Strawberry Studies: Screening of Germplasm and Identification of Quantitative Trait Loci for Necrotrophic and Hemibiotrophic Resistance to Anthracnose Diseases, and Validation of a Set of SSR Fingerprinting Markers.
Fecha
2019-12-13
Tipo
tesis doctoral
Autores
Chacón Jiménez, José Guillermo
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Resumen
La fresa (Fragaria ×ananassa) es un cultivo de importancia global, aunque su compleja composición genómica ha hecho que la mejora convencional sea difícil. Descubrimientos recientes y las nuevas herramientas para estudiar el genoma de la fresa han posibilitado estudios más profundos que en el pasado. La fresa se ha embarcado en una nueva era de descubrimientos con la aplicación de estudios genéticos y genómicos y, la implementación de la mejora asistida por marcadores moleculares y genómica, particularmente para evaluar los rasgos más caros y complejos de estudiar, tales como la resistencia a enfermedades.
Las enfermedades conocidas como antracnosis producidas por los patógenos Colletotrichum spp. son problemáticos en la producción de la fresa. C. acutatum es responsible por la antracnosis de la pudrición de la fruta (por sus siglas en inglés “AFR”), y C. gloeosporioides produce la antracnosis de la pudrición de la corona (por sus siglas en inglés “ACR”) en la fresa. Estos patógenos también tienen un estado biotrófico en las hojas de las plantas, conocido como infección hemibiotrófica (por sus siglas en inglés “HBI”). Aunque la resistencia a las fases neurotróficas es importante, la resistencia a la HBI es igualmente o más importante aún. El objetivo principal de este proyecto es la identificación de los loci de rasgos cuantitativos (por sus siglas en inglés “QTL”) relacionados a la resistencia a AFR, ACR y las HBI utilizando una población biparental de 280 plantas de semilla propagadas clonalmente in vitro. La afectación de AFR y ACR fue evaluada en condiciones de campo, y las HBI de ambos patógenos fueron evaluadas en platas que crecieron en el fitotrón de NCSU. El contenido de flavonoides y antocianinas fue medido in hojas maduras. Estos compuestos son conocidos como componentes importantes en las vías de resistencia a enfermedades.
No se observe correlación entre los seis rasgos (resistencia a ACR, AFR o HBI para ambos patógenos, contenido de flavonoides y antocianinas) evaluados en plantas clonales, lo que sugiere que hay diferentes mecanismos de resistencia en los diferentes sistemas de patogenicidad. La heredabilidad en sentido Amplio (H2) de los rasgos fue calculada utilizando un modelo lineal mixto. La H2 para la resistencia a AFR fue de 0,47 y de 0,25 para ACR. La evaluación de campo de la población de plantas resulta en la identificación de 14 plantas resistentes a ACR y 8 a AFR. Se encontraron dos grupos de 11 genotipos distintos que mostraron una alta resistencia a HBI por C. gloeosporioides y C. acutatum.
Se utilizaron secuencia de ADN cortas (conocidas en ingles como “reads”) obtenidas por medio de secuenciamiento con representación reducida con el protocolo omeSeq, para el descubrimiento de marcadores tipo polimorfismos de un solo nucleótido (por sus siglas en inglés “SNP”) con un modelo poliploide, incluyendo marcadores 2x y 4x. Un estudio de asociación a lo ancho del genoma (por sus siglas en inglés “GWAS”) fue hecho utilizando un modelo 4x y resultó en la identificación de dos QTLs para la resistencia a AFR, uno el cromosoma 2 y otro en el cromosoma 6. Para el HBI de C. acutatum se encontró un QTL en el cromosoma 4. Los resultados para C. gloeosporioides HBI suportan un QTL en el cromosoma 5 y dos para C. acutatum HBI en los cromosomas 2 y 6.
En coordinación con el Repositorio Nacional de Germoplasma Clonal (por sus siglas en inglés “NCGR”) del Departamento de Agricultura de Estados Unidos, Servicios de Investigación Agricultural (por sus siglas en inglés “USDA-ARS”) se validó la utilidad de un grupo de seis marcadores tipo secuencias simples repetidas (por sus siglas en inglés “SSR”), que se podrían usar en la caracterización genética del germoplasma de fresa. Un total de 186 accesiones fueron evaluadas con este grupo de SSRs, incluyendo accesiones de cultivares del NCGR, y 4 cultivares y 128 selecciones avanzadas del programa de mejora genética de fresa de la Universidad Estatal de Carolina del Norte (por sus siglas en inglés “NCSU”). El genotipeo fue realizado en un multiplex con los seis pares de imprimadores marcados con tintes fluorescentes en una sola reacción. Los resultados muestran una diversidad de alelos ligeramente inferior que, en otros estudios, con un total de 52 alelos detectados, un mínimo de seis y un máximo de 11 por marcador, y un promedio de 3,15 alelos por marcados por accesión. El grupo de marcadores es suficientemente robusto para identificar 184 accesiones con patrones alélicos únicos. Solo un par de accesiones no se pudieron resolver. Nuestros resultados indican que este grupo de marcadores SSR evaluado en este estudio puede utilizarse para identificar accesiones basado en sus patrones de alelos.
The strawberry (Fragaria ×ananassa) is an important global crop, nonetheless it has a complex genomic composition that has made traditional breeding challenging. Recent discoveries in the description and tools to study the strawberry genome are enabling in-depth studies not possible in the past. The application of genetic and genomic studies, and the implementation of marker and genomic assisted breeding, particularly for the more expensive and complex traits to evaluate such as disease resistance is embarking on a new era of discovery. The anthracnose diseases produced by Colletotrichum spp. pathogens are problematic in strawberry production. C. acutatum is responsible for anthracnose fruit rot (AFR), and C. gloeosporioides, produces anthracnose crown rot (ACR) in strawberry. This pathogen also has biotrophic stage in the plant leaves, known as hemibiotrophic infection (HBI). Although resistance to the necrotrophic phase is important, the HBI resistance is equally or more important. The main objectives of this project were the identification of QTLs related to the resistance to AFR, ACR and HBI using a biparental population of 280 seedlings clonally propagated in vitro. The AFR and the ACR were evaluated under field conditions, and the HBI for both pathogens was evaluated in plants grown in the NCSU phytotron. Flavonoid and anthocyanin content was also measured in mature leaves. These compounds are known to be important components in disease resistance pathways. No correlation was observed among the six traits (ACR, AFR, HBI resistance to both pathogens, flavonoid and anthocyanin content) evaluated in clonal plants, suggesting that there are different mechanisms of resistance in the different pathogen systems. The broad sense heritability (H2) of the traits was calculated using mixed linear models. The H2 for AFR was 0.47 and 0.25 for ACR. Field screening of the seedling population resulted in the identification of 14 and 8 highly resistant genotypes to ACR and AFR, respectively. There were two distinct groups of 11 genotypes that displayed a high level of HBI for C. gloeosporioides and C. acutatum. Reads generated by reduced representation sequencing with the omeSeq protocol were used to discover and call SNP markers with a polyploid model, including 2x and 4x markers. A genome wide association study (GWAS) was performed using a 4x model and it resulted in the identification of two QTLs for AFR resistance, one on chromosome 2 and another on chromosome 6. For C. acutatum HBI resistance we found one QTL on chromosome 4. The results for C. gloeosporioides HBI support a QTL on chromosome 5 and another two for C. acutatum HBI on chromosomes 2 and 6. In cooperation with the US Department of Agriculture Agricultural Research Service (USDA-ARS) National Germplasm Clonal Repository (NCGR) we validated the utility of a set of six Single Sequence Repeats (SSR) markers that could be used for genetic characterization of strawberry germplasm. A total of 186 accessions were tested with a set of SSRs, including cultivar accessions from the NCGR, and 4 cultivars and 128 advanced selections from the NCSU strawberry breeding program. The genotyping was performed in multiplex with the six pairs of fluorescent dye tagged primers in one reaction. The results showed a slightly lower diversity of alleles than in other studies, with a total of 52 alleles detected, a minimum of 6 and a maximum of 11 per marker, and average of 3.15 alleles per marker per accession. The primer set is robust enough to identify 184 of the accessions with unique allelic patterns. Only one pair of accessions were unresolved. Our results indicate that the set of SSR markers evaluated in this study can be used for individual accession identification based on their allelic patterns.
The strawberry (Fragaria ×ananassa) is an important global crop, nonetheless it has a complex genomic composition that has made traditional breeding challenging. Recent discoveries in the description and tools to study the strawberry genome are enabling in-depth studies not possible in the past. The application of genetic and genomic studies, and the implementation of marker and genomic assisted breeding, particularly for the more expensive and complex traits to evaluate such as disease resistance is embarking on a new era of discovery. The anthracnose diseases produced by Colletotrichum spp. pathogens are problematic in strawberry production. C. acutatum is responsible for anthracnose fruit rot (AFR), and C. gloeosporioides, produces anthracnose crown rot (ACR) in strawberry. This pathogen also has biotrophic stage in the plant leaves, known as hemibiotrophic infection (HBI). Although resistance to the necrotrophic phase is important, the HBI resistance is equally or more important. The main objectives of this project were the identification of QTLs related to the resistance to AFR, ACR and HBI using a biparental population of 280 seedlings clonally propagated in vitro. The AFR and the ACR were evaluated under field conditions, and the HBI for both pathogens was evaluated in plants grown in the NCSU phytotron. Flavonoid and anthocyanin content was also measured in mature leaves. These compounds are known to be important components in disease resistance pathways. No correlation was observed among the six traits (ACR, AFR, HBI resistance to both pathogens, flavonoid and anthocyanin content) evaluated in clonal plants, suggesting that there are different mechanisms of resistance in the different pathogen systems. The broad sense heritability (H2) of the traits was calculated using mixed linear models. The H2 for AFR was 0.47 and 0.25 for ACR. Field screening of the seedling population resulted in the identification of 14 and 8 highly resistant genotypes to ACR and AFR, respectively. There were two distinct groups of 11 genotypes that displayed a high level of HBI for C. gloeosporioides and C. acutatum. Reads generated by reduced representation sequencing with the omeSeq protocol were used to discover and call SNP markers with a polyploid model, including 2x and 4x markers. A genome wide association study (GWAS) was performed using a 4x model and it resulted in the identification of two QTLs for AFR resistance, one on chromosome 2 and another on chromosome 6. For C. acutatum HBI resistance we found one QTL on chromosome 4. The results for C. gloeosporioides HBI support a QTL on chromosome 5 and another two for C. acutatum HBI on chromosomes 2 and 6. In cooperation with the US Department of Agriculture Agricultural Research Service (USDA-ARS) National Germplasm Clonal Repository (NCGR) we validated the utility of a set of six Single Sequence Repeats (SSR) markers that could be used for genetic characterization of strawberry germplasm. A total of 186 accessions were tested with a set of SSRs, including cultivar accessions from the NCGR, and 4 cultivars and 128 advanced selections from the NCSU strawberry breeding program. The genotyping was performed in multiplex with the six pairs of fluorescent dye tagged primers in one reaction. The results showed a slightly lower diversity of alleles than in other studies, with a total of 52 alleles detected, a minimum of 6 and a maximum of 11 per marker, and average of 3.15 alleles per marker per accession. The primer set is robust enough to identify 184 of the accessions with unique allelic patterns. Only one pair of accessions were unresolved. Our results indicate that the set of SSR markers evaluated in this study can be used for individual accession identification based on their allelic patterns.
Descripción
Esta tesis fue patrocinada por medio de la Fundación Fulbright que financió mis estudios, así como fondos de investigación aportados por la Asociación de Poductores de Fresa de Carolina del Norte y de Escuela de Grduados de la Universidad Estatal de Carolina del Norte.
Palabras clave
Fresa, Antracnosis, Resistencia genética, QTL, SSR