Marker Assisted Breeding Support for Tropical Horticulture and Forestry in far north Queensland

Molecular marker development for tropical fruit and forestry species.

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.

Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1 B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

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.

Identification of I-7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes

The tomato I-3 and I-7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I-3 has been identified previously on chromosome 7 and encodes an S-receptor-like kinase, but little is known about I-7. Molecular markers have been developed for the marker-assisted breeding of I-3, but none are available for I-7. We used an RNA-seq and single nucleotide polymorphism (SNP) analysis approach to map I-7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I-7 as a gene encoding a leucine-rich repeat receptor-like protein (LRR-RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I-7, like many other LRR-RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I-7 gene for Fol resistance, we found that I-7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I-7-mediated resistance, unlike I-2- or I-3-mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1-independent pathway for resistance activation. The identification of I-7 has allowed us to develop molecular markers for marker-assisted breeding of both genes currently known to confer Fol race 3 resistance (I-3 and I-7). Given that I-7-mediated resistance is not suppressed by Avr1, I-7 may be a useful addition to I-3 in the tomato breeder's toolbox.

Applications of pedigree-based genome mapping in wheat and barley breeding programs.

The aim of the pedigree-based genome mapping project is to investigate and develop systems for implementing marker assisted selection to improve the efficiency of selection and increase the rate of genetic gain in breeding programs. Pedigree-based whole genome marker application provides a vehicle for incorporating marker technologies into applied breeding programs by bridging the gap between marker-trait association and marker implementation. We report on the development of protocols for implementation of pedigree-based whole genome marker analysis in breeding programs within the Australian northern winter cereals region. Examples of applications from the Queensland DPI&F wheat and barley breeding programs are provided, commenting on the use of microsatellites and other types of molecular markers for routine genomic analysis, the integration of genotypic, phenotypic and pedigree information for targeted wheat and barley lines, the genomic impacts of strong selection pressure in case study pedigrees, and directions for future pedigree-based marker development and analysis.

Rice molecular breeding laboratories in the genomics era: Current status and future considerations

Using DNA markers in plant breeding with marker-assisted selection (MAS) could greatly improve the precision and efficiency of selection, leading to the accelerated development of new crop varieties. The numerous examples of MAS in rice have prompted many breeding institutes to establish molecular breeding labs. The last decade has produced an enormous amount of genomics research in rice, including the identification of thousands of QTLs for agronomically important traits, the generation of large amounts of gene expression data, and cloning and characterization of new genes, including the detection of single nucleotide polymorphisms. The pinnacle of genomics research has been the completion and annotation of genome sequences for indica and japonica rice. This information-coupled with the development of new genotyping methodologies and platforms, and the development of bioinformatics databases and software tools-provides even more exciting opportunities for rice molecular breeding in the 21st century. However, the great challenge for molecular breeders is to apply genomics data in actual breeding programs. Here, we review the current status of MAS in rice, current genomics projects and promising new genotyping methodologies, and evaluate the probable impact of genomics research. We also identify critical research areas to "bridge the application gap" between QTL identification and applied breeding that need to be addressed to realize the full potential of MAS, and propose ideas and guidelines for establishing rice molecular breeding labs in the postgenome sequence era to integrate molecular breeding within the context of overall rice breeding and research programs.

A marker-assisted backcross approach for developing submergence-tolerant rice cultivars

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC2F2 generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC3F2 double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3-3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program.

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message “To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.” Abstract Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.

Breeding and fecundity of the endemic Australian gudgeon, sleepy cod Oxyeleotris lineolatus (Steindachner 1867) (Eleotridae)

Sleepy cod (Oxyeleotris lineolatus Steindachner) is a tropical species of eleotrid native to northern Australia. A related species, sand or marbled goby, is the highest priced freshwater fish in Asia, and a market for a similar fish exists in expatriate Chinese communities. Sleepy cod breed when minimum temperatures reach 24 °C for more than 3 days. During the breeding season the genital papilla is broad and flattened in females compared to the triangular papilla of males and juveniles. Spawning pairs were usually of approximately equal size. Females could spawn up to 10 times during one breeding season. Wet weather increased the frequency of spawning. Eggs were usually laid hanging from the underside of a surface. Most spawning occurred between 05:00 and 10:00 h. Females attended egg masses immediately after spawning, after which males cared for eggs until hatching, 3–5 days later. Agitation of the egg mass was essential for development. The mean number of eggs per spawning was 43 130. Larvae commenced feeding 2–5 days after hatching, on plankton from 100 to 250 m in size. A spawning trap used to collect egg masses is described. The breeding biology of sleepy cod is considered to be an adaptation to the monsoonal tropics.

Standardising bull breeding soundness evaluations and reporting in Australia

There is substantial variation in bull breeding soundness evaluation procedures and reports in Australia; the situation is compounded by difficulties in interpretation and the validity of many reports. In an effort to overcome this, the scientific literature was reviewed [Fordyce G. In: Fordyce G, editor. Bull fertility: selection and management in Australia. Eight Mile Plains, Australia: Australian Cattle Vets; 2002] and the needs of stakeholders were considered in preparing a manual, Evaluating and Reporting Bull Fertility [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] that outlined standards for assessing and reporting bull breeding soundness. A new recording and reporting system, called Bull Reporter, is based on standards from this manual and groups bull fertility traits into five summary categories: Scrotum, Physical, Crush-side Semen, Sperm Morphology, and Serving. The client will generally select which categories they wish to have included in the evaluation to suit their specific purposes. While there is adequate room for comments, the veterinarian is not required to make an overall judgment of whether the bull has normal capacity to sire calves under natural mating management, but ensures the standards for each selected category are met. Professional, standardised, easy-to-read reports are produced either electronically [Entwistle KW, Fordyce G. Evaluating and reporting bull fertility. Eight Mile Plains, Australia: Australian Cattle Vets; 2003.] or manually. A bull owner or their agent signs the certificate to affirm that bulls have not undergone procedures to rectify faults which may have otherwise caused them to fail the standards. An accreditation system for assessing sperm morphology was established because of its demonstrated relationship with pregnancy rates and because of the difficulties in achieving consistent and accurate assessments among laboratories. It is considered that Bull Reporter is applicable to beef and dairy bulls across all levels of management, genotypes and environments throughout Australia, with substantial potential for application elsewhere in the world.

Identification of QTL for sugar-related traits in a sweet x grain sorghum (Sorghum bicolor L. Moench) recombinant inbred population

QTL for stem sugar-related and other agronomic traits were identified in a converted sweet (R9188) × grain (R9403463-2-1) sorghum population. QTL analyses were conducted using phenotypic data for 11 traits measured in two field experiments and a genetic map comprising 228 SSR and AFLP markers grouped into 16 linkage groups, of which 11 could be assigned to the 10 sorghum chromosomes (SBI-01 to SBI-10). QTL were identified for all traits and were generally co-located to five locations (SBI-01, SBI-03, SBI-05, SBI-06 and SBI-10). QTL alleles from R9188 were detected for increased sucrose content and sugar content on SBI-01, SBI-05 and SBI-06. R9188 also contributed QTL alleles for increased Brix on SBI-05 and SBI-06, and increased sugar content on SBI-03. QTL alleles from R9403463-2-1 were found for increased sucrose content and sucrose yield on SBI-10, and increased glucose content on SBI-07. QTL alleles for increased height, later flowering and greater total dry matter yield were located on SBI-01 of R9403463-2-1, and SBI-06 of R9188. QTL alleles for increased grain yield from both R9403463-2-1 and R9188 were found on SBI-03. As an increase in stem sugars is an important objective in sweet sorghum breeding, the QTL identified in this study could be further investigated for use in marker-assisted selection of sweet sorghum.

The accuracy of varietal selection using factor analytic models for multi-environment plant breeding trials

Modeling of cultivar x trial effects for multienvironment trials (METs) within a mixed model framework is now common practice in many plant breeding programs. The factor analytic (FA) model is a parsimonious form used to approximate the fully unstructured form of the genetic variance-covariance matrix in the model for MET data. In this study, we demonstrate that the FA model is generally the model of best fit across a range of data sets taken from early generation trials in a breeding program. In addition, we demonstrate the superiority of the FA model in achieving the most common aim of METs, namely the selection of superior genotypes. Selection is achieved using best linear unbiased predictions (BLUPs) of cultivar effects at each environment, considered either individually or as a weighted average across environments. In practice, empirical BLUPs (E-BLUPs) of cultivar effects must be used instead of BLUPs since variance parameters in the model must be estimated rather than assumed known. While the optimal properties of minimum mean squared error of prediction (MSEP) and maximum correlation between true and predicted effects possessed by BLUPs do not hold for E-BLUPs, a simulation study shows that E-BLUPs perform well in terms of MSEP.

Strawberry breeding in a subtropical environment

Strawberry breeding aims to provide cultivars that maximise consumer satisfaction and producer profitability in a changing environment. In this paper some concepts of profitability, consumer satisfaction and sustainability are explored for a subtropical climate using Queensland Australia, and Florida USA, as examples. The typical production environment is annual autumn planting of bare rooted runners into polythene covered raised beds at about 40000 plants/ha. Harvesting is late autumn to early spring, with fruit arriving at the major markets up to 2000km away from the production area within 1-4 days of harvest. The basic premise in the breed-big work is that consumers must enjoy the experience of eating strawberries, and that perceived flavour, sweetness, and juiciness are the major contributors to this experience. Using market chain information, we developed a basic value model comprised of costs, returns, and sustainability of market. To this basic outline are applied operational descriptors, such as 'speed of harvest', and associated plant characteristics, such as 'fruit display'. The expression of each plant characteristic is ascribed a value or level and together numerically describe the phenotype. This description is mathematically manipulated to provide a 'value index' for the cultivar. Nine cultivars including 'Strawberry Festival', 'Kabarla', 'DPI Rubygem' and 'Sweet Charlie' are described, and environmental issues that may impact on the subtropical strawberry breeding objectives are discussed. Product differentiation and the use of exotic germplasm as a new source of genes for flavour and resistance to disease and environmental stress will likely be the cornerstones of future progress in subtropical strawberry breeding. This approach should satisfy both consumers and producers.

Use of part records in Merino breeding programs - The inheritance of wool growth and fibre traits during different times of the year to determine their value in Merino breeding programs

Fibre diameter can vary dramatically along a wool staple, especially in the Mediterranean environment of southern Australia with its dry summers and abundance of green feed in spring. Other research results have shown a very low phenotypic correlation between fibre diameter grown between seasons. Many breeders use short staples to measure fibre diameter for breeding purposes and also to promote animals for sale. The effectiveness of this practice is determined by the relative response to selection by measuring fibre traits on a full 12 months wool staple as compared to measuring them only on part of a staple. If a high genetic correlation exists between the part record and the full record, then using part records may be acceptable to identify genetically superior animals. No information is available on the effectiveness of part records. This paper investigated whether wool growth and fibre diameter traits of Merino wool grown at different times of the year in a Mediterranean environment, are genetically the same trait, respectively. The work was carried out on about 7 dyebanded wool sections/animal.year, on ewes from weaning to hogget age, in the Katanning Merino resource flocks over 6 years. Relative clean wool growth of the different sections had very low heritability estimates of less than 0.10, and they were phenotypically and genetically poorly correlated with 6 or 12 months wool growth. This indicates that part record measurement of clean wool growth of these sections will be ineffective as indirect selection criteria to improve wool growth genetically. Staple length growth as measured by the length between dyebands, would be more effective with heritability estimates of between 0.20 and 0.30. However, these measurements were shown to have a low genetic correlation with wool grown for 12 months which implies that these staple length measurements would only be half as efficient as the wool weight for 6 or 12 months to improve total clean wool weight. Heritability estimates of fibre diameter, coefficient of variation of fibre diameter and fibre curvature were relatively high and were genetically and phenotypically highly correlated across sections. High positive phenotypic and genetic correlations were also found between fibre diameter, coefficient of variation of fibre diameter and fibre curvature of the different sections and similar measurements for wool grown over 6 or 12 months. Coefficient of variation of fibre diameter of the sections also had a moderate negative phenotypic and genetic correlation with staple strength of wool staples grown over 6 months indicating that coefficient of variation of fibre diameter of any section would be as good an indirect selection criterion to improve stable strength as coefficient of variation of fibre diameter for wool grown over 6 or 12 months. The results indicate that fibre diameter, coefficient of variation of fibre diameter and fibre curvature of wool grown over short periods of time have virtually the same heritability as that of wool grown over 12 months, and that the genetic correlation between fibre diameter, coefficient of variation of fibre diameter and fibre curvature on part and on full records is very high (rg > 0.85). This indicates that fibre diameter, coefficient of variation of fibre diameter and fibre curvature on part records can be used as selection criteria to improve these traits. However, part records of greasy and clean wool growth would be much less efficient than fleece weight for wool grown over 6 or 12 months because of the low heritability of part records and the low genetic correlation between these traits on part records and on wool grown for 12 months.