petuniaâ Ă â hybrida), derived from a hybrid between P. axillaris and P. integrifolia, is one of the most economically important bedding plant crops and Petunia spp. serve as model systems for investigating the mechanisms underlying diverse mating systems and pollination syndromes. In addition, we have previously described genetic variation and quantitative trait loci (QTL) related to petunia development rate and morphology, which represent important breeding targets for the floriculture industry to improve crop production and performance. Despite the importance of petunia as a crop, the floriculture industry has been slow to adopt marker assisted selection to facilitate breeding strategies and there remains a limited availability of sequences and molecular markers from the genus compared to other economically important members of the Solanaceae family such as tomato, potato and pepper. Results Here we report the de novo assembly, annotation and characterization ...
Introduction Anthracnose caused by CoUetotrichum lindemuthianum (Sacc. and Magn.) Scrib. is one o... more Introduction Anthracnose caused by CoUetotrichum lindemuthianum (Sacc. and Magn.) Scrib. is one of the most widespread and economically important fungal diseases of common bean {Phaseolus vulgaris LO« Genetic resistance is the most effective method of control of anthracnose in common bean where ten major resistance genes have been characterized. Inheritance of anthracnose resistance in the differential cultivar Widusa has not been fully characterized. To facilitate the use of Widusa as a source of resistance to anthracnose, we investigated its mode of inheritance in a series of allelism tests with previously characterized resistance genes.
ABSTRACT Stevia rebaudiana is a small perennial shrub used for centuries as a bio-sweetener and f... more ABSTRACT Stevia rebaudiana is a small perennial shrub used for centuries as a bio-sweetener and for its medicinal properties. Stevia accumulates high concentrations of diterpene steviol glycosides which contribute to its high-potency sweetening with no calorific value. The development of molecular markers linked to glycoside and agronomic traits will facilitate early selection for breeding improved varieties. We developed a reference transcriptome from RNA seq data. A total of 194,965,048 paired-end reads of 100bp length were obtained from a bulked RNA sample of diverse tissue types. After processing, 171,503,922 reads were used to construct a reference transcriptome. This de novo assembly was then mined for simple-sequence repeat (SSR) markers. A single locus isoform was selected and the remaining 48,238 transcripts were used to identify SSRs using the MISA software package. A total of 5,876 SSRs were identified in 4,879 transcripts. Primers were designed for 4,193 SSR. In silico PCR was performed on these primers to remove primers that may produce multiple amplicons. The remaining 3,396 SSR primers were then compared to two other de novo assemblies. Those present in all three assemblies (1,063) were selected for evaluation. Additional SSRs were mined from publicly available ESTs. We tested 148 primers pairs on stevia DNA and found over 95% successful amplicon production. These markers were used to evaluate the level of genetic variation in our germplasm and will be utilized in future genetic mapping efforts.
Gene duplication is an important mechanism for evolution of new genes. In plants, a special group... more Gene duplication is an important mechanism for evolution of new genes. In plants, a special group of transposable elements, called Pack-MULEs or transduplicates, is able to duplicate and amplify genes or gene fragments on a large scale. Despite the abundance of Pack-MULEs, the functionality of these duplicates is not clear. Here, we present a comprehensive analysis of expression and purifying selection on 2809 Pack-MULEs in rice (Oryza sativa), which are derived from 1501 parental genes. At least 22% of the Pack-MULEs are transcribed, and 28 Pack-MULEs have direct evidence of translation. Chimeric Pack-MULEs, which contain gene fragments from multiple genes, are much more frequently expressed than those derived only from a single gene. In addition, Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs is associated with a reduction in expression of both the Pack-MULEs and their parental genes. Furthermore, an assessment of the selection pressure on t...
Glycosylation is a key modification that contributes to determine bioactivity and bioavailability... more Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extr...
Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet c... more Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate speci...
Resistance to anthracnose in common bean is conditioned primarily by nine major independent genes... more Resistance to anthracnose in common bean is conditioned primarily by nine major independent genes, Co-1 to Co-10 as the Co-3/Co-9 genes are allelic. With the exception of the recessive co-8 gene, all other nine are dominant genes and multiple alleles exist at the Co-1, Co-3 and Co-4 loci. A reverse of dominance at the Co-1 locus suggests that an order of dominance exists among individual alleles at this locus. The nine resistance genes Co-2 to Co-10 are Middle American in origin and Co-1 is the only locus from the Andean gene pool. Seven resistance loci have been mapped to the integrated bean linkage map and Co-1 resides on linkage group B1; Co-2 on B11, Co-3 on B4; Co-4 on B8; Co-6 on B7; and Co-9 and Co-10 are located on B4 but do not appear to be linked. Three Co-genes map to linkage groups B1, B4 and B11 where clusters with genes for rust resistance are located. In addition, there is co-localization with major resistance genes and QTL that condition partial resistance to anthrac...
Anthracnose is a devastating fungal disease of common bean (Phaseolus vulgaris L.), the causal ag... more Anthracnose is a devastating fungal disease of common bean (Phaseolus vulgaris L.), the causal agent of which is Colletotrichum lindemuthianum. MSU 7-1 is a common bean breeding line that contains the Co-52 and Co-7 anthracnose resistance genes. In this study, phenotypic analyses were conducted in an F2 population derived from the Mexico 222 (S) x MSU 7-1 (R) cross that was inoculated with race 64 of C. lindemuthianum. Segregation analysis revealed a 3R:1S ratio, indicating that only Co-52 confers resistance to race 64 once the Co-7 gene has been defeated by this race. The molecular marker g12333250, in linkage group Pv07, was linked in coupling phase to Co-52 at a distance of 1.2 cM. The presence or absence of this marker was also determined in the G 2333, TU, H1 and PI 207262 cultivars. It was found that the g12333250 molecular marker was present in G 2333 and absent in the other cultivars. Because the allele and marker are physically linked in a cis configuration, the Co-52 resis...
Work flow used for assemblies allowing incorporation of reads from all 5 tissue libraries while r... more Work flow used for assemblies allowing incorporation of reads from all 5 tissue libraries while removing redundancy. Figure S2. Distributions of the number of isoforms for each transcript. Figure S3. SNP discovery pipeline with reads aligned to P. axillaris reference transcriptome assembly. Figure S4. Gene Ontology (GO) categories of the unigenes and unigenes with SNPs. Figure S5. Gene Ontology (GO) categories of the unigenes with more than ten SNPs. Distribution of the GO categories assigned to the unigenes were annotated in three categories: cellular components, molecular functions, and biological processes. Table S1. Primer sequences used for SNP validation by Sanger sequencing. Table S2. Primer sequences for CAPS markers developed from plastochron-related genes. (DOCX 782Â kb)
Background Petunia (Petuniaâ Ă â hybrida), derived from a hybrid between P. axillaris and P. inte... more Background Petunia (Petuniaâ Ă â hybrida), derived from a hybrid between P. axillaris and P. integrifolia, is one of the most economically important bedding plant crops and Petunia spp. serve as model systems for investigating the mechanisms underlying diverse mating systems and pollination syndromes. In addition, we have previously described genetic variation and quantitative trait loci (QTL) related to petunia development rate and morphology, which represent important breeding targets for the floriculture industry to improve crop production and performance. Despite the importance of petunia as a crop, the floriculture industry has been slow to adopt marker assisted selection to facilitate breeding strategies and there remains a limited availability of sequences and molecular markers from the genus compared to other economically important members of the Solanaceae family such as tomato, potato and pepper. Results Here we report the de novo assembly, annotation and characterization ...
Introduction Anthracnose caused by CoUetotrichum lindemuthianum (Sacc. and Magn.) Scrib. is one o... more Introduction Anthracnose caused by CoUetotrichum lindemuthianum (Sacc. and Magn.) Scrib. is one of the most widespread and economically important fungal diseases of common bean {Phaseolus vulgaris LO« Genetic resistance is the most effective method of control of anthracnose in common bean where ten major resistance genes have been characterized. Inheritance of anthracnose resistance in the differential cultivar Widusa has not been fully characterized. To facilitate the use of Widusa as a source of resistance to anthracnose, we investigated its mode of inheritance in a series of allelism tests with previously characterized resistance genes.
ABSTRACT Stevia rebaudiana is a small perennial shrub used for centuries as a bio-sweetener and f... more ABSTRACT Stevia rebaudiana is a small perennial shrub used for centuries as a bio-sweetener and for its medicinal properties. Stevia accumulates high concentrations of diterpene steviol glycosides which contribute to its high-potency sweetening with no calorific value. The development of molecular markers linked to glycoside and agronomic traits will facilitate early selection for breeding improved varieties. We developed a reference transcriptome from RNA seq data. A total of 194,965,048 paired-end reads of 100bp length were obtained from a bulked RNA sample of diverse tissue types. After processing, 171,503,922 reads were used to construct a reference transcriptome. This de novo assembly was then mined for simple-sequence repeat (SSR) markers. A single locus isoform was selected and the remaining 48,238 transcripts were used to identify SSRs using the MISA software package. A total of 5,876 SSRs were identified in 4,879 transcripts. Primers were designed for 4,193 SSR. In silico PCR was performed on these primers to remove primers that may produce multiple amplicons. The remaining 3,396 SSR primers were then compared to two other de novo assemblies. Those present in all three assemblies (1,063) were selected for evaluation. Additional SSRs were mined from publicly available ESTs. We tested 148 primers pairs on stevia DNA and found over 95% successful amplicon production. These markers were used to evaluate the level of genetic variation in our germplasm and will be utilized in future genetic mapping efforts.
Gene duplication is an important mechanism for evolution of new genes. In plants, a special group... more Gene duplication is an important mechanism for evolution of new genes. In plants, a special group of transposable elements, called Pack-MULEs or transduplicates, is able to duplicate and amplify genes or gene fragments on a large scale. Despite the abundance of Pack-MULEs, the functionality of these duplicates is not clear. Here, we present a comprehensive analysis of expression and purifying selection on 2809 Pack-MULEs in rice (Oryza sativa), which are derived from 1501 parental genes. At least 22% of the Pack-MULEs are transcribed, and 28 Pack-MULEs have direct evidence of translation. Chimeric Pack-MULEs, which contain gene fragments from multiple genes, are much more frequently expressed than those derived only from a single gene. In addition, Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs is associated with a reduction in expression of both the Pack-MULEs and their parental genes. Furthermore, an assessment of the selection pressure on t...
Glycosylation is a key modification that contributes to determine bioactivity and bioavailability... more Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extr...
Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet c... more Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate speci...
Resistance to anthracnose in common bean is conditioned primarily by nine major independent genes... more Resistance to anthracnose in common bean is conditioned primarily by nine major independent genes, Co-1 to Co-10 as the Co-3/Co-9 genes are allelic. With the exception of the recessive co-8 gene, all other nine are dominant genes and multiple alleles exist at the Co-1, Co-3 and Co-4 loci. A reverse of dominance at the Co-1 locus suggests that an order of dominance exists among individual alleles at this locus. The nine resistance genes Co-2 to Co-10 are Middle American in origin and Co-1 is the only locus from the Andean gene pool. Seven resistance loci have been mapped to the integrated bean linkage map and Co-1 resides on linkage group B1; Co-2 on B11, Co-3 on B4; Co-4 on B8; Co-6 on B7; and Co-9 and Co-10 are located on B4 but do not appear to be linked. Three Co-genes map to linkage groups B1, B4 and B11 where clusters with genes for rust resistance are located. In addition, there is co-localization with major resistance genes and QTL that condition partial resistance to anthrac...
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