Molecular markers and plant breeding

The achievements of modern biotechnology allow to modernize significantly the traditional plant breeding. The use of molecular codominant markers reduces considerably the quantity of breeding material and promotes the selection of genotypes, which posses desirable genes in the homozygous state. Molecular marking systems of agronomically important simple and quantitative traits have been developed using mono- and multiloci systems. Markers of the plant type and development rate, alleles of the storage protein genes, Wx-genes, short-stem genes, etc., have been created and tested at the South Biotecnology Center, National Academy of Agricultural Sciences. The technology of the application of DNA-typing for the identification and registration of varieties that has been developed in the South Biotecnology Center is of great importance for the systematization of germplasm sources and the protection of the rights of breeders.     

Molecular tools for breeding basidiomycetes.

The industrial production of edible basidiomycetes is increasing every year as a response to the increasing public demand of them because of their nutritional properties. About a dozen of fungal species can be currently produced for food with sound industrial and economic bases. Notwithstanding, this production is threatened by biotic and abiotic factors that make it necessary to improve the fungal strains currently used in industry. Breeding of edible basidiomycetes, however, has been mainly empirical and slow since the genetic tools useful in the selection of the new genetic material to be introduced in the commercial strains have not been developed for these fungi as it was for other organisms. In this review we will discuss the main genetic factors that should be considered to develop breeding approaches and tools for higher basidiomycetes. These factors are (i) the genetic system controlling fungal mating; (ii) the genomic structure and organisation of these fungi; and (iii) the identification of genes involved in the control of quantitative traits. We will discuss the weight of these factors using the oyster mushroom Pleurotus ostreatus as a model organism for most of the edible fungi cultivated industrially.     

Molecular breeding of carotenoid biosynthetic pathways

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.     

Molecular marker-assisted breeding options for maize improvement in Asia

Maize is one of the most important food and feed crops in Asia, and is a source of income for several million farmers. Despite impressive progress made in the last few decades through conventional breeding in the “Asia-7” (China, India, Indonesia, Nepal, Philippines, Thailand, and Vietnam), average maize yields remain low and the demand is expected to increasingly exceed the production in the coming years. Molecular marker-assisted breeding is accelerating yield gains in USA and elsewhere, and offers tremendous potential for enhancing the productivity and value of Asian maize germplasm. We discuss the importance of such efforts in meeting the growing demand for maize in Asia, and provide examples of the recent use of molecular markers with respect to (i) DNA fingerprinting and genetic diversity analysis of maize germplasm (inbreds and landraces/OPVs), (ii) QTL analysis of important biotic and abiotic stresses, and (iii) marker-assisted selection (MAS) for maize improvement. We also highlight the constraints faced by research institutions wishing to adopt the available and emerging molecular technologies, and conclude that innovative models for resource-pooling and intellectual-property-respecting partnerships will be required for enhancing the level and scope of molecular marker-assisted breeding for maize improvement in Asia. Scientists must ensure that the tools of molecular marker-assisted breeding are focused on developing commercially viable cultivars, improved to ameliorate the most important constraints to maize production in Asia.     

Molecular markers in breeding for virus resistance in barley

The soil-borne barley yellow mosaic virus disease (BaMMV, BaYMV, BaYMV-2) and the aphid-transmitted barley yellow dwarf virus (BYDV) are serious threats to winter barley cultivation. Resistance to barley yellow mosaic virus disease has been identified in extensive screening programmes and several recessive resistance genes have been mapped, e.g. rym4, rym5, rym9, rym11, rym13. In contrast to barley yellow mosaic virus disease, no complete resistance to BYDV is known in the barley gene pool, but tolerant accessions have been identified and QTL for BYDV-tolerance have been detected on chromosomes 2HL and 3HL. The use of resistance and tolerance in barley breeding can be considerably improved today by molecular markers (RFLPs, RAPDs, AFLPs, SSRs, STSs, SNPs), as they facilitate (i) efficient genotyping and estimation of genetic diversity; (ii) reliable selection on a single plant level independent of symptom expression in the field (iii) acceleration of back crossing procedures; (iv) pyramiding of resistance genes; (v) detection of QTL and marker-based combination of positive alleles; and (vi) isolation of resistance genes via map-based cloning.     

Molecular breeding of a biotin-hyperproducing Serratia marcescens strain.

We previously reported that an acidomycin-resistant mutant of Serratia marcescens Sr41, SB304, and a mutant that was derived from SB304 and was resistant to a higher concentration of acidomycin, SB412, produced 5 and 20 mg of D-biotin, respectively, per liter of a medium containing sucrose and urea (N. Sakurai, Y. Imai, M. Masuda, S. Komatsubara, and T. Tosa, Appl. Environ. Microbiol. 59:2857-2863, 1993). In order to increase the productivity of D-biotin, the biotin (bio) operons were cloned from strains SB412, SB304, and 8000 (wild-type strain), and pLGM412, pLGM304, and pLGW101, respectively, were obtained through subcloning. These plasmids harbored 7.2-kb DNA fragments coding for the bioABFCD genes on a low-copy-number vector and were introduced into SB304, SB412, and 8000. Among the resulting recombinant strains, SB412(pLGM304) exhibited the highest D-biotin production (200 mg/liter) in the production medium. The plasmid was stably maintained in cells. Unexpectedly, SB412(pLGM412) grew very slowly, and the D-biotin productivity of this recombinant strain was not evaluated because pLGM412 was unstable.     

Molecular breeding strategies for the modification of lipid composition

Summary Lipids are key components of all living cells. Acyl lipids and sterols provide the matrix of the biological membranes that both define the boundaries of cells and organelles, and act as sites for the trafficking of molecules within and into/out of cells. Lipids are also important metabolic intermediates and the most efficient form of energy storage that is available to a cell. It is the latter, energy-storing function that is of most relevance to this review. Storage lipids are accumulated in abundance in many of our most important crops, including maize, soybean, rapeseed, and oil palm, giving rise to a commerical sector valued at over $50 billion/year. Because the storage lipids of the major global oil crops have a relatively restricted composition, there is great interest in using all available breeding technologies, whether traditional or modern, to enhance the variation in lipid quality in existing crops and/or to domesticate new crops that already accumulate useful novel lipids. Over the past few decades, there has been a great deal of effort to manipulate fatty acid composition in order to produce novel lipids, especially for industrial applications. However, these attempts, many based on genetic engineering, have met with only limited commercial success-to date. More recently, there has been a resurgence of interest in the modification of both acyl and non-acyl lipids to enhance the nutritional quality of plant oils. In this review, we will examine the background to plant lipid modification and some of the latest developments, with a particular focus on edible oils.     

Molecular breeding of polymerases for resistance to environmental inhibitors

Potent inhibitors limit the use of PCR assays in a wide spectrum of specimens. Here, we describe the engineering of polymerases with a broad resistance to complex environmental inhibitors using molecular breeding of eight different polymerase orthologues from the genus Thermus and directed evolution by CSR in the presence of inhibitors. Selecting for resistance to the inhibitory effects of Neomylodon bone powder, we isolated 2D9, a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. 2D9 displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. The selected polymerase promises to have utility in PCR-based applications in a wide range of fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.     

Molecular breeding of polymerases for amplification of ancient DNA

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.     

Molecular breeding of switchgrass for use as a biofuel crop

Switchgrass (Panicum virgatum L.) is projected to become one of the main herbaceous, biofuel crops in United States. This status was the result of several years of research; much it sponsored by the United States Department of Energy (DOE). Literature documenting fundamental aspects of switchgrass taxonomy, genetics, breeding, management, physiology, and use is now available and form the basis for protocols to establish and manage the crop, as well as efforts to develop improved cultivars. Future improvement will include production of high yielding hybrids and the use of genomic and transgenic biotechnologies to enhance both productivity and chemical composition. Reducing bioconversion recalcitrance via reduction of lignin content is an example of projected future research in this area.     

Molecular approaches in pig breeding to improve meat quality.

This article reviews the advances in molecular genetics that have led to the identification of genes and markers associated with meat quality in pig. The development of a considerable number of annotated livestock genome sequences represents an incredibly rich source of information that can be used to identify candidate genes responsible for complex traits and quantitative trait loci effects. In pig, the huge amount of information emerging from the study of the genome has helped in the acquisition of new knowledge concerning biological systems and it is opening new opportunities for the genetic selection of this specie. Among the new fields of genomics recently developed, functional genomics and proteomics that allow considering many genes and proteins at the same time are very useful tools for a better understanding of the function and regulation of genes, and how these participate in complex networks controlling the phenotypic characteristics of a trait. In particular, global gene expression profiling at the mRNA and protein level can provide a better understanding of gene regulation that underlies biological functions and physiology related to the delivery of a better pig meat quality. Moreover, the possibility to realize an integrated approach of genomics and proteomics with bioinformatics tools is essential to obtain a complete exploitation of the available molecular genetics information. The development of this knowledge will benefit scientists, industry and breeders considering that the efficiency and accuracy of the traditional pig selection schemes will be improved by the implementation of molecular data into breeding programs.     

Molecular breeding of a novel herbicide-tolerant rice by gene targeting

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.     

Molecular plant breeding: achievements in green biotechnology and future perspectives

Since one decade ago, transgenic crop plants are globally grown; in 2004, it was estimated to cover a total of 81 Mio ha in 17 countries. At present, four plant species (soybean, maize, cotton and rapeseed) dominate with two traits (herbicide tolerance and insect resistance). The traits on which research concentrates and the constructs which might come next onto the market are outlined. The procedure on how to clone such genes of interest, e.g. via map-based cloning, and some other helpful approaches of green biotechnology, like high throughput techniques and functional markers, are summarised, and a rough calculation about the market value of transgenic crops in US dollars is quoted.