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.

Molecular Breeding for Complex Adaptive Traits: How Integrating Crop Ecophysiology and Modelling Can Enhance Efficiency

Progress in crop improvement is limited by the ability to identify favourable combinations of genotypes (G) and management practices (M) in relevant target environments (E) given the resources available to search among the myriad of possible combinations. To underpin yield advance we require prediction of phenotype based on genotype. In plant breeding, traditional phenotypic selection methods have involved measuring phenotypic performance of large segregating populations in multi-environment trials and applying rigorous statistical procedures based on quantitative genetic theory to identify superior individuals. Recent developments in the ability to inexpensively and densely map/sequence genomes have facilitated a shift from the level of the individual (genotype) to the level of the genomic region. Molecular breeding strategies using genome wide prediction and genomic selection approaches have developed rapidly. However, their applicability to complex traits remains constrained by gene-gene and gene-environment interactions, which restrict the predictive power of associations of genomic regions with phenotypic responses. Here it is argued that crop ecophysiology and functional whole plant modelling can provide an effective link between molecular and organism scales and enhance molecular breeding by adding value to genetic prediction approaches. A physiological framework that facilitates dissection and modelling of complex traits can inform phenotyping methods for marker/gene detection and underpin prediction of likely phenotypic consequences of trait and genetic variation in target environments. This approach holds considerable promise for more effectively linking genotype to phenotype for complex adaptive traits. Specific examples focused on drought adaptation are presented to highlight the concepts.

Molecular Breeding for Rainfed Lowland Rice in the Mekong Region

In the past 20 years, the rice-breeding program in Thailand had little success in developing new cultivars to replace Kao Dawk Mali 105 (KDML105) and Kao Khor 6 (RD6) for the tainted lowland rice environments. The main reason for the poor adoption of new cultivars by farmers is the susceptibility to diseases and unacceptable grain qualities. The conventional breeding program also takes at least 15 years from initial crossing to the release of new cultivars. A new breeding strategy can be established to shorten the period for cultivar improvement by using marker-assisted selection (MAS), rapid generations advance (RGA), and early generation testing in multi-locations for grain yield and qualities. Four generation of MAS backcross breeding were conducted to transfer genes and QTL for bacterial blight resistance (BLB), submergence tolerance (SUB), brown plant hopper resistance (BPH) and blast resistance (BL) into KDML105. Selected backcross lines, introgressed with target gene/QTL, were tolerant to SUB and resistant to BLB, BPH and BL. The agronomic performance and grain quality of these lines were as good as or better than KDML105.

Molecular breeding for rainfed lowland rice in the Mekong Region

In the past 20 years, the rice-breeding program in Thailand had little success in developing new cultivars to replace Kao Dawk Mali 105 (KDML105) and Kao Khor 6 (RD6). Main reason is a poor adoption of new cultivars by farmers due to poor adaptation of new cultivars to the rainfed environments, susceptibility to diseases and insect pests and unacceptable grain qualities. The conventional breeding program also takes at least 15 years for releasing new cultivars. New breeding strategy can be established to shorten period for cultivar improvement by using marker-assisted selection (MAS), rapid generations advance (RGA), early generation testing in multi-locations for grain yield and qualities. Four generation of MAS backcross breeding were conducted to transfer gene and QTL for bacterial blight resistance (BLB), submergence tolerance (SUB), brown planthopper resistance (BPH) and blast resistance (BL) into KDML105. Selected backcross lines, introgressed with target gene/QTL, were tolerant to SUB and resistant to BLB, BPH and BL. The agronomic performance and grain quality of these lines were as good as or better than KDML105.

DNA markers linked to the R(2) rust resistance gene in sunflower (Helianthus annuus L.) facilitate anticipatory breeding for this disease variant.

Pre-emptive breeding for host disease resistance is an effective strategy for combating and managing devastating incursions of plant pathogens. Comprehensive, long-term studies have revealed that virulence to the R (2) sunflower (Helianthus annuus L.) rust resistance gene in the line MC29 does not exist in the Australian rust (Puccinia helianthi) population. We report in this study the identification of molecular markers linked to this gene. The three simple sequence repeat (SSR) markers ORS795, ORS882, and ORS938 were linked in coupling to the gene, while the SSR marker ORS333 was linked in repulsion. Reliable selection for homozygous-resistant individuals was efficient when the three markers, ORS795, ORS882, and ORS333, were used in combination. Phenotyping for this resistance gene is not possible in Australia without introducing a quarantinable race of the pathogen. Therefore, the availability of reliable and heritable DNA-based markers will enable the efficient deployment of this gene, permitting a more effective strategy for generating sustainable commercial cultivars containing this rust resistance gene.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Improving taro (Colocasia esculenta var. esculenta) production using biotechnological approaches

Taro (Colocasia esculenta L. Schott) is an important crop worldwide but is of particular significance in many Pacific Island countries where it forms part of the staple diet and serves as an export commodity. Escalating pest and disease problems are jeopardizing taro production with serious implications to food security and trade. Biotechnological approaches to addressing pest and disease problems, such as somatic embryogenesis and transgenesis, are potentially viable options. However, despite biotechnological advancements in higher profile agronomic crops, such progress in relation to Colocasia esculenta var. esculenta has been slow. This paper reviews taro biology, highlights the cultural and economic significance of taro in Pacific Island countries and discusses the progress made towards the molecular breeding of this important crop to date.

Biopharming the SimpliRED™ HIV diagnostic reagent in barley, potato and tobacco

This is the first report of an antibody-fusion protein expressed in transgenic plants for direct use in a medical diagnostic assay. By the use of gene constructs with appropriate promoters, high level expression of an anti-glycophorin single-chain antibody fused to an epitope of the HIV virus was obtained in the leaves and stems of tobacco, tubers of potato and seed of barley. This fusion protein replaces the SimpliRED™ diagnostic reagent, used for detecting the presence of HIV-1 antibodies in human blood. The reagent is expensive and laborious to produce by conventional means since chemical modifications to a monoclonal antibody are required. The plant-produced fusion protein was fully functional (by ELISA) in crude extracts and, for tobacco at least, could be used without further purification in the HIV agglutination assay. All three crop species produced sufficient reagent levels to be superior bioreactors to bacteria or mice, however barley grain was the most attractive bioreactor as it expressed the highest level (150 μg of reagent g-1), is inexpensive to produce and harvest, poses a minuscule gene flow problem in the field, and the activity of the reagent is largely undiminished in stored grain. This work suggests that barley seed will be an ideal factory for the production of antibodies, diagnostic immunoreagents, vaccines and other pharmaceutical proteins.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbil-gus plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs. Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T 1 generation.

Ectopic overexpression of a mungbean vacuolar Na+/H+ antiporter gene (VrNHX1) leads to increased salinity stress tolerance in transgenic Vigna unguiculata L. Walp

Salinity is a major threat to sustainable agriculture worldwide. Plant NHX exchangers play an important role in conferring salt tolerance under salinity stress. In this study, a vacuolar Na+/H+ antiporter gene VrNHX1 (Genbank Accession No. JN656211.1) from mungbean (Vigna radiata) was introduced into cowpea (Vigna unguiculata) by the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot hybridization confirmed the stable integration of VrNHX1 into the cowpea genome. Comparative expression analysis by semi-quantitative RT-PCR revealed higher expression of VrNHX1 in transgenic cowpea plants than wild-type. Under salt stress conditions, T2 transgenic 35S:VrNHX1 cowpea lines exhibited higher tolerance to 200 mM NaCl treatment than wild-type. Furthermore, T2 transgenic 35S:VrNHX1 lines maintained a higher K+/Na+ ratio in the aerial parts under salt stress and accumulated higher [Na+] in roots than wild-type. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content and proline, ascorbate and chlorophyll contents in T2 transgenic 35S:VrNHX1 lines.

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.

Congruence in QTL for adventitious rooting in Pinus elliottii x Pinus caribaea hybrids resolves between and within-species effects

Targeting between-species effects for improvement in synthetic hybrid populations derived from outcrossing parental tree species may be one way to increase the efficacy and predictability of hybrid breeding. We present a comparative analysis of the quantitative trait loci (QTL) which resolved between from within-species effects for adventitious rooting in two populations of hybrids between Pinus elliottii and P. caribaea, an outbred F1 (n=287) and an inbred-like F2 family (n=357). Most small to moderate effect QTL (each explaining 2-5% of phenotypic variation, PV) were congruent (3 out of 4 QTL in each family) and therefore considered within-species effects as they segregated in both families. A single large effect QTL (40% PV) was detected uniquely in the F2 family and assumed to be due to a between-species effect, resulting from a genetic locus with contrasting alleles in each parental species. Oligogenic as opposed to polygenic architecture was supported in both families (60% and 20% PV explained by 4 QTL in the F 2 and F1 respectively). The importance of adventitious rooting for adaptation to survive water-logged environments was thought in part to explain oligogenic architecture of what is believed to be a complex trait controlled by many hundreds of genes.

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.