Rapid evolution in plant chitinases: Molecular targets of selection in plant-pathogen coevolution

Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

How Escherichia coli can bias the results of molecular cloning: Preferential selection of defective genomes of hepatitis C virus during the cloning procedure

Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression. These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected. The aim of the present study was to express HCV structural proteins. However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins. The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription–PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector. The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins. Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products. Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis. The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72–92%). Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity. It can also be an impediment to the construction of infectious viral clones.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Trait physiology and crop modelling as a framework to link phenotypic complexity to underlying genetic systems

New tools derived from advances in molecular biology have not been widely adopted in plant breeding for complex traits because of the inability to connect information at gene level to the phenotype in a manner that is useful for selection. In this study, we explored whether physiological dissection and integrative modelling of complex traits could link phenotype complexity to underlying genetic systems in a way that enhanced the power of molecular breeding strategies. A crop and breeding system simulation study on sorghum, which involved variation in 4 key adaptive traits-phenology, osmotic adjustment, transpiration efficiency, stay-green-and a broad range of production environments in north-eastern Australia, was used. The full matrix of simulated phenotypes, which consisted of 547 location-season combinations and 4235 genotypic expression states, was analysed for genetic and environmental effects. The analysis was conducted in stages assuming gradually increased understanding of gene-to-phenotype relationships, which would arise from physiological dissection and modelling. It was found that environmental characterisation and physiological knowledge helped to explain and unravel gene and environment context dependencies in the data. Based on the analyses of gene effects, a range of marker-assisted selection breeding strategies was simulated. It was shown that the inclusion of knowledge resulting from trait physiology and modelling generated an enhanced rate of yield advance over cycles of selection. This occurred because the knowledge associated with component trait physiology and extrapolation to the target population of environments by modelling removed confounding effects associated with environment and gene context dependencies for the markers used. Developing and implementing this gene-to-phenotype capability in crop improvement requires enhanced attention to phenotyping, ecophysiological modelling, and validation studies to test the stability of candidate genetic regions.

Mapping the I-3 gene for resistance to Fusarium wilt in tomato: application of an I-3 marker in tomato improvement and progress towards the cloning of I-3

Fusarium wilt of tomato, caused by the fungal pathogen, Fusarium oxysporum f. sp. lycopersici (Fol), is an economically damaging disease that results in huge losses in Australia and other countries worldwide. The I-3 gene, which confers resistance to Fol race 3, has been described in wild tomato, Lycopersicon pennellii, accessions LA716 and PI414773. We are pursuing the isolation of I-3 from LA716 by map-based cloning. We have constructed a high-resolution map of the I-3 region and have identified markers closely flanking I-3 as well as markers co-segregating with I-3. In addition, construction of a physical map based on these markers has been initiated. This review describes the context of our research and our progress towards isolating the I-3 gene. It also describes some important practical outcomes of our work, including the development and use of a PCR-based marker for marker-assisted selection for I-3, and the finding that the I-3 gene from LA716 is different to that from PI1414773, which we have now designated I-7. Tomato varieties combining I-3 and I-7 have been developed and are currently being introduced into commercial production to further safeguard tomato crops against Fusarium wilt.

Trait physiology and crop modelling to link phenotypic complexity to underlying genetic systems

New tools derived from advances in molecular biology have not been widely adopted in plant breeding because of the inability to connect information at gene level to the phenotype in a manner that is useful for selection. We explore whether a crop growth and development modelling framework can link phenotype complexity to underlying genetic systems in a way that strengthens molecular breeding strategies. We use gene-to-phenotype simulation studies on sorghum to consider the value to marker-assisted selection of intrinsically stable QTLs that might be generated by physiological dissection of complex traits. The consequences on grain yield of genetic variation in four key adaptive traits – phenology, osmotic adjustment, transpiration efficiency, and staygreen – were simulated for a diverse set of environments by placing the known extent of genetic variation in the context of the physiological determinants framework of a crop growth and development model. It was assumed that the three to five genes associated with each trait, had two alleles per locus acting in an additive manner. The effects on average simulated yield, generated by differing combinations of positive alleles for the traits incorporated, varied with environment type. The full matrix of simulated phenotypes, which consisted of 547 location-season combinations and 4235 genotypic expression states, was analysed for genetic and environmental effects. The analysis was conducted in stages with gradually increased understanding of gene-to-phenotype relationships, which would arise from physiological dissection and modelling. It was found that environmental characterisation and physiological knowledge helped to explain and unravel gene and environment context dependencies. We simulated a marker-assisted selection (MAS) breeding strategy based on the analyses of gene effects. When marker scores were allocated based on the contribution of gene effects to yield in a single environment, there was a wide divergence in rate of yield gain over all environments with breeding cycle depending on the environment chosen for the QTL analysis. It was suggested that knowledge resulting from trait physiology and modelling would overcome this dependency by identifying stable QTLs. The improved predictive power would increase the utility of the QTLs in MAS. Developing and implementing this gene-to-phenotype capability in crop improvement requires enhanced attention to phenotyping, ecophysiological modelling, and validation studies to test the stability of candidate QTLs.

Molecular modelling assisted design and synthesis of cyclothialidine derivatives as DNA gyrase inhibitors

Since cyclothialidine was discovered as the most active DNA gyrase inhibitor in 1994, enormous efforts have been devoted to make it into a commercial medicine by a number of pharmaceutical companies and research groups worldwide. However, no serious breakthrough has been made up to now. An essential problem involved with cyclothialidine is that though it demonstrated the potent inhibition of DNA gyrase, it showed little activity against bacteria. This probably is attributable to its inability to penetrate bacterial cell walls and membranes. We applied the TSAR programme to generate a QSAR equation to the gram-negative organisms. In that equation, LogP is profoundly indicated as the key factor influencing the cyclothialidine activity against bacteria. However, the synthesized new analogues have failed to prove that. In the structure based drug design stage, we designed a group of open chain cyclothialidine derivatives by applying the SPROUT programme and completed the syntheses. Improved activity is found in a few analogues and a 3D pharmacophore of the DNA gyrase B is proposed to lead to synthesis of the new derivatives for development of potent antibiotics.

Organic matter dynamics in the Florida coastal Everglades: A molecular marker and isotopic approach

Everglades National Park (ENP) is about to undergo the world's largest wetland restoration with the aim of improving the quality, timing and distribution of water flow. The changes in water flow are hypothesized to alter the nutrient fluxes and organic matter (OM) dynamics within ENP, especially in the estuarine areas. This study used a multi-proxy approach of molecular markers and stable δ 13C isotope measurements, to determine the present day OM dynamics in ENP. ^ OM dynamics in wetland soils/sediments have proved to be difficult to understand using traditional geochemical approaches. These are often inadequate to describe the multitude of OM sources (e.g. higher land plant, emergent vegetation, submerged vegetation) to the soils/sediments and the complex diagenetic processes that can alter the OM characteristics. A multi-proxy approach, however, that incorporates both molecular level and bulk parameter information is ideal to comprehend complex OM dynamics in aquatic environments. Therefore, biomass-specific molecular markers or proxies can be useful in tracing the sources and processing of OM. This approach was used to examine the OM dynamics in the two major drainage basins, Shark River Slough and Taylor River Slough, of ENP. Freshwater to marine transects were sampled in both systems for soils/sediments and suspended particulate organic matter (SPOM) to be characterized through bulk OM analyses, lipid biomarker determinations (e.g. sterols, fatty acids, hydrocarbons and triterpenoids) and compound-specific stable carbon isotope (δ 13C) determinations. ^ One key accomplishment of the research was the assessment of a molecular marker proxy (Paq) to distinguish between emergent/higher plant vegetation from submerged vegetation within ENP. This proxy proved to be quite useful at tracing OM inputs to the soils/sediments of ENP. A second key accomplishment was the development of a 3-way model using vegetation specific molecular markers. This novel, descriptive model was successfully applied to the estuarine areas of Taylor and Shark River sloughs, providing clear evidence of mixing of freshwater, estuarine and marine derived OM in these areas. In addition, diagenetic transformations of OM in these estuaries were found to be quite different between Taylor and Shark Rivers, and are likely a result of OM quality and hydrological differences. ^

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangifera indica) transcriptome for mapping and estimation of genetic diversity

The development of molecular markers for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here our identification of thousands of unambiguous molecular markers that can be easily assayed across genotypes of the species. With origin centered in Southeast Asia, mangos are grown throughout the tropics and subtropics as a nutritious fruit that exhibits remarkable intraspecific phenotypic diversity. With the goal of building a high density genetic map, we have undertaken discovery of sequence variation in expressed genes across a broad range of mango cultivars. A transcriptome sequence reference was built de novo from extensive sequencing and assembly of RNA from cultivar 'Tommy Atkins'. Single nucleotide polymorphisms (SNPs) in protein coding transcripts were determined from alignment of RNA reads from 24 mango cultivars of diverse origins: 'Amin Abrahimpur' (India), 'Aroemanis' (Indonesia), 'Burma' (Burma), 'CAC' (Hawaii), 'Duncan' (Florida), 'Edward' (Florida), 'Everbearing' (Florida), 'Gary' (Florida), 'Hodson' (Florida), 'Itamaraca' (Brazil), 'Jakarata' (Florida), 'Long' (Jamaica), 'M. Casturi Purple' (Borneo), 'Malindi' (Kenya), 'Mulgoba' (India), 'Neelum' (India), 'Peach' (unknown), 'Prieto' (Cuba), 'Sandersha' (India), 'Tete Nene' (Puerto Rico), 'Thai Everbearing' (Thailand), 'Toledo' (Cuba), 'Tommy Atkins' (Florida) and 'Turpentine' (West Indies). SNPs in a selected subset of protein coding transcripts are currently being converted into Fluidigm assays for genotyping of mapping populations and germplasm collections. Using an alternate approach, SNPs (144) discovered by sequencing of candidate genes in 'Kensington Pride' have already been converted and used for genotyping.

QTL mapping for reaction to Phaeosphaeria leaf spot in a tropical maize population

Phaeosphaeria leaf spot (PLS) is an important disease in tropical and subtropical maize (Zea mays, L.) growing areas, but there is limited information on its inheritance. Thus, this research was conducted to study the inheritance of the PLS disease in tropical maize by using QTL mapping and to assess the feasibility of using marker-assisted selection aimed to develop genotypes resistance to this disease. Highly susceptible L14-04B and highly resistant L08-05F inbred lines were crossed to develop an F(2) population. Two-hundred and fifty six F(2) plants were genotyped with 143 microsatellite markers and their F(2:3) progenies were evaluated at seven environments. Ten plants per plot were evaluated 30 days after silk emergence following a rating scale, and the plot means were used for analyses. The heritability coefficient on a progeny mean basis was high (91.37%), and six QTL were mapped, with one QTL on chromosomes 1, 3, 4, and 6, and two QTL on chromosome 8. The gene action of the QTL ranged from additive to partial dominance, and the average level of dominance was partial dominance; also a dominance x dominance epistatic effect was detected between the QTL mapped on chromosome 8. The phenotypic variance explained by each QTL ranged from 2.91 to 11.86%, and the joint QTL effects explained 41.62% of the phenotypic variance. The alleles conditioning resistance to PLS disease of all mapped QTL were in the resistant parental inbred L08-05F. Thus, these alleles could be transferred to other elite maize inbreds by marker-assisted backcross selection to develop hybrids resistant to PLS disease.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.

Evaluating plant breeding strategies by simulating gene action and dryland environment effects

Functional genomics is the systematic study of genome-wide effects of gene expression on organism growth and development with the ultimate aim of understanding how networks of genes influence traits. Here, we use a dynamic biophysical cropping systems model (APSIM-Sorg) to generate a state space of genotype performance based on 15 genes controlling four adaptive traits and then search this spice using a quantitative genetics model of a plant breeding program (QU-GENE) to simulate recurrent selection. Complex epistatic and gene X environment effects were generated for yield even though gene action at the trait level had been defined as simple additive effects. Given alternative breeding strategies that restricted either the cultivar maturity type or the drought environment type, the positive (+) alleles for 15 genes associated with the four adaptive traits were accumulated at different rates over cycles of selection. While early maturing genotypes were favored in the Severe-Terminal drought environment type, late genotypes were favored in the Mild-Terminal and Midseason drought environment types. In the Severe-Terminal environment, there was an interaction of the stay-green (SG) trait with other traits: Selection for + alleles of the SG genes was delayed until + alleles for genes associated with the transpiration efficiency and osmotic adjustment traits had been fixed. Given limitations in our current understanding of trait interaction and genetic control, the results are not conclusive. However, they demonstrate how the per se complexity of gene X gene X environment interactions will challenge the application of genomics and marker-assisted selection in crop improvement for dryland adaptation.

Mapeo de QTL para Mal de Río Cuarto en maíz. QTL mapping for tolerance to Mal de Río Cuarto disease in maize.

El Mal de Río Cuarto (MRC) es una enfermedad del maíz (Zea mays L.), endémica de ciertas zonas de la Argentina y constituye la patología más importante de este cultivo por la severidad de los daños y por la creciente difusión del área geográfica afectada. El empleo de la resistencia genética bajo un manejo integrado de la enfermedad, constituye la estrategia más económica y ambientalmente sustentable para lograr el incremento y la estabilidad en la producción de los cultivos de maíz, reduciendo el uso nocivo de agroquímicos. La reacción a la enfermedad MRC se encuentra influida por una fuerte interacción genotipo-ambiente que dificulta el mejoramiento genético. En los ensayos multiambientales la inconsistencia de la respuesta y la inestabilidad de los genotipos ensayados hace dificultoso llevar a cabo una buena selección basada en los síntomas de la enfermedad. Para contribuir a aumentar la eficacia de los métodos de mejoramiento es posible implementar programas en los que se incluyan herramientas biotecnológicas como los marcadores moleculares, que permiten reducir el efecto ambiental y contribuyen a soslayar, al menos en parte, los inconvenientes planteados por los efectos de la interacción genotipo-ambiente facilitando la identificación de los genotipos buscados. La selección fenotípica realizada convencionalmente, puede ser complementada con la selección asistida por marcadores (marker-assisted selection MAS) consistente en utilizar la información genética que brindan marcadores moleculares de ADN, tales como los SSR, asociados a loci o segmentos cromosómicos (quantitative trait loci QTL) que confieran resistencia a MRC. Si bien los resultados preliminares obtenidos por nuestro grupo de trabajo señalan la presencia de posibles QTLs e informan que la reacción frente a la enfermedad tiene una moderada heredabilidad y una substancial variación debida a la interacción genotipo-ambiente, es necesario confirmar los resultados relativos a los parámetros poblacionales y obtener una mejor delimitación de las regiones genómicas identificadas. Con la finalidad de aumentar la eficiencia de los programas de mejoramiento en el desarrollo de genotipos tolerantes mediante la selección asistida por marcadores utilizando una población de mapeo F2 con un fondo genético diferente y líneas endocriadas recombinantes evaluadas en ensayos multiambientales, se proponen los siguientes objetivos (i) estudiar la forma de herencia de la reacción frente a MRC, (ii) identificar nuevos QTLs asociados a este carácter y (iii) verificar la consistencia de los QTLs identificados previamente.

Transferring QTL technology to the pig breeding industry (PigQTech) - a demonstration project

The aim of the project has been to demonstrate how the farm animal breeding industry can utilise gene mapping technology to accelerate genetic improvement. Previous theoretical studies had suggested that the use of marker assisted selection could potentially increase the annual improvement for quantitative traits like backfat with about 10% and for more difficult traits such as meat quality and reproduction by as much as 40-60% compared with existing technology. The work has comprised two major tasks: 1. Commercially relevant populations have been screened for segregation at QTLs identified in experimental populations. The aim has been to establish optimal strategies for QTL detection in commercial pig populations and the extent to which QTLs explaining major phenotypic differences between divergent lines used in experimental studies also explain quantitative variation within commercial lines. The results are important for specifying future strategies for finding economically valuable QTLs. 2. Marker assisted backcrossing has been used to demonstrate how a QTL allele can be introgressed from one breed to another. The work has focused on the major fatness QTL on pig chromosome 4 previously identified in a wild pig/Large White intercross. The end result was not designed to be a commercially viable product in its own right, but the process has validated a number of points of major importance for the exploitation of QTLs in livestock.

Short communication. SNP analyses of the 5HT1R and STAT genes in Pacific white shrimp, Litopenaeus vannamei

La industria de la producción de camarón es una de las industrias acuícolas que se encuentra en más crecimiento en la actualidad. Los estudios para encontrar marcadores genéticos son muy efectivos para la mejora de sus propiedades y de gran interés para los productores de camarón. En este trabajo se utilizaron seis individuos de una población de Litopenaeus vannamei, donde se encontraron cuatro polimorfismos de nucleótido único (SNPs) en el gen 5HT1R (5-hidroxitriptamina receptor1) y un SNP en el gen STAT (transductor de señal y activador de la transcripción). Sin embargo, el polimorfismo en el gen STAT resultó ser homocigoto en una población diferente utilizada para análisis de asociación. Los presentes análisis revelaron que el alelo C, en dos polimorfismos SNP (C109T y C395G) del gen 5HT1R, tiende a estar asociado con el aumento del peso corporal. Consideramos que hay necesidad de hacer nuevos estudios utilizando una muestra más amplia y diversa de la población en cuestión.