Genetic map construction and QTL mapping of resistance to blackleg (Leptosphaeria maculans) disease in Australian canola (Brassica napus L.) cultivars.

Genetic map construction and identification of quantitative trait loci (QTLs) for blackleg resistance were performed for four mapping populations derived from five different canola source cultivars. Three of the populations were generated from crosses between single genotypes from the blackleg-resistant cultivars Caiman, Camberra and (AV)Sapphire and the blackleg-susceptible cultivar Westar(10). The fourth population was derived from a cross between genotypes from two blackleg resistant varieties (Rainbow and (AV)Sapphire). Different types of DNA-based markers were designed and characterised from a collection of 20,000 EST sequences generated from multiple Brassica species, including a new set of 445 EST-SSR markers of high value to the international community. Multiple molecular genetic marker systems were used to construct linkage maps with locus numbers varying between 219 and 468, and coverage ranging from 1173 to 1800 cM. The proportion of polymorphic markers assigned to map locations varied from 70 to 89% across the four populations. Publicly available simple sequence repeat markers were used to assign linkage groups to reference nomenclature, and a sub-set of mapped markers were also screened on the Tapidor x Ningyou (T x N) reference population to assist this process. QTL analysis was performed based on percentage survival at low and high disease pressure sites. Multiple QTLs were identified across the four mapping populations, accounting for 13-33% of phenotypic variance (V (p)). QTL-linked marker data are suitable for implementation in breeding for disease resistance in Australian canola cultivars. However, the likelihood of shifts in pathogen race structure across different geographical locations may have implications for the long-term durability of such associations.

Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (Lolium perenne L.).

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species.

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

Individual and multi-environment combined analyses identify QTLs for morphogenetic and reproductive development traits in white clover (Trifolium repens L.).

White clover (Trifolium repens L.) is a key component legume of temperate pasture agriculture and an important target for molecular marker-assisted plant breeding. A genetic map of white clover has been used to assess genetic control of agronomically important traits that vary in the F2(I.4RxI.5J) mapping family. Phenotypic analysis was performed for a range of vegetative morphogenesis traits (such as leaf area, internode length, plant height and plant spread) and reproductive morphogenesis and development traits (such as flowering date, floral intensity and seed yield), with both spatial and temporal replication. A multi-environment combined analysis (combined analysis) has been performed for traits assessed across multiple experimental datasets in order to identify consistent genetic effects. Quantitative trait locus (QTLs) were detected for the majority of traits, and the locations and magnitudes of QTL effects were compared between individual and combined analyses. This molecular genetic dissection of agronomic traits in white clover provides the basis for equivalent studies in more complex populations, design of marker-assisted selection strategies and comparative genetics with model legume species. Selection for QTLs derived from the combined analysis will permit robust improvement of phenotypic traits over different environments.

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation.

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.

Molecular cloning and genetic mapping of perennial ryegrass casein protein kinase 2 alpha-subunit genes.

The alpha-subunit of the casein protein kinase CK2 has been implicated in both light-regulated and circadian rhythm-controlled plant gene expression, including control of the flowering time. Two putative CK2alpha genes of perennial ryegrass (Lolium perenne L.) have been obtained from a cDNA library constructed with mRNA isolated from cold-acclimated crown tissue. The genomic organisation of the two genes was determined by Southern hybridisation analysis. Primer designs to the Lpck2a-1 and Lpck2a-2 cDNA sequences permitted the amplification of genomic products containing large intron sequences. Amplicon sequence analysis detected single nucleotide polymorphisms (SNPs) within the p150/112 reference mapping population. Validated SNPs, within diagnostic restriction enzyme sites, were used to design cleaved amplified polymorphic sequence (CAPS) assays. The Lpck2a-1 CAPS marker was assigned to perennial ryegrass linkage group (LG) 4 and the Lpck2a-2 CAPS marker was assigned to LG2. The location of the Lpck2a-1 gene locus supports the previous conclusion of conserved synteny between perennial ryegrass LG4, the Triticeae homoeologous group 5L chromosomes and the corresponding segment of rice chromosome 3. Allelic variation at the Lpck2a-1 and Lpck2a-2 gene loci was correlated with phenotypic variation for heading date and winter survival, respectively. SNP polymorphism may be used for the further study of the role of CK2alpha genes in the initiation of reproductive development and winter hardiness in grasses.

QTL analysis and comparative genomics of herbage quality traits in perennial ryegrass (Lolium perenne L.).

Genetic control of herbage quality variation was assessed through the use of the molecular marker-based reference genetic map of perennial ryegrass (Lolium perenne L.). The restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP) and genomic DNA-derived simple sequence repeat-based (SSR) framework marker set was enhanced, with RFLP loci corresponding to genes for key enzymes involved in lignin biosynthesis and fructan metabolism. Quality traits such as crude protein (CP) content, estimated in vivo dry matter digestibility (IVVDMD), neutral detergent fibre content (NDF), estimated metabolisable energy (EstME) and water soluble carbohydrate (WSC) content were measured by near infrared reflectance spectroscopy (NIRS) analysis of herbage harvests. Quantitative trait locus (QTL) analysis was performed using single-marker regression, simple interval mapping and composite interval mapping approaches, detecting a total of 42 QTLs from six different sampling experiments varying by developmental stage (anthesis or vegetative growth), location or year. Coincident QTLs were detected on linkage groups (LGs) 3, 5 and 7. The region on LG3 was associated with variation for all measured traits across various experimental datasets. The region on LG7 was associated with variation for all traits except CP, and is located in the vicinity of the lignin biosynthesis gene loci xlpomt1 (caffeic acid-O-methyltransferase), xlpccr1 (cinnamoyl CoA-reductase) and xlpssrcad 2.1 (cinnamyl alcohol dehydrogenase). Comparative genomics analysis of these gene classes with wheat (Triticum aestivum L.) provides evidence for conservation of gene order over evolutionary time and the basis for cross-specific genetic information transfer. The identification of co-location between QTLs and functionally associated genetic markers is critical for the implementation of marker-assisted selection programs and for linkage disequilibrium studies, which will enable future improvement strategies for perennial ryegrass.

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.).

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Genetic linkage mapping of an annual x perennial ryegrass population.

Annual (Lolium multiflorum Lam.) and perennial ( L. perenne L.) ryegrass are two common forage and turfgrass species grown throughout the world. Perennial ryegrass is most commonly used for turfgrass purposes, and contamination by annual ryegrass, through physical seed mixing or gene flow, can result in a significant reduction in turfgrass quality. Seed certifying agencies in the United States currently use a test called seedling root fluorescence (SRF) to detect contamination between these species. The SRF test, however, can be inaccurate and therefore, the development of additional markers for species separation is needed. Male and female molecular-marker linkage maps of an interspecific annual x perennial ryegrass mapping population were developed to determine the map location of the SRF character and to identify additional genomic regions useful for species separation. A total of 235 AFLP markers, 81 RAPD markers, 16 comparative grass RFLPs, 106 SSR markers, 2 isozyme loci and 2 morphological characteristics, 8-h flowering, and SRF were used to construct the maps. RFLP markers from oat and barley and SSR markers from tall fescue and other grasses allowed the linkage groups to be numbered, relative to the Triticeae and the International Lolium Genome Initative reference population P150/112. The three-generation population structure allowed both male and female maps to be constructed. The male and female maps each have seven linkage groups, but differ in map length with the male map being 537 cm long and the female map 712 cm long. Regions of skewed segregation were identified in both maps with linkage groups 1, 3, and 6 of the male map showing the highest percentage of skewed markers. The (SRF) character mapped to linkage group 1 in both the male and female maps, and the 8-h flowering character was also localized to this linkage group on the female map. In addition, the Sod-1 isozyme marker, which can separate annual and perennial ryegrasses, mapped to linkage group 7. These results indicate that Lolium linkage groups 1 and 7 may provide additional markers and candidate genes for use in ryegrass species separation.

Genetic and physical analysis of a single Festuca pratensis chromosome segment substitution in Lolium perenne.

Molecular marker analysis and genomic in situ hybridisation (GISH) were used to examine the process of chromosome segment introgression in BC2 diploid hybrids (2n=2x=14) between Lolium perenne and Festuca pratensis. Two genotypes having what appeared to be the same, single, introgressed chromosome segment of F. pratensis in the L. perenne background were crossed with diploid L. perenne to produce a recombinant series for the introgressed region. Physical and genetic analysis of this series showed that, while recombination seemed to be possible at all points along the chromosome arm, the rate of recombination varied depending on relative position: more recombination was detected in the interstitial region as compared with the centromeric or telomeric regions. The implications of these results for the use of GISH and molecular marker analysis in the measurement of linkage drag in backcross breeding programmes is discussed.

Probing meiosis in hybrids of Lolium (Poaceae) with a discriminatory repetitive genomic sequence.

A moderately repetitive genomic DNA sequence (designated pLPBB2-123) derived from Lolium perenne L. (Poaceae) is considerably more abundant in the genome of this species than in that of the closely related L. temulentum. The repetitive sequence probe is clearly able to discriminate between the genomic DNA of both species in Southern analysis, and effectively 'paints' only the chromosome set of L. perenne in diploid and triploid hybrids with L. temulentum. Fluorescence in situ hybridisation of this sequence onto homologous chromosomes during meiosis I of the hybrids shows that the sequence is evenly distributed along all of the chromosomes of L. perenne and appears to have little effect on the structural integrity or recombination potential of hybrid bivalents. Discrimination between chromatin of different parental origin in hybrid bivalents shows for the first time a progressive relaxation of relational coiling of homoeologues throughout meiotic prophase. It also highlights structural irregularities that can now be unequivocally assigned to the longer chromosomes of L. temulentum. The advantages of the use of specific differentially amplified sequences instead of whole genome probes are discussed within the context of introgression breeding programmes within the Lolium/Festuca complex.

Retrotransposon evolution in diverse plant genomes.

Retrotransposon or retrotransposon-like sequences have been reported to be conserved components of cereal centromeres. Here we show that the published sequences are derived from a single conventional Ty3-gypsy family or a nonautonomous derivative. Both autonomous and nonautonomous elements are likely to have colonized Poaceae centromeres at the time of a common ancestor but have been maintained since by active retrotransposition. The retrotransposon family is also present at a lower copy number in the Arabidopsis genome, where it shows less pronounced localization. The history of the family in the two types of genome provides an interesting contrast between "boom and bust" and persistent evolutionary patterns.

De novo evolution of satellite DNA on the rye B chromosome.

The most distinctive region of the rye B chromosome is a subtelomeric domain that contains an exceptional concentration of B-chromosome-specific sequences. At metaphase this domain appears to be the physical counterpart of the subtelomeric heterochromatic regions present on standard rye chromosomes, but its conformation at interphase is less condensed. In this report we show that the two sequence families that have been previously found to make up the bulk of the domain have been assembled from fragments of a variety of sequence elements, giving rise to their ostensibly foreign origin. A single mechanism, probably based on synthesis-dependent strand annealing (SDSA), is responsible for their assembly. We provide evidence for sequential evolution of one family on the B chromosome itself. The extent of these rearrangements and the complexity of the higher-order organization of the B-chromosome-specific families indicate that instability is a property of the domain itself, rather than of any single sequence. Indirect evidence suggests that particular fragments may have been selected to confer different properties on the domain and that rearrangements are frequently selected for their effect on DNA structure. The current organization appears to represent a transient stage in the evolution of a conventional heterochromatic region from complex sequences.

Chromosome pairing in Lolium perenne x L. temulentum diploid hybrids: genetic and cytogenetic evaluation.

A Lolium perenne genotype (E5/2/5/10), which had been selected for low chiasma frequency over a number of generations and which was suspected of containing one or two heterozygous dominant genes with a significant effect on chiasma frequency, was crossed with L. temulentum (Ba3081) to create a hybrid population of 47 diploid plants. The mean chiasma or paired arm (PA) frequency of homoeologous chromosomes at meiosis in the population was 9.1/cell (1.3 PA/chromosome pair) with a distribution skewed towards high PA frequency. More than 90% of the hybrid chromosomes paired at meiosis in spite of the disparity in chromosome length and DNA quantity between the two species. Overall, the distribution of PAs between chromosomes for a given number of PAs/cell favoured the production of rod bivalents over ring bivalents and univalents, indicating that there is a mechanism present that maximizes the total number of bivalent associations formed. Molecular marker analysis using AFLPs and isoenzymes did not identify any clear major gene effect on PA frequency in the hybrid population. It was concluded that the control of PA frequency in E5/2/5/10 was not a simple genetic mechanism.

Molecular cytogenetic characterisation of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale).

The terminal heterochromatic segments of the long arms of 20 rye B-chromosomes were isolated by means of laser microdissection technology. Also the remaining portions of the long arms, along with the short arms of the same chromosomes were isolated. Each sample was used for degenerate oligonucleotide primer-polymerase chain reaction (DOP-PCR) amplification reactions. The resulting products were used as probes for chromosome in situ hybridisation experiments, and in Southern hybridisation to digests of 0B and +B DNA. Competition hybridisation of these probes with 0B DNA allowed the detection of B-specific sequences. The terminal heterochromatin of the rye B-chromosome contains both B-specific sequences and sequences also present on the A-chromosomes of rye. The B-specific D1100 family is the major repeat species located in the terminal heterochromatin. Primers designed to the cloned sequence (E1100) were used to search for related low copy sequences in 0B DNA. The sequences of the PCR products revealed no similarities to that of the clone E1100 except for the primer sequences. The possible origin of this sequence is discussed in the context of models for the evolution of the rye B-chromosome.

Molecular analysis of the structure of the maize B-chromosome.

The overall composition of the maize B is similar to that of the standard chromosome complement (A-chromosomes). This has been demonstrated by the use of several methods including: (a) genomic in situ hybridization (GISH), (b) analysis of highly repetitive sequences by the comparison of restriction digests of 0B and +B DNA and (c) the characterization of middle-repetitive cloned sequences. By the use of the technique of random amplification of polymorphic DNA-polymerase chain reaction, we have identified a B-specific repetitive sequence family. Sequence analysis of the B-specific clone reveals a relationship to the PREM-1 family of maize retroelements, which are preferentially transcribed in pollen. These results suggest an internal origin of the B-chromosome from within the maize genome, but demonstrate also that specific sequences can evolve by rapid processes of genomic turnover. Models for the origin of the maize B are discussed in the context of the present data.

Analysis of rye B-chromosome structure using fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) has been used to analyse the structure of the rye B chromosome. Genomic in situ hybridization (GISH) demonstrates the high level of overall similarity between A and B chromosomes of rye, as well as the presence of a number of specific sequences. The B-specific repeat families D1100 and E3900 have been analysed in terms of their physical location and possible contiguity. Rye Bs contain members of the rye-specific dispersed repetitive family R173, as well as centromeric regions similar to those of the As. The B chromosomes analysed in our study lack detectable rDNA sequences. Anomalous results have been obtained with a number of subtelomeric repetitive probes from rye. Bs usually lack these sequences, but evidence is presented that in some cases A-B translocation events may relocate such sequences from the As to the Bs. These data are discussed in the context of current models for the origin of the B chromosome.

Use of random PCR (RAPD) technology to analyse phylogenetic relationships in the Lolium/Festuca complex.

The RAPD PCR technique has been employed to investigate phylogenetic relationships between species of the genera Lolium and Festuca. Several decamer primers were used to generate patterns from groups of genotypes of several different species. The degree of band sharing was used to evaluate genetic distances between species and to construct a phylogenetic tree which is in good overall agreement with classical taxonomy, but contains a number of novel insights. The degree of homoplasy inherent in this approach has been investigated using Southern hybridization. These results are discussed in the context of current work in molecular biosystematics.

Identification of the E3900 family, a second family of rye B chromosome specific repeated sequences.

A second family of highly repeated sequences has been identified on the B chromosome of rye (Secale cereale). The E3900 family was detected as a variant band in EcoRI digests of +B DNA. A clone of the basic repeat of the family was obtained, and the organization of the family was investigated by genomic hybridization. The E3900 family has no apparent homology to the A chromosome sequences of rye or other members of the Gramineae. The family has been localized by in situ hybridization to the end of the long arm of the rye B chromosome. The previously characterized E1100 sequence shows in situ hybridization to the same location as the E3900 family. These results are discussed in light of current theories of the origin of B chromosomes.

Analysis of the regulatory elements of the Escherichia coli uvrC gene by construction of operon fusions.

The regulatory region of the Escherichia coli uvrC gene has been analysed by the subcloning of appropriate restriction fragments into the promoter probe vector pPV502. A series of plasmids carrying operon fusions to the gene for chloramphenicol acetyltransferase (cat) has been constructed. Three promoters capable of controlling uvrC have been identified (P1, P2 and P3), the majority of transcription being derived from the most distal of these promoters (P1). Transcription termination apparently plays some role in the control of the gene through premature termination of the P1-, but not the P2- or P3-derived transcripts. In addition, a promoter acting in the opposite direction to uvrC transcription has been detected. The activity of each of the promoters has been assayed under normal and SOS-inducing conditions. The uvrC gene is not apparently under the control of the recA-lexA regulatory circuit, unlike uvrA and uvrB. A series of recombinant plasmids carrying a 1.9 kb Bg/II fragment encoding most of the uvrC gene has been constructed. The properties of these plasmids suggest that the six amino acids at the carboxy-terminus of the uvrC gene product are not critical for DNA repair activity.