Genetic variation within two sympatric spotted gum eucalypts exceeds between taxa variation

Population substructure and hybridization, among other factors, have the potential to cause erroneous associations in linkage disequilibrium (LD) mapping. Two closely related spotted gum eucalypts, Corymbia variegata and C. henryi (Myrtaceae) occur in sympatry in the east coast of Australia and potentially interbreed. They are morphologically similar but are distinguished as separate species based on capsule and foliage size. To determine whether they hybridize in nature and its implications for LD mapping, we investigated the level of molecular divergence between the two species at two sympatric locations separated by 300 kilometres. Very few individuals of intermediate morphology were identified, despite the two species occurring only metres apart. Analysis of genetic structure using 12 microsatellite loci showed that genetic differentiation between populations of the same species at different locations (FST = 0.07 for both species; p = 0.0001) was significantly higher than that observed between species at each location (mean FST = 0.02 and 0.04 for Cherry tree and Bunyaville respectively; p = 0.0001; all Mann-Whitney U-test p ≤ 0.01). No species-specific alleles or significant allele frequency differences were detected within a site, suggesting recurrent local gene flow between the two species. The lack of significant allele frequency differences implies no population stratification along taxonomic lines. This suggested that there is little concern for cryptic hybridization when sampling from sites of sympatry for LD mapping.

DArT markers: Diversity analyses and mapping in Sorghum bicolor

The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm. Results: A genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM. Conclusion: We have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.

Detecting quantitative trait loci affecting beef tenderness on bovine chromosome 7 near calpastatin and lysyl oxidase

From a study of 3 large half-sib families of cattle, we describe linkage between DNA polymorphisms on bovine chromosome 7 and meat tenderness. Quantitative trait loci (QTL) for Longissimus lumborum peak force (LLPF) and Semitendonosis adhesion (STADH) were located to this map of DNA markers, which includes the calpastatin ( CAST) and lysyl oxidase (LOX) genes. The LLPF QTL has a maximum lodscore of 4.9 and allele substitution of approximately 0.80 of a phenotypic standard deviation, and the peak is located over the CAST gene. The STADH QTL has a maximum lodscore of 3.5 and an allele substitution of approximately 0.37 of a phenotypic standard deviation, and the peak is located over the LOX gene. This suggests 2 separate likelihood peaks on the chromosome. Further analyses of meat tenderness measures in the Longissimus lumborum, LLPF and LL compression (LLC), in which outlier individuals or kill groups are removed, demonstrate large shifts in the location of LLPF QTL, as well as confirming that there are indeed 2 QTL on bovine chromosome 7. We found that both QTL are reflected in both LLPF and LLC measurements, suggesting that both these components of tenderness, myofibrillar and connective tissue, are detected by both measurements in this muscle.

DNA markers linked to yield, yield components, and morphological traits in autotetraploid lucerne (Medicago sativa L.)

We have mapped and identified DNA markers linked to morphology, yield, and yield components of lucerne, using a backcross population derived from winter-active parents. The high-yielding and recurrent parent, D, produced individual markers that accounted for up to 18% of total yield over 6 harvests, at Gatton, south-eastern Queensland. The same marker, AC/TT8, was consistently identified at each individual harvest, and in individual harvests accounted for up to 26% of the phenotypic variation for yield. This marker was located in linkage group 2 of the D map, and several other markers positively associated with yield were consistently identified in this linkage group. Similarly, markers negatively associated with yield were consistently identified in the W116 map, W116 being the low-yielding parent. Highly significant positive correlations were observed between total yield and yield for harvests 1-6, and between total yield and stem length, tiller number, leaf yield/plant, leaf yield/5 stems, stem yield/plant, and stem yield/5 stems. Highly significant QTL were located for all these characters as well as for leaf shape and pubescence.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.

A new semi-dwarfing gene identified by molecular mapping of quantitative trait loci in barley.

Semi-dwarfing genes have been widely used in spring barley (Hordeum vulgare L.) breeding programs in many parts of the world, but the success in developing barley cultivars with semi-dwarfing genes has been limited in North America. Exploiting new semi-dwarfing genes may help in solving this dilemma. A recombinant inbred line population was developed by crossing ZAU 7, a semi-dwarf cultivar from China, to ND16092, a tall breeding line from North Dakota. To identify quantitative trait loci (QTL) controlling plant height, a linkage map comprised of 111 molecular markers was constructed. Simple interval mapping was performed for each of the eight environments. A consistent QTL for plant height was found on chromosome 7HL. This QTL is not associated with maturity and rachis internode length. We suggest the provisional name Qph-7H for this QTL. Qph-7H from ZAU 7 reduced plant height to about 3/4 of normal; thus, Qph-7H is considered a semi-dwarfing gene. Other QTLs for plant height were found, but their expression was variable across the eight environments tested.

QTL mapping of multiple foliar disease and root-lesion nematode resistances in wheat.

A genetic linkage map, based on a cross between the synthetic hexaploid CPI133872 and the bread wheat cultivar Janz, was established using 111 F1-derived doubled haploid lines. The population was phenotyped in multiple years and/or locations for seven disease resistance traits, namely, Septoria tritici blotch (Mycosphaeralla graminicola), yellow leaf spot also known as tan spot (Pyrenophora tritici-repentis), stripe rust (Puccinia striiformis f. sp. tritici), leaf rust (Puccinia triticina), stem rust (Puccinia graminis f. sp. tritici) and two species of root-lesion nematode (Pratylenchyus thornei and P. neglectus). The DH population was also scored for coleoptile colour and the presence of the seedling leaf rust resistance gene Lr24. Implementation of a multiple-QTL model identified a tightly linked cluster of foliar disease resistance QTL in chromosome 3DL. Major QTL each for resistance to Septoria tritici blotch and yellow leaf spot were contributed by the synthetic hexaploid parent CPI133872 and linked in repulsion with the coincident Lr24Sr24/ locus carried by parent Janz. This is the first report of linked QTL for Septoria tritici blotch and yellow leaf spot contributed by the same parent. Additional QTL for yellow leaf spot were detected in 5AS and 5BL. Consistent QTL for stripe rust resistance were identified in chromosomes 1BL, 4BL and 7DS, with the QTL in 7DS corresponding to the Yr18Lr34/ region. Three major QTL for P. thornei resistance (2BS, 6DS, 6DL) and two for P. neglectus resistance (2BS, 6DS) were detected. The recombinants combining resistance to Septoria tritici blotch, yellow leaf spot, rust diseases and root-lesion nematodes from parents CPI133872 and Janz constitute valuable germplasm for the transfer of multiple disease resistance into new wheat cultivars.

Quantitative and molecular genetics of juvenile wood traits in radiata and slash/Caribbean pines.

The Juvenile Wood Initiative (JWI) project has been running successfully since July 2003 under a Research Agreement with FWPA and Letters of Association with the consortium partners STBA (Southern Tree Breeding Association), ArborGen and FPQ (Forestry Plantations Queensland). Over the last five and half years, JWI scientists in CSIRO, FPQ, and STBA have completed all 12 major milestones and 28 component milestones according to the project schedule. We have made benchmark progress in understanding the genetic control of wood formation and interrelationships among wood traits. The project has made 15 primary scientific findings and several results have been adopted by industry as summarized below. This progress was detailed in 10 technical reports to funding organizations and industry clients. Team scientists produced 16 scientific manuscripts (8 published, 1 in press, 2 submitted, and several others in the process of submission) and 15 conference papers or presentations. Primary Scientific Findings. The 15 major scientific findings related to wood science, inheritance and the genetic basis of juvenile wood traits are: 1. An optimal method to predict stiffness of standing trees in slash/Caribbean pine is to combine gravimetric basic density from 12 mm increment cores with a standing tree prediction of MoE using a time of flight acoustic tool. This was the most accurate and cheapest way to rank trees for breeding selection for slash/Caribbean hybrid pine. This method was also recommended for radiata pine. 2. Wood density breeding values were predicted for the first time in the STBA breeding population using a large sample of 7,078 trees (increment cores) and it was estimated that selection of the best 250 trees for deployment will produce wood density gains of 12.4%. 3. Large genetic variation for a suite of wood quality traits including density, MFA, spiral grain, shrinkage, acoustic and non-acoustic stiffness (MoE) for clear wood and standing trees were observed. Genetic gains of between 8 and 49% were predicted for these wood quality traits with selection intensity between 1 to 10% for radiata pine. 4. Site had a major effect on juvenile-mature wood transition age and the effect of selective breeding for a shorter juvenile wood formation phase was only moderate (about 10% genetic gain with 10% selection intensity, equivalent to about 2 years reduction of juvenile wood). 5. The study found no usable site by genotype interactions for the wood quality traits of density, MFA and MoE for both radiata and slash/Caribbean pines, suggesting that assessment of wood properties on one or two sites will provide reliable estimates of the genetic worth of individuals for use in future breeding. 6. There were significant and sizable genotype by environment interactions between the mainland and Tasmanian regions and within Tasmania for DBH and branch size. 7. Strong genetic correlations between rings for density, MFA and MoE for both radiata and slash/Caribbean pines were observed. This suggests that selection for improved wood properties in the innermost rings would also result in improvement of wood properties in the subsequent rings, as well as improved average performance of the entire core. 8. Strong genetic correlations between pure species and hybrid performance for each of the wood quality traits were observed in the hybrid pines. Parental performance can be used to identify the hybrid families which are most likely to have superior juvenile wood properties of the slash/Caribbean F1 hybrid in southeast Queensland. 9. Large unfavourable genetic correlations between growth and wood quality traits were a prominent feature in radiata pine, indicating that overcoming this unfavourable genetic correlation will be a major technical issue in progressing radiata pine breeding. 10. The project created the first radiata pine 18 k cDNA microarray and generated 5,952 radiata pine xylogenesis expressed sequence tags (ESTs) which assembled into 3,304 unigenes. 11. A total of 348 genes were identified as preferentially expressed genes in earlywood or latewood while a total of 168 genes were identified as preferentially expressed genes in either juvenile or mature wood. 12. Juvenile earlywood has a distinct transcriptome relative to other stages of wood development. 13. Discovered rapid decay of linkage disequilibrium (LD) in radiata pine with LD decaying to approximately 50% within 1,700 base pairs (within a typical gene). A total of 913 SNPS from sequencing 177,380 base pairs were identified for association genetic studies. 14. 149 SNPs from 44 genes and 255 SNPs from a further 51 genes (total 95 genes) were selected for association analysis with 62 wood traits, and 30 SNPs were shortlisted for their significant association with variation of wood quality traits (density, MFA and MoE) with individual significant SNPs accounting for between 1.9 and 9.7% of the total genetic variation in traits. 15. Index selection using breeding objectives was the most profitable selection method for radiata pine, but in the long term it may not be the most effective in dealing with negative genetic correlations between wood volume and quality traits. A combination of economic and biological approaches may be needed to deal with the strong adverse correlation.

Location of major effect genes in sorghum (Sorghum bicolor (L.) Moench).

Major effect genes are often used for germplasm identification, for diversity analyses and as selection targets in breeding. To date, only a few morphological characters have been mapped as major effect genes across a range of genetic linkage maps based on different types of molecular markers in sorghum (Sorghum bicolor (L.) Moench). This study aims to integrate all available previously mapped major effect genes onto a complete genome map, linked to the whole genome sequence, allowing sorghum breeders and researchers to link this information to QTL studies and to be aware of the consequences of selection for major genes. This provides new opportunities for breeders to take advantage of readily scorable morphological traits and to develop more effective breeding strategies. We also provide examples of the impact of selection for major effect genes on quantitative traits in sorghum. The concepts described in this paper have particular application to breeding programmes in developing countries where molecular markers are expensive or impossible to access.

Fifteen microsatellite loci for the jungle perch, Kuhlia rupestris

We developed and optimized 15 polymorphic microsatellite loci in the jungle perch, Kuhlia rupestris. Loci were screened in a single population (n = 24) from Fraser Island, Queensland, Australia. Number of alleles per locus ranged from 3 to 19 and observed heterozygosity from 0.25 to 1. No significant linkage disequilibrium was detected between any pair of loci. Genotype proportions for these loci in the population sampled were in Hardy–Weinberg equilibrium.

Integrating sorghum whole genome sequence information with a compendium of sorghum QTL studies reveals uneven distribution of QTL and of gene-rich regions with significant implications for crop improvement.

A comprehensive analysis was conducted using 48 sorghum QTL studies published from 1995 to 2010 to make information from historical sorghum QTL experiments available in a form that could be more readily used by sorghum researchers and plant breeders. In total, 771 QTL relating to 161 unique traits from 44 studies were projected onto a sorghum consensus map. Confidence intervals (CI) of QTL were estimated so that valid comparisons could be made between studies. The method accounted for the number of lines used and the phenotypic variation explained by individual QTL from each study. In addition, estimated centimorgan (cM) locations were calculated for the predicted sorghum gene models identified in Phytozome (JGI GeneModels SBI v1.4) and compared with QTL distribution genome-wide, both on genetic linkage (cM) and physical (base-pair/bp) map scales. QTL and genes were distributed unevenly across the genome. Heterochromatic enrichment for QTL was observed, with approximately 22% of QTL either entirely or partially located in the heterochromatic regions. Heterochromatic gene enrichment was also observed based on their predicted cM locations on the sorghum consensus map, due to suppressed recombination in heterochromatic regions, in contrast to the euchromatic gene enrichment observed on the physical, sequence-based map. The finding of high gene density in recombination-poor regions, coupled with the association with increased QTL density, has implications for the development of more efficient breeding systems in sorghum to better exploit heterosis. The projected QTL information described, combined with the physical locations of sorghum sequence-based markers and predicted gene models, provides sorghum researchers with a useful resource for more detailed analysis of traits and development of efficient marker-assisted breeding strategies.

Characterization of north-eastern Australian environments using APSIM for increasing rainfed maize production

Recurring water stresses are a major risk factor for rainfed maize cropping across the highly diverse agro-ecological environments of Queensland (Qld) and northern New South Wales (NNSW). Enhanced understanding of such agro-ecological diversity is necessary to more consistently sample target production environments for testing and targeting release of improved germplasm, and to improve the efficiency of the maize pre-breeding and breeding programs of Qld and New South Wales. Here, we used the Agricultural Production Systems Simulator (APSIM) – a well validated maize crop model to characterize the key distinctive water stress patterns and risk to production across the main maize growing regions of Qld and NNSW located between 15.8° and 31.5°S, and 144.5° and 151.8°E. APSIM was configured to simulate daily water supply demand ratios (SDRs) around anthesis as an indicator of the degree of water stress, and the final grain yield. Simulations were performed using daily climatic records during the period between 1890 and 2010 for 32 sites-soils in the target production regions. The runs were made assuming adequate nitrogen supply for mid-season maize hybrid Pioneer 3153. Hierarchical complete linkage analyses of the simulated yield resulted in five major clusters showing distinct probability distribution of the expected yields and geographic patterns. The drought stress patterns and their frequencies using SDRs were quantified using multivariate statistical methods. The identified stress patterns included no stress, mid-season (flowering) stress, and three terminal stresses differing in terms of severity. The combined frequency of flowering and terminal stresses was highest (82.9%), mainly in sites-soils combinations in the west of Qld and NNSW. Yield variability across the different sites-soils was significantly related to the variability in frequencies of water stresses. Frequencies of water stresses within each yield cluster tended to be similar, but different across clusters. Sites-soils falling within each yield cluster therefore could be treated as distinct maize production environments for testing and targeting newly developed maize cultivars and hybrids for adaptation to water stress patterns most common to those environments.

Genetic Structure of Cochliobolus sativus Populations Sampled from Roots and Leaves of Barley and Wheat in North Dakota

Common root rot (CRR) and spot blotch, caused by Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dast., are important diseases of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) worldwide. However, the population biology of C. sativus is still poorly understood. In this study, the genetic structure of three C. sativus populations, consisting of isolates sampled respectively from barley leaves (BL), barley roots (BR) and wheat roots (WR) in North Dakota, was analysed with amplified fragment length polymorphism (AFLP) markers. A total of 127 AFLP loci were generated among 208 C. sativus isolates analysed with three primer combinations. Gene diversity (H = 0.277-0.335) were high in all three populations. Genetic variation among C. sativus individuals within population accounted for 74%, whereas 26% of the genetic variation was explained among populations. Genetic differentiation was high (empty set PT = 0.261, corrected G ''(st)= 0.39), whereas gene flow (Nm) ranged from 1.27 to 1.56 among the three populations analysed. The multilocus linkage disequilibrium (LD) ((r) over bard = 0.0760.117) was moderate in C. sativus populations. Cluster analyses indicate that C. sativus populations differentiated according to the hosts (barley and wheat) and tissues (root and leaf) although generalists also exist in North Dakota. Crop breeding may benefit from combining genes for resistance against both specialists and generalists of C. sativus.

Association genetics in Corymbia citriodora subsp variegata identifies single nucleotide polymorphisms affecting wood growth and cellulosic pulp yield

Wood is an important biological resource which contributes to nutrient and hydrology cycles through ecosystems, and provides structural support at the plant level. Thousands of genes are involved in wood development, yet their effects on phenotype are not well understood. We have exploited the low genomic linkage disequilibrium (LD) and abundant phenotypic variation of forest trees to explore allelic diversity underlying wood traits in an association study. Candidate gene allelic diversity was modelled against quantitative variation to identify SNPs influencing wood properties, growth and disease resistance across three populations of Corymbia citriodora subsp. variegata, a forest tree of eastern Australia. Nine single nucleotide polymorphism (SNP) associations from six genes were identified in a discovery population (833 individuals). Associations were subsequently tested in two smaller populations (130160 individuals), validating our findings in three cases for actin 7 (ACT7) and COP1 interacting protein 7 (CIP7). The results imply a functional role for these genes in mediating wood chemical composition and growth, respectively. A flip in the effect of ACT7 on pulp yield between populations suggests gene by environment interactions are at play. Existing evidence of gene function lends strength to the observed associations, and in the case of CIP7 supports a role in cortical photosynthesis.

Genetic Structure of Cochliobolus sativus Populations Sampled from Roots and Leaves of Barley and Wheat in North Dakota

Common root rot (CRR) and spot blotch, caused by Cochliobolus sativus (Ito and Kurib.) Drechsl. ex Dast., are important diseases of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) worldwide. However, the population biology of C. sativus is still poorly understood. In this study, the genetic structure of three C. sativus populations, consisting of isolates sampled respectively from barley leaves (BL), barley roots (BR) and wheat roots (WR) in North Dakota, was analysed with amplified fragment length polymorphism (AFLP) markers. A total of 127 AFLP loci were generated among 208 C. sativus isolates analysed with three primer combinations. Gene diversity (H = 0.277-0.335) were high in all three populations. Genetic variation among C. sativus individuals within population accounted for 74%, whereas 26% of the genetic variation was explained among populations. Genetic differentiation was high (empty set PT = 0.261, corrected G ''(st)= 0.39), whereas gene flow (Nm) ranged from 1.27 to 1.56 among the three populations analysed. The multilocus linkage disequilibrium (LD) ((r) over bard = 0.0760.117) was moderate in C. sativus populations. Cluster analyses indicate that C. sativus populations differentiated according to the hosts (barley and wheat) and tissues (root and leaf) although generalists also exist in North Dakota. Crop breeding may benefit from combining genes for resistance against both specialists and generalists of C. sativus.