The taxonomy of helminthosporium species

ABSTRACT

In Australia, fungal pathogens in particular Bipolaris sorokiniana impact heavily on the grains industry and have caused significant losses especially in barley. Therefore identifying new allelic combinations in barley lines through molecular analysis expressing durability to fungal pathogens will aid in breeding efficacy. To accomplish this, testing and selection of both the desirable phenotypes in the field and their genotypes in the laboratory will ensure any desired agronomical type is maintained. This study aimed to establish molecular markers that will be useful for selecting for resistance to this important fungal disease while maintaining quality traits, but the search for and exploitation of resistance genes has continued to be an equally important endeavour. In the present study, 180 doubled haploid lines derived from the cross of VB9524/ND11231*12 were screened for linkage to available SSR markers. Quantitative trait locus (QTL) mapping and classical genetic linkage approaches were used to identify and map markers for resistance to Bipolaris sorokiniana. The DArt based simple sequence repeat (SSR) marker bPb3865 and SSR- scssr10559 were mapped to the 18-0 double haploid (DH) progeny of the ND/VB cross. This association, by means of marker regression showed a significant relationship between the ND parental genotype and the major QTL identified on the 3H chromosome.

Acknowledgements

I would like to firstly thank God, my parents, Beth and Werner, and the rest of the family for their patience and understanding. Moreover I extend my thanks to the whole team at the Centre for Systems Biology led by Mark Sutherland for support and a great work environment. And to Anke, my supervisor, I appreciate all the effort you put in this year.

HAPTER 1: LITERITURE REVIEW

INTRODUCTION-

Being an agronomically important foliar pathogen, Bipolaris sorokiniana (Bs), the causal agent of spot blotch of barley is found in many growing regions of the world (Almgren et al. 1999). In Australia, fungal infection by Bs causes significant disease in barley and consequently a decrease in the plant biomass especially around SE Qld and NE NSW. At present the region requires resistant cultivars which until recently was hampered by a lack of information regarding host / pathogen interactions.

Recent studies have tested and identified several N. American lines showing resistance to Bs conferred in adult barley on the 3HS and 7H chromosomes, and on the 3HS chromosome in seedlings. The resistance was validated against all Australian Bs fungal pathotypes tested within the geographical regions mentioned. As a consequence the current study analysed molecular markers on 3 American pedigrees crossed to local cultivars to determine similarities in resistance. Given these comparisons, haplotypes established similarities among the respective populations; in terms of known resistant loci in local germplasm.

The first of the 3 American pedigrees ND11231-12 originated from North Dakota University, Fargo, USA and was first developed as a source of low grain protein seed. The Australia barley line VB9524 was developed by the Department of Primary Industries, Victoria, Australia, as an advanced selection line from a cross between Arapiles and Franklin. As a result the DH barley cross ND11321-12/VB9524 consisting of 180 lines was developed using Anther Culture (Emebiri, Platz & Moody 2005).

The second of the American pedigrees, the line WPG 8412, was developed in Winnipeg Canada, and crossed to the Australian line Lindwall. A 186 DH lines were developed with all the progeny pre-screened phenotypically for plant resistance to spot blotch. The final American pedigree TR251 was crossed, again using DH technique, to the West Australian line Gairdner, not previously grown in Australia and since have been introduced into the germplasm collection at the QDPI&F Hermitage Research Station in Warwick and have been screened for field resistance to Bs.

During 2004-2006, planting and harvesting of both ND11231 – 12 / VB9524 and TR251 / Gairdner Double Haploid (DH) barley populations took place at the Hermitage Research Station, Warwick. The field trials provided information regarding adult spot blotch resistance, with seedlings screened for resistance to spot blotch for the duration of the trials, using the rating scale developed by Fetch and Steffenson (Fetch & Steffenson 1999). This data was used to generate genetic linkage based on marker orders that were determined using MapManager QTX, and RECORD (Bovill 2007). Map Manager QTX is software that analyses genetic mapping experiments. It includes functions for mapping both Mendelian and quantitative trait loci. QTX is a superior version of Map Manager QT (Lehmensiek et al. 2005).

Barley

Barley (Hordeum vulgare) is an innovative success story in Australian agriculture. This success was achieved through experimentation on soils and climates, and secondly to trialling promising cultivars with strong marketable characteristics. In general Barley cultivation is essentially restricted to temperate and cooler geographical regions and at higher altitudes in the tropics (Badr et al. 2000). In Australia cultivation has been concentrated to the northern parts of NSW and the southern central regions of Queensland, SA and to a lesser extent WA (figure 1).

The black shaded areas are the regions of Australia that cultivate Barley is grown (figure source: www.AustralianAgriculture.com).

Climate is a main factor in the distribution of barley in Australia and also is the leading feature for its susceptibility to disease (Steffenson, Hayes & Kleinhofs 1996). The main uses for barley in Australia and internationally are shown in the following figures and include feed for livestock and Malt for beer:

There has been a significant increase in the local cultivation of barley since 2006/7. All figures 2,3 & 4 are adapted from data sourced from the Australian Bureau of statistics (ABS) 2008.

“The Species?

The genus Hordeum is made up of 32 wild species belonging to the tribe Triticeae and family Poaceae-which includes the meadow grasses (Orabi et al., 2007). As a member of the Gramineae (grasses), barley, from an ecological viewpoint belongs to the most successful family of flowering plants (Kellogg 1998). Grasses are the most important plant to humans with the family including the cereal crops barley, rice, maize, wheat, and sorghum. Principally their success comes from the source of fibre and energy their grains provide. The lineage is made up of approximately 10, 000 species manifested around 70 million years ago (Gale & Devos 1998). Archaeological evidence confirmed agricultural practices involving barley going back Ca.10, 000 years BP (Harlan and Zohary, 1966). More over this practice was said to have occurred in the region of the Middle East known as the ‘fertile crescent’ (Orabi et al. 2007). Other regions which have been included in early barley agriculture were the Far East (Southeast Asia) and Meso America (Blattner & Badani Méndez 2001; Gupta et al. 2003; Harlan & Zohary 1966; Kolodinska Brantestam et al. 2007).

As a self pollinator (autogamous), barley becomes genetically fixed towards homozygosity over time; meaning, eventually genomes (2n) would consist of identical alleles. Currently this feature is common to the cereals-wheat (7n); rice (n) and barley (2n) (Badr et al. 2000).

The barley genome

Cultivated barley, H. vulgare subsp spontaneum is a diploid (2n), and unlike the closely related allopolyploid wheat, comprises two sets of seven chromosomes (x) (2n = 2x = 14). Barley is estimated to have a genome that extends over 5.5 x 109 Base pairs (Bp) in length, of which 76% been found repetitive (Varshney, Hoisington & Tyagi 2006). This repetition is typically due to the large proportion of repeated simple nucleotide sequences (SSR) which are found either singly or in tandem, and are interspersed throughout the genome in clusters, in particular flanking genes (Tautz & Renz 1984). These sequences are commonly called satellites or simple sequence repeats. Overall average gene densities throughout barley genomes range from one gene per 123-212 Kb, assuming random gene distribution.

Double Haploids (DH)

One of the most significant biotechnological advances in commercial plant breeding has been the development of in vitro techniques to produce haploid plants that can be used to generate homozygous lines.

Scientifically altering germplasm e.g. producing DHs using F1 generated lines is a platform commonly used in Australia for scientific research purposes. Anther and microspore culture, are the two main methods employed in the production of a doubled haploid (DH) barley plants. (Kuhlmann & Foroughi-Wehr 1989). Anther culture (androgenesis) is a method that can generate a haploid plant from a pollen microspore. Whole anthers are placed on a nutrient culture medium, and some of the microspores within the anther modify their developmental pathway to form embryos rather than mature pollen. Thus immature pollen (microspore) is the starting material for androgenesis.

Pollen selection, at the correct developmental stage, is crucial, and may vary from species to species (Davies & Morton 1998). In addition, some individual genotypes may not be amenable to anther culture, or may require specific pre-treatment for their development (Zivy et al. 1992). In cereals colchicine is a common pre-treatment to aid the spontaneous doubling of chromosomes. Anther culture in barley can produce a significant number of diploids without the use of colchicine (Redha et al. 1998). Double haploids allow novel allele combinations, particularly ones involving recessive characters (traits) to be readily assessed in mature plants (Soriano et al. 2007).

These powerful tools (DHs), for the use in plant breeding and genetic analyses achieve complete homozygosity in one generation which expedites plant breeding strategies by providing inbred lines, of genetic choice, for assessment (Patel, Reinbergs & Fejer 1985). The successful use of a DH lines in a breeding program requires that they are analogous to conventionally derived lines, in agronomic performance (fitness) and stability (disease resistance).

FUNGAL PATHOGENS AND BARLEY

Fungal pathogens

Commercially fungal disease impacts and decreases both the physiological performance and plant biomass of major cereal crops and results in yield reductions in terms of harvested seed (Bilgic, Steffenson & Hayes 2005). Barley susceptibility to fungal disease causing pathogens is highly dependent on growing environments, genetic make-up of the variety and fungal pathogenicity (Fetch & Steffenson 1999; Kumar et al. 2001; Valjavec-Gratian & Steffenson 1997).

Global regions that had been found to have higher occurrences of fungal susceptibility based on environmental factors included those of high temperature; humidity and poor drainage. In Australia regions of northern NSW and southern QLD Darling Downs are most affected by fungal disease for these very reasons and moreover, rain and heavy dew provide ideal conditions for a primary infection. Genetic information regarding the make-up of the hosts’ resistance gene repertoire is aiding the production of more disease resistant barley lines in Australian breeding programs, however on a global scale the information relating to the variability of virulence of a pathogen between isolates is an essential requirement for future selection trials, particularly for programs involved in diverse global regions.

Bipolaris sorokiniana

The fungus Bipolaris sorokiniana (anamorphic) causes the disease spot blotch (Kumar et al., 2002) on barley (Hordeum vulgare L.). Necrotic spots on the leaf surface characterise the phenotypic appearance of the disease (Almgren et al., 1999). Infection occurs following the germination of conidiospores on the leaf surface, and the direct penetration of hyphae through the cuticle of the host plant into the mesophyll tissue of the leaf forming a colony (Alcorn 1988);(Kumar et al. 2001).

N.B.: Magnified Conidia of B. sorokiniana, showing brown colouring and multiple septations (Figure source-Knight 2007 thesis)

Following infiltration, Bipolaris sorokiniana produces toxins that interact with host membranes, causing cell death through metabolites leaking to surrounding tissue, and breaking down biological processes (Kumar et al. 2002). The toxins described below are responsible for and induce the necrosis of both leaf material and chlorophyll in plant tissue.

Bipolaris sorokiniana is an asexual fungus belonging to:

  • Class: Loculoascomycetes
  • Order: Pleosporales
  • Family: Pleosporaceae

The three main phytotoxins produced by Bipolaris sorokiniana are:

  • Pre-helminthosporol -Most prolific; weakens cells, inhibits enzyme activity
  • Helminthosporol -Increases the permeability of membranes
  • Sorokinianin – Inhibits barley seed germination

The fungus reproduces by means of conidia which develop from conidiophores (figure 5). Conidiophores are plain usually tubular, erect and septate. The sexual state of the fungus (Jana & Bailey 1995) has, on the odd occasion been encountered in nature.

Astoundingly a single spore can travel up to 1000 km on prevailing wind currents (Bilgic, Steffenson & Hayes 2005). This highlights the need for “elite? host germplasm that confers a resistance to all regional pathotypes of the pathogen (Almgren et al. 1999; Kumar et al. 2002; Valjavec-Gratian & Steffenson 1997; Zhong & Steffenson 2002).

Spot blotch

The spot blotch infection, caused by Bipolaris sorokiniana, begins as a browny/ black spot on leaf sheaths that progress up the leaves during crop development. Depending on this progression, complete loss of biomass is possible, resulting in major yield and biomass reductions, especially on seedling barley plants (Kumar et al. 2002). Adult plants that are susceptible to the disease have lesions that continue to coalesce forming blotches that envelop and destroy large sections of the leaf. Furthermore, severely infected leaves are found to senesce prematurely (Valjavec-Gratian & Steffenson 1997). On the whole infectivity is usually directly related to the pathogenicity of the individual fungal isolate in question.

Pathogenicity studies of fungal isolates on barley are generally based on exposing sets of barley varieties of varying resistances to a range of pathogen isolates with the intention of evaluating their susceptibilities (Fetch & Steffenson 1999). In the case of the spot blotch, caused by the fungal pathogen Bipolaris sorokiniana, an internationally agreed set of barley lines is yet to be established, hampering the characterization of resistances present in Australian barley germplasm.

Resistance in barley to fungal disease is characterized based on the severity of the infection using the scale of infection response (IR) 1 – 9. The 1-9 rating scale detailed by Fletch and Steffenson (1999) is based on the presence of necrosis and chlorosis and the relative size of spot blotch lesions observed on the second leaves of barley seedlings. These infection responses (IRs) are classified into three general categories of low (IRs 1 to 3), intermediate (IRs 4 and 5) and high (IRs 6 to 9). Low IRs (Figure 6-1; 2; 3) show small necrotic lesions with little or no marginal chlorosis. Intermediate IRs display medium-sized necrotic lesions, with distinct chlorosis at margins, while high IRs (7; 8; 9 –Figure 6) appear as large necrotic lesions with distinct margins and expanding chlorosis. These responses are critical for the use in QTL mapping studies, which associate genetic components being the DNA to phenotypic appearances of a barley accession.

The photo shows the different barley IRs; 1 being highly resistant and 9 highly susceptible. In figure 6 below the leaf on the far right shows a higher susceptibility compared to the far left leaf, with visual disease scores of 9 and 1 respectively.

Barley accessions, over time, have been selected for by means of their phenotypic attributes. Consequently the molecular revolution and the birth of DNA markers provided the platform for genotypic attributes to be associated with already known phenotypes. DNA markers are reliable tools that associate genes and traits, and are a critical step in generating a map of any species based on their DNA and polygenic trait loci.

Quantitative Trait Loci (QTL)

Quantitative Trait Loci (QTL) is a term given to the number of gene combinations possibly expressed simultaneously in a given genome, and at different intensities. The intensity is dependent on and is defined as Quantitative-continuous and measurable; Trait- characteristic expressed by a gene; and Loci- a chromosomal location. QTL analysis is a reliable method for estimating genetic diversity and is crucial for studying populations especially for significant traits i.e. grain quality. A classical approach to the identification of loci involved in complex polygenic (Quantitative) traits consists of screening large numbers of individuals from segregating populations with a suite of markers that are evenly distributed throughout the genome, with subsequent statistical analyses indentifying regions of the genome that are involved in the trait. (Igartua et al. 2000)(Marquez-Cedillo et al. 2000).

Diversity arrays

Diversity Array Technology (DArT) is a new DNA hybridisation-based genotyping technology that allows whole genome scanning, for identifying common genetic components, using a microarray platform. Crop improvement has relied on the effective use of genetic diversity. Molecular marker technologies are promising to increase the efficiency of managing genetic diversity in breeding programmes. Numerous marker technologies have been developed over the last few decades. The most commonly used systems, used have included restriction fragment length polymorphism (RFLP) and microsatellites or simple sequence repeats (SSR). Using these technologies has aided other methods that can manage large scale diversity screening for particular loci (Wenzl et al., 2004). Such technologies have included Diversity arrays technology (DArT) which can type hundreds to thousands of genomic loci in parallel to determine similarities geno-typically which is then compared to conventional phenotypic screening. Moreover DArT was developed as a hybridisation-based alternative marker system for genomic DNA, similar in principle to the microarray platforms for large scale assessment of gene expression.

Doubled haploid lines are benchmark in verifying the Mendelian behaviour of DArT markers. Adding DArT markers to an already established data set of conventional markers (RFLP, SSR) further aids the verification of their linkage to agronomically important traits. A comparison of logarithm of the odds (LOD) scores, call rates and the degree of genome coverage will then indicate the quality and information content of the DArT data comparable to that of the combined original SSR/RFLP data set (Wenzl et al. 2004). This allows high-throughput screening of hundreds of molecular markers simultaneously and is particularly suited to genome-wide analysis.

MOLECULAR MARKERS AND LINKAGE MAPPING

Molecular Markers

Complete genetic maps, based on DNA markers, have been developed for all seven barley chromosomes and consequently been used in QTL analysis for many agronomical important traits (Kleinhofs et al. 1993). An important use of markers has been marker assisted selection (MAS),which is making possible the identification of markers linked to commercially important traits such as disease resistance (Graner & Tekauz 1996), response or tolerance to abiotic stress (Forster et al. 2000) and seed or feed quality traits (Han et al. 1997).

Molecular markers can used to easily discern phenotypic traits by:

  • Probing DNA-marking chromosomal regions
  • And by acting as beacons, which spatially identifies DNA segments that are specific to the markers that compliment their DNA.

Molecular Markers may be applied for a number of purposes:

  • Genetic identity - Parentage (maternity and paternity)
  • Differentiation of geographic populations
  • Phylogenetic relationships of species, family, genera, orders, phyla
  • Differentiation of Populations for various traits including disease resistance, drought tolerance etc

Ideal Marker Properties:

  • Highly polymorphic
  • Co-dominant inheritance (resolution of homozygous and heterozygous states of diploid organisms)
  • Frequent occurrence in a genome
  • Easily accessible (availability)
  • Simple and rapid assay
  • High reproducibility between samples and laboratories

Restriction fragment length polymorphisms

Restriction fragment length polymorphisms (RFLP) are naturally occurring Mendelian characteristics and are the basis to the development of the earliest type of DNA marker used. They arise through DNA rearrangements due to evolutionary processes, such as point mutations, which arise within and alter the restriction enzyme (RE) recognition site sequence. Altered genomic combinations (in RE sequences); are created by independent chromosomal pairing, or to a lesser extent through crossing-over between alleles on chromosomes during meiosis Gave rise to the first marker analysis based on length polymorphisms. RFLP analysis, makes use of restriction enzyme-digested genomic DNA and resolved by gel electrophoresis, with specific banding patterns blotted (transferred) onto a nitrocellulose membrane then visualized by hybridization with labelled probe/s. RFLPs are a reliable marker for use in linkage analysis and breeding and can easily determine if a linked trait is present in a homozygous or heterozygous state in an individual (figure 7).

The diagram details the intricate process of RFLP analysis showing the RE digestion, Blotting (3) to visualising specific bands bound to probes figure source: Genomes 2.

However, their usefulness has been hindered, due to the large amount of DNA required for restriction digestion and Southern blotting. Additionally the need for radioactive isotope makes the analysis somewhat expensive and hazardous. Furthermore the assay is time-consuming and labour-intensive.

Lately, uses for RFLP markers that are specifically linked to a desired trait can be converted into PCR-based sequence tag-site (STS) marker which is based on the nucleotide sequence of the marker producing a polymorphic band pattern for a specific amplicon. Using this technique, removes the need for hybridization procedures involved in RFLP analysis and as a result STS markers are on the increase. Other non-labour intensive marker systems that are robust and reproducible include simple sequence repeats or “microsatellites? a more common term.

Microsatellites

Microsatellite markers were first utilized in studies of humans (Tautz 1989) and later in plant studies (Morgante & Olivieri 1993). The advantage of microsatellite markers are that they are co-dominant and reveal multiple alleles at a single locus. (Venter et al. 2001). Microsatellites, or simple sequence repeats (SSRs), are stretches of DNA consisting of tandemly repeated short units of 1–6 base pairs in length. They are highly variable in the number of repeats they contain and are co-dominantly inherited, which makes them ideal genetic resources, to investigate for polymorphic marker content (Ramsay, Luke et al. 1999). Microsatellites are distributed throughout eukaryotic genomes, within and between coding regions and are in abundance at centromeric and telomeric regions respectively (Gadaleta et al. 2007; Ramsay, Luke et al. 1999; Struss & Plieske 1998). Microsatellites are simple to use, cheaper and more reliable than RFLPs, as RFLPs are very time consuming and technically challenging- creating a higher risk for experimental error, jeopardising reproducibility between laboratories.

A downside to generating genotypes by screening barley or any plant with a DNA marker, which is near but not actually in a gene, is the error caused when a recombination event occurs between the marker and the actual gene of interest. This error is directly proportional to the distance that the marker is from the gene.

A Marker, being 1cM from a gene is said to have a 1% chance that the linkage between it and the gene will be broken; alternatively there is a 99% chance that the marker and the gene will be linked following meiosis. The closer the marker is to the gene the more accurate the analysis is, yet the best marker is one that in essence hybridizes to the particular gene in question. These recombination events, depicted through markers, serve one main purpose-generating genetic linkage maps. Maps are based on genotypes that are determined by PCR and resolved through the mobility in gel electrophoresis of PCR amplicons of DNA of a given length. The mobility of single strands can vary considerably as a result of only small changes in length (Lehmensiek et al. 2005; Marquez-Cedillo et al. 2000; Zhong & Steffenson 2002).

Barley Haplotyping

The haploid genotype (n), of any organism, in genetics is described by the term haplotype. A haplotype (Greek haploos = single) is a combination of alleles at multiple loci that are transmitted together on the same chromosome between related species. Genotypes based on marker alleles at different loci make a haplotype. Identical genotypes thus confer linking of genetic characteristics during meiosis and that related individuals share the characteristic (Landwehr et al. 2007).

Generally, distinct haplotypes exist in barley between both the disease susceptible and resistant accession, and that on this basis, haplotypes are the differentiating factor used to experimentally select for resistant barley line/s using a gene pyramid (Landwehr et al. 2007). A haplotype based analysis is more informative than a single marker in associating an allele with a trait, and has more power in analysing associations with phenotypes between cultivars. (Rafalski 2002).

Genetic / linkage mapping

The idea that individual genes occupy regular positions on chromosomes was one of the great insights of early genetics, with the very first genetic map published in 1913 by Alfred H. Sturtevant, who was working on fruit flies in the laboratory of Thomas H. Morgan at Columbia University (Sturtevant 1913).

A linkage map is a genetic map of a species or experimental population that shows the position of its known genes and/or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome (Pozzi et al.). The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. Historically, the markers originally used were detectable phenotypes (enzyme production, eye colour) derived from coding DNA sequences; eventually, confirmed or assumed non-coding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used.

In individuals of experimental populations or species, several phenotypes or traits occur randomly with respect to one another in a process known as independent assortment. As a consequence scientists understand that independent assortment occurs when the genes affecting phenotypes are found on different chromosomes or separated by a great enough distance on the same chromosome that recombination occurs at least half of the time. An exception to independent assortment develops when genes appear near one another on the same chromosome as described earlier. When genes occur close to each other on the same chromosome, they are usually inherited as a single unit (meiotic recombination rates are low). Genes inherited in this way are said to be linked, - multiple genes in the same chromosomal region, and are often referred to as "linkage groups? (Li et al. 2003; Michalatos-Beloin et al. 1996; Ramsay, L. et al. 2000).

LOD scores and linkage

A LOD is the likelihood of odds logarithm. Simply it is a mathematical tool that scores the likelihoods of marker loci linked on chromosomes. The likelihood is the probability of observing particular data given a specific value of the parameter(s) of the underlying hypothesized statistical process (probability distribution function). The usual likelihood used in linkage analysis is the binomial as applied to the recombination fraction, between a success and a failure being linked or not between progeny in a SRS of a population.

By convention, a LOD score greater than 3.0 is considered evidence for linkage. (A score of 3.0 means the likelihood of observing the given pedigree if the two loci are not linked is less than 1 in 1000). On the other hand, a LOD score less than 2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. LOD scores play a significant role in ordering and aligning markers on a linkage map.LOD scores are used to assess the linkage of traits to a particular chromosomal region.

PROJECT OUTLINE

Project Aims

This project aims to further increase SSR marker densities on a variety of barley genetic maps of local DH barley lines. And to confirm the haplotype for the resistant loci linked to Bipolaris sorokiniana. The polymorphic markers were then screened across each population.

Eight parental cultivars were screened, which included the 3 crosses mentioned previously and a further 2 independent lines Bowman and Morex, to confirm the haplotype/s of the resistant loci linked to Bipolaris sorokiniana.

The 3HS chromosome arm in the barley doubled haploid population ND11231– 12 / VB9524 contributes to resistance to spot blotch (caused by Bipolaris sorokiniana), in adult barley plants in the field (Bovill et al. 2007). While a genetic map is available for this population, there are very few SSR polymorphic markers which map to the 3HS arm, making an accurate location of the resistance locus along the arm very difficult to determine. Further polymorphic markers on chromosome 3HS in the ND11231 – 12 / VB9524 will be sought through screening article databases for any new sets of simple sequence repeat (SSR) markers that have become available.

Secondly, sources of resistance to this disease that may be unrelated to the North Dakota line ND112, have recently become available. A line developed in Winnipeg, Canada WPG 8412, which shows good resistance to spot blotch has been crossed to the Australian susceptible line Lindwall to produce a DH population in which the progeny have been screened phenotypically for adult plant resistance to spot blotch. Another population, TR251/Gairdner, will also be screened with the generated polymorphic markers from the haplotyping to determine whether there is linkage to known regions of resistance from the ND/VB-WPG source.

Project Significance

Genetic information on the variability of resistance in barley to the pathogen Bipolaris sorokiniana will aid in developing a marker selection system. This system will allow breeders to optimise selection, pyramid genes from different sources and ultimately produce varieties which provide useful resistance against spot blotch yet retaining viable marketable characteristics in specific regions of Northern NSW and SE QLD.

CHAPTER 2 MATERIALS AND METHODS

General Introduction

Currently the genetic basis and its involvement in the disease spot blotch is poorly understood. Complicating matters are the variations in virulence of identified fungal pathotypes which hinders absolute quantification of this disease both in the glasshouse and field. At present any increase in the understanding of the genetics to resistance against spot blotch is entirely attributable to the advances through technologies such as molecular markers.

Marker assisted selection (MAS) essentially targets loci, around and in close proximity to desired genes. MAS has allowed the early selection of traits before phenotypic evaluation is possible simplifying selecting traits that are often difficult to score. Several requirements must be fulfilled before markers can be used in selection. Of these close linkage between markers and the target gene (Wricke, Dill & Enft 1996) is vital.

A number of studies in the USA (Steffenson, Hayes & Kleinhofs 1996) using DH populations, have observed that seedling resistance to spot blotch was inherited and governed by a single gene (Rcs5) on the short arm of chromosome 7H. In other studies this has been challenged. A report by (Bilgic, Steffenson & Hayes 2005), compared the differential expression of spot blotch resistance in several DH populations, the results showed that in 75% of the populations seedling resistance was conditioned by a number of genes.

The major QTL which was identified near the Rcs5 gene on chromosome 7H explains 30% of the phenotypic variance and another two minor QTLs identified; one near the centromeric region of chromosome 7H and one on the short arm of chromosome 3H explaining 19% of the phenotypic variation.

This chapter details the materials and methods used to validate SSR markers to the QTLs associated with resistance to Bipolaris sorokiniana.

Trial Data History

Jessica Bovill, former USQ Masters Student in conjunction with the GRDC provided the 2004/06 field and 2005/06 seedling trial data for ND11231 – 12 / VB9524 and TR251 / Gairdner Double Haploid (DH) populations used in this study. This work is based on her final curation map, and the marker regression for the season 2004/6 barley field trial data in map manager QTX was the foundation to which candidate markers were determined.

Regression, as described by (Kearsey & Hyne 1994) is a result of the additive difference between marker genotype means, at a locus, against a function of the recombination frequency between that locus and a putative QTL. The marker regression for the DNA region, contributing resistance to spot blotch, was defined through the highest association between marker and trait (highlighted locus/MapManager QTX). This method, among several, to determine marker associations was used in this study. The highlighted locus is the chromosomal position to have the greatest association to the QTL. These locations including the immediate flanking regions were used as parameters to anchor markers to regions on the 7H and 3H regions of the published consensus maps. The highlighted locus HvGLTP4x3D001 from the TR/G marker regression was used to aid in the development of the markers for the 3H region of ND/VB.

Plant Materials

This study utilized three DH Barley populations developed through crossing North American pedigrees (ND; TR251 and WPG) with Australian varieties (VB; Lindwall and Gairdner). The North American pedigrees were established cultivars that show resistant to Bipolaris sorokiniana. The Australian varieties, on the other hand, displayed greater susceptibility. Table 1 details the three populations.

other cultivars- Morex and Bowman were included during the haplotyping.

Seed germinations

In order to apply DNA molecular marker technology, clean DNA was required. DNA must be extracted from each individual in the mapping population including the parents (Collard et al. 2005).

While DNA was already available from the TR251 x Gairdner population, it was necessary to extract DNA from individuals in the ND/VB and WPG/Lindwall populations. Individual lines from both populations were germinated on tissue in 24-well ELISA plates under moist conditions at 25oC until approximately 2.5 inches of primary leaf blade was visible (Figure 8). At this stage the plates were transferred to the molecular laboratory, where under ice cold conditions, approximately 50ug of leaf was collected, chopped up and transferred to a clean 1ml microfuge tube.

Pictured is germinating barley at 4 days of incubation. 3 seeds were placed in each well and kept moist until ready to harvest (Photo-Chris Leist).

DNA Extraction

DNA extraction followed the Wizard Genomic DNA method, developed by Promega®. The full extraction protocol is given in Appendix 1.

The 1ml microfuge tubes containing the leaf matter, together with mechanical force in a tissue-lyser (Qiagen®), released the DNA into solution. This lysate, following incubation @ 65oC, had its RNA removed (RNase) and consequently with further incubation all proteins were precipitated with protein precipitation solution and pelleted by centrifugation. All the supernatant was retained and added to tubes containing 100% isopropyl alcohol. Thus, following two final rounds of centrifugation, the first to pellet the DNA and the second to wash it, using 70% ethanol, the DNA was rehydrated in 100uL of H2O once the supernatant had been discarded.

DNA Quantification

A Random DNA Sample was taken from all three populations. Each random sample was subsequently combined with dye and run @ 90 volts through a 1% Agarose gel electrophoresis. After 20 -30 minutes, depending on the visible band front. Ethidium Bromide (EtBr an intercalating agent) in the presence of UV, generated base pair luminescence that was visibly quantified using a GelDoc system (Bio-Rad). A further SRS, containing both the agarose quantified DNA samples and those that were in the original set, were taken from all three populations.

Candidate Markers & PCR

The molecular map for the ND/VB population consisted of a high density of AFLP markers. Chromosomes 3HS and 7H were considered significant in the expression of resistance, based on QTL analysis. These regions were screened with a total of 28 SSR markers and 2 Dart derived SSRs. Candidate SSR markers were selected from published barley consensus maps. Primers were synthesized by Invitrogen (Australia). Thermal cycling conditions for SSR markers were given by GrainGenes.

PCR was conducted on candidate markers. Annealing temperature was the only setting to differ between the markers. DNA samples were amplified by Polymerase Chain Reaction (PCR). PCR was conducted in a 10 µL multiplex 96 well reaction containing: 500 nM of each primer; 1.5 mM MgCl2; 200 µM of each dNTP; 1 x PCR buffer; and 0.5 U Taq DNA polymerase. Thermocylcing was carried out with an initial 5 min 94°C denaturation step, followed by 30-45 cycles of: 94°C for up to a minute; 50-60°C (depending on the annealing temperature) for 60 s and 72°C for a minute.

Electrophoresis

Electrophoresis of PCR product was performed using the Corbett Gelscan 2000. A gel mix of 15 mL containing poly-acrylamide, and 10 x TBE (890 mM Tris, 890 mM boric acid, and 20 mM EDTA) was prepared with MilliQ water. 100 µL of ammonium persulfate (10% w/v) and 10 µL of TEMED were added to the gel mix following de-gassing. The gel mixture was poured between two glass plates (spaced 0.1 mm apart); the upper plate, with the well indentations, had previously been treated with bind silane. 0.4 µL of PCR product was loaded on to the gel and the gel was run at 1200mV for between 15-35 minutes depending of band visibility. Images were saved and later viewed to genotype in TIFF format.

Linkage analysis and map curation

Polymorphic SSR markers were identified by screening across all six parents (haplotyping). Following, the parental analysis the entire mapping population from each cross was screened with all indentified polymorphic markers. Typically, the markers segregate in a Mendelian fashion (DH=1:1), allowing parental and recombinant lines to be genotyped (Martin, Williams & Tanksley 1991) and through a Marker regression the marker-trait associations were determined indentifying the relationship with a QTL.

The final step in the construction of the linkage map is to analyse the linkage of markers to determine whether an association exists between the markers and a quantitative trait. The linkage analysis of markers was performed by the computer program, MapManager QTX (Collard et al. 2005).

CHAPTER 3 RESULTS

General introduction

The fungal pathogen Bipolaris sorokiniana (teleomorph-Cochliobolus sativus), on barley, stress causing the foliar disease spot blotch (SB). SB poses serious disease constraints to barley production in warmer growing regions of the world, with estimated yield losses ranging from 30-70%. While chemical treatments e.g. fungicide may aid in controlling spot blotch infections, the most effective and environmentally friendly approach to control this disease is breeding for varieties with natural resistance. In Australia, no commercially available varieties offer resistance to SB. This study has sought to further establish molecular markers, in particular simple sequence repeat markers, that will be useful for selecting for resistance to this important fungal disease.

The double haploid (DH) population ND11231-12/VB9524 consists of 180 lines and was developed by Emebiri et al. (2005) to investigate and identify regions of the barley genome that influence variations in grain protein concentration. ND11231-12, which shows a high level of resistance to spot blotch, originated from breeding programs at the North Dakota University, Fargo, USA. ND11231-12 is a narrow leafed two-row barley line derived from Karl and sister line Logan, another source of the low-protein genes which was released from the North Dakota in 1995. The susceptible line VB9524 was developed by the Department of Primary Industries, Victoria, Australia, and is an advanced selection from a cross of Arapiles with Franklin (Emebiri et al., 2005). The Department of Primary Industries, Victoria, Australia, kindly supplied seed from this DH population.

At present of all the alleles identified in wild barley, only 40-56% exists in global germplasm collections. This leaves a large proportion of genes that could be used in cultivated barley that would otherwise benefit breeders (Ellis et al. 2000). In Australia barley cultivars susceptible to fungal diseases- such as spot blotch, caused by the fungus Bipolaris sorokiniana, have been crossed to disease resistant North American cultivars. The outcomes of these crosses have provided parental types derived from pedigrees that are known to be inherently low in grain protein and thus have marketable attributes.

As a result more efficient tools have been employed, to capture all the successes of selection lines, such as the sophisticated methods designed for the production of double haploids from elite parent lines. DH production is a relatively new technique to assist and accelerate barley (Hordeum vulgare L.) breeding (Kuhlmann & Foroughi-Wehr 1989). This method completely guarantees all the progeny to share either genetic makeup from whichever parent donated their haploid genome.

This chapter at first will detail the results beginning with parental haplotyping Then flanking marker validation through to development. The latter part looks at the map curation.

Haplotyping

The eight parentals were screened with the respective markers. The following figure shows their genetic relationship.

A= Male parental inheritance-yellow; B=Female-red. P- denotes dominant marker present, M-missing. Fragment sizes are given in base pairs (bp).

Each colour individually represents the parental genotype based on the marker specified. It can be seen that the marker bpb 3865, derived from DArt sequences, was the marker of choice to map our populations as according to the haplotyping which showed the right fragment size and locus.

Linkage association

The table shows the final links report for the 2 markers bPb-3865 and scssr10559. Their LOD scores show the association of the highlighted locus XP13M61-168.

The major 7H QTL identified in the expression of both seedling and field resistance to spot blotch is highly associated with the regions between the SSR markers EBmag794 and Ebmac603. The highlighted locus within this region was Est31>2 which shows the greatest association (table 2) to the trait with flanking markers HVM4 above and EBmac603 below.

The 3H region in the ND11231-12/VB9524 population is linked to the AFLP marker XP13M61-168, in addition to chromosome 3HS, a region on the 7HS chromosome arm inherited from ND11231-12, has a significant effect on the expression of field spot blotch resistance. Table 2 shows the percent (%) variance explained by a given marker.

The major 3H QTL identified in the expression of field resistance to spot blotch is highly associated with the region marked by P13M61-168. The highlighted locus, being the region with the highest association to the trait, was validated using the statistic “stat? (Table 2). The “stat’ is measured as a LRS score and when multiplied by 0.217, gives the LOD statistic.

Using TR/G as a background population the marker HvLGTP4x3D001 showed the greatest association (table 3) to the trait with flanking markers GBM1159 above and scssr10559 below.

Linkage mapping

A total of 28 microsatellite markers were developed to detect polymorphism between the three DH populations and their parents. Of the 28 developed 12 markers were polymorphic/ and showed the parental segregating haplotype (Figure 10). From these 12, markers bPb-3865 and scssr10559 were mapped in the ND/VB population within the region flanking the highlighted locus HvGLTP4x3D001 sourced through the TRr251 marker regression. The overall map distances for the population was 210.2cM. A further 7 markers were screened across the WPG/Lindwall population; 4 on the 3H short arm, and 3 on the 7H chromosome. However due to time restraints only genotyping and linkage analysis was performed on this population.

Shown here is the original linkage map for ND/VB, also shown is the revised map with markers bPb3865 and marker scssr10559 flanking marker P13M61-168. The QTL is denoted by the black line on the left of the figure. Marker bPb3865 is shown to closely flank the QTL.

Each fragment depending on parental type was labelled either A or B. The first two bands in the gel are from each parent, the first at approx.200Bp as referenced by the dark banding of the ladder on the left with the second band length at approx.210Bp. The segregation can be seen between both loci in a predicted 1/1 ratio, differentiated by fragment migration distance.

LOD = Likelihood of odds ratio; VAR = Percent of phenotypic variation explained. The The construction of the genetic map and genetic linkage was based on informative markers/alleles. Each marker was scored, A for maternally inherited loci and those paternally inherited were typed B. The Chi-square statistic was p=0.01 and an additive regression model having no control over other QTLs was used. A P-value of 0.01 indicates a 1% probability that these results would have been obtained in the absence of a marker-trait association; the lower the P-value, the higher the probability that a QTL truly exists in the region of the marker (Han et al. 1997). Chi square analysis determined the significance of the observed genetic segregation ratios of alleles compared to their expected. A total of 12 double cross-over were removed from ND/VB.

CHAPTER 4 DISCUSSION

Discussion

As a step towards further understanding the structural genomics of the locally bred cultivars we generated several SSR markers that showed linkage to significant chromosomal regions known to contribute resistance to bipolaris sorokiniana and incorporated them into existing DH barley genetic maps.

Previously identified on the 3HS chromosome as responsible for the expression of field spot blotch resistance contained few SSR markers. A total of 28 SSR markers which were located within 20cM from the trait were screened across ND, VB, TR251, Gairdner, Lindwall and Winnipeg parents in order to map regions with SSR-PCR-based markers. Of the markers screened, very low levels of polymorphism were observed. The SSR markers scssrr10559 and bPb3865 mapped to the short arm of 3H in ND/VB, the marker trait association was good explained by a LOD >3.

Both populations TR/G and WPG/L were excluded in the final results due to several factors. Firstly when the data for the TR/G population was analysed using MapManager a high number of double cross over’s were encountered. This was attributed to contaminated DNA and operator error. With the WPG/L population, as table 2 shows, a rather low percent of phenotypic variation was shown by the markers employed. As a result both the TR/G and WPG/L data was excluded from examination.

The use of F-1 derived DH crosses for identifying QTL controlling traits that have agronomical and malting importance in crop species is well documented (Paterson et al. 2000). These estimates of QTL effects are based on a range of genetic reference populations and analytic procedures. The key advantages for barley’s use in QTL analysis is that many cross combinations show meaningful intraspecific polymorphism for agronomical important traits (Kleinhofs et al. 1993). Secondly, genetic reference populations of doubled haploids can be readily created, producing populations that simplify map construction and QTL estimation (Hayes et al. 1993).

A QTL may be the target for map-based cloning or molecular marker-assisted selection (MAS). In the latter case, if linkages can be established between genes controlling quantitative traits and molecular markers, indirect selection may provide greater gains than direct selection. Direct selection, as the name suggests, selects significant agronomically important phenotypes through field trial observations. This can prove costly and labour intensive. As a result, molecular technology has advanced at such a rate that the option of indirectly selecting favourable phenotypes through genotypic analysis has gained popularity(Holton et al. 2002; Steffenson, Hayes & Kleinhofs 1996).

Good map curation techniques significantly improve the quality of genetic maps. Lehmensiek et al. (2005) showed that map curation, through the reordering of markers and editing marker data for double crossovers, improves map resolution and the magnitude of the marker-trait association; both of which have significant impacts on QTL detection. In the case of the original ND/VB map (Emebiri et al., 2005), the addition of further SSR markers, the reordering of markers using the program Links Report and the removal of doubled crossovers, greatly improved the quality of the genetic map and significantly decreased the overall map distance and average chromosome length.

Marker order has been shown to substantially impact on QTL detection of quantitative traits (Collard et al., 2005). The links report, allowing manual marker ordering, is viable for small datasets, however the program RECORD orders markers by minimising recombination events and is capable of dealing with large datasets for the construction of high density genetic linkage maps (Van Os et al., 2005).

In addition to marker order, genotypic errors can significantly effect linkage map construction and overall map distances (Collard et al., 2005; Lehmensiek et al., 2005). Studies by (Hackett & Broadfoot) have shown, through the analysis of simulation data, that non-random typing errors resulted in large increases in map length and missing values had less of an impact on map distances. The removal of suspected genotyping errors by changing double crossovers between markers less that 25 cM apart to a missing score also had a significant impact on chromosomal length. Markers that increase the distance of chromosomes are generally disregarded.

QTL analysis aims to detect an association between markers and a QTL or gene controlling the phenotypic expression of a quantitative trait. Markers are used to partition the mapping population into different genotypic classes according to the genotype at a particular marker locus. As a result it can be determined whether a significant difference exists between the classes with respect to the trait being measured (Martin et al. 1993). A significant difference between phenotypic means of the groups indicates that QTLs controlling the trait are linked to the marker locus used to partition the mapping population (Collard et al. 2005). This was shown with our 2 markers developed for the ND/VB DH cross.

Future Directions

Marker based identification of agronomical important traits in populations barley is now the way of the future. As technology advances, more cost effective ways of generating genetic analysis arises. At the beginning of the millennium AFLPs and RFLPs were still common practise in breeding programs. More recently the cost effective and robust nature of SSR markers has led the way for cereal crop genetics. At present comparative genomics, together with single nucleotide polymorphic markers are revolutionising cereal breeding. It is apparent that SNP markers are becoming increasingly useful in cereal genetics (Landwehr et al. 2007). The decreasing cost of SNP genotyping is also likely to make genetic map construction feasible, although it should be remembered that SNPs are not ideal for mapping in outbred populations. Cheaper SNP genotyping should also result in the wider uptake of population genomics.

Both the WPG/L and TR/G populations need further analysis. The errors in genotyping and the poor DNA quality led to the exclusion of these results.

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