Characterization of Novel Sorghum \u3ci\u3ebrown midrib\u3c/i\u3e Mutants from an EMS-Mutagenized Population

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles

Characterization of Novel Sorghum \u3ci\u3ebrown midrib\u3c/i\u3e Mutants from an EMS-Mutagenized Population

Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles

Diagnóstico molecular y estudio de la epidemiologia del fitoplasma de la hoja pequeña de la Gliricidia (Gliricidia sepium)

71 p.En el madreado se ha detectado una enfermedad asociada con un fitoplasma, llamada enfermedad de la Hoja Pequeña de la Gliricidia (EHPG). Se caracterizaron los síntomas que consisten en: reducción del tamaño de los foliolos, distorsión foliar, proliferación de brotes y brotes débiles. El síntoma amarillamiento no resultó confiable para la identificación de la enfermedad en el campo. Una enfermedad de esta naturaleza puede causar graves pérdidas económicas ya que debido al declinamiento gradual de los árboles es fácil que pase desapercibida hasta encontrarse en un nivel de severidad y expansión grandes, Como primer paso para estudiar la epidemiologia de la enfermedad he necesario optimizar una técnica apropiada para la detección del patógeno. Se utilizó la técnica molecular para amplificar ácidos nucleicos, conocida como Reacción en Cadena de la Polimerasa (PCR, por sus siglas en inglés, Polymerase Chain Reaction). Se realizó la optimización de los factores críticos del proceso. Entre los principales se incluyen: el método de extracción y máximo tiempo de almacenaje de las muestras frescas, la concentración de los componentes esenciales para la reacción y las temperaturas óptimas de las tres fases de cada ciclo. Con la técnica optimizada se llevaron a cabo estudios de transmisión de la enfermedad. Se encontró transmisión a través de estacas. Aunque el nivel de severidad de la enfermedad afecta negativamente el enraizamiento, estacas de plantas asintomáticas infectadas y de plantas con síntomas moderados enraízan y presentan infección del patógeno en las nuevas plantas. Se detectó ADN del fitoplasma en plántulas germinadas de semillas de plantas enfermas. Esto sugiere una posible transmisión a través de semillas. Los insectos Empoasca hastosa, Lopidea murray, Alconeura sp. , Ollariamus sp. , Hydatothrips sp se identificaron como posibles vectores de EHPG, ya que se le detectó ADN del fitoplasma en sus tejidos.1. índice de cuadros 2. Índice de figuras 3. Índice de anexos 4. Introducción 5. Revisión de literatura 6. Materiales y métodos 7. Resultados y discusión 8. Conclusiones 9. Recomendaciones 10. Bibliografía 11. Anexo

Genetic dissection of sorghum traits important for biofuel production

Sorghum is a promising bioenergy feedstock due to its high yield potential and tolerance to stresses. Genetic variants with altered lignin composition and sweet stems are of particular interest, but the genetic and molecular basis for these traits are not well understood and hampers breeding efforts. Efficiency of conversion of lignocellulosic biomass is affected by lignin content and composition. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue and altered lignin content. Genetic and chemical approaches were used to identify four bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 allelic groups. The changes in subunit lignin composition were characterized for each of the groups to aid in the identification of the genes underlying the mutations. Enzymatic saccharification of stover demonstrated that the mutations in the bmr2, bmr6 and bmr12 groups can increase glucose yields up to 25% compared to wild-type isolines. Lignin subunit composition of the bmr6 group is consistent with reduction of cinnamyl alcohol dehydrogenase (CAD) activity. Analysis of the sorghum genome revealed 14 CAD-like genes. Based on their phylogenetic relationship and the identification of non-conservative mutations in three allelic bmr6 lines, SbCAD2 was identified as the Bmr6 gene. The effects of combining the bmr6 mutation with the sweet stem trait were investigated. The presence of the sweet stem helped reduce the biomass yield penalty associated with the bmr6 mutation. The sweet stem trait and the bmr6 mutation act in an additive manner to reduce lignin content in the stover. This is likely to improve both the saccharification properties of the biomass and forage quality. However, their combination also resulted in increased stalk lodging. Markers associated with agronomic and biomass characteristics were identified to assist in the breeding of bioenergy sorghum lines

Plagas de cultivos tropicales II

10 P.Actualmente esta presente en todas las zonas tropicales y subtropicales del mundo. Es cultivada comercialmente en lugares tan diversos como la India, Hawái, Florida, Puerto Rico, Pakistán, México y Suramérica. El fruto se consume fresco y procesado. Para consumo fresco se prefiere variedades dulces con menores cantidades de ácido ascórbico (0.35% de acidez). Para fines de procesamiento se utiliza en dulces, jugos y mermeladas, y se necesita alrededor de 2.5% de acidez. Otros usos incluyen la madera, con la cual se fabrican artesanías y además tiene usos medicinales. La corteza es usada como antidiarreico y ulceras al igual que las hojas

Using Genotyping by Sequencing to Map Two Novel Anthracnose Resistance Loci in Sorghum bicolor

Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar ‘Bk7’, a biparental mapping population of F3:4 and F4:5 sorghum lines was generated by crossing ‘Bk7’ with the susceptible inbred ‘Early Hegari-Sart’. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from ‘Bk7’. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. Genetic analysis following an independent selection experiment of lines derived from a cross between ‘Bk7’ and sweet sorghum ‘Mer81-4’ narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases

\u3ci\u3eBrown midrib2 (Bmr2)\u3c/i\u3e encodes the major 4-coumarate: coenzyme A ligase involved in lignin biosynthesis in sorghum (\u3ci\u3eSorghum bicolor\u3c/i\u3e (L.) Moench)

Successful modification of plant cell-wall composition without compromising plant integrity is dependent on being able to modify the expression of specific genes, but this can be very challenging when the target genes are members of multigene families. 4-coumarate:CoA ligase (4CL) catalyzes the formation of 4-coumaroyl CoA, a precursor of both flavonoids and monolignols, and is an attractive target for transgenic down-regulation aimed at improving agro-industrial properties. Inconsistent phenotypes of transgenic plants have been attributed to variable levels of down-regulation of multiple 4CL genes. Phylogenetic analysis of the sorghum genome revealed 24 4CL(-like) proteins, five of which cluster with bona fide 4CLs from other species. Using a map-based cloning approach and analysis of two independent mutant alleles, the sorghum brown midrib2 (bmr2) locus was shown to encode 4CL. In vitro enzyme assays indicated that its preferred substrate is 4-coumarate. Missense mutations in the two bmr2 alleles result in loss of 4CL activity, probably as a result of improper folding as indicated by molecular modeling. Bmr2 is the most highly expressed 4CL in sorghum stems, leaves and roots, both at the seedling stage and in pre-flowering plants, but the products of several paralogs also display 4CL activity and compensate for some of the lost activity. The contribution of the paralogs varies between developmental stages and tissues. Gene expression assays indicated that Bmr2 is under auto-regulatory control, as reduced 4CL activity results in over-expression of the defective gene. Several 4CL paralogs are also up-regulated in response to the mutation

Identification and Characterization of Four Missense Mutations in \u3ci\u3eBrown midrib\u3c/i\u3e 12 (\u3ci\u3eBmr12\u3c/i\u3e), the Caffeic \u3ci\u3eO\u3c/i\u3e-Methyltranferase (COMT) of Sorghum

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum