Supporting conventional breeding

Screening

Breeding programmes often grow thousands, or millions, of individual plants to increase the probability of identifying individual plants with specific gene combinations; this requires new tools, some biotechnological, for plant selection.

Isozyme markers were used in the 1980s to hasten the introgression of monogenic traits from wild germplasm into a cultivated background, a process now known as marker assisted selection (MAS) and now based around the direct detection of variation in DNA sequences (Table 12.1). This can be used to indirectly select traits by detecting genetic variation closely linked to underlying genes.

marker-assisted backcrossing The use of molecular markers is justified when conventional phenotypic trait selection is difficult, or is dependent on specific environments or developmental stages that influence the expression of the target phenotype (Xu and Crouch, 2008). MAS can hasten backcross-ing and is useful in maintaining recessive alleles.

Example 10: Marker-assisted backcrossing (MABC) of Sub1, a major quantitative trait locus (QTL) on chromosome 9 of rice, has improved submergence tolerance of 'Swarna', a cultivar widely grown in flood-prone regions of Asia (Neeraja et al., 2007). Simple sequence repeat (SSR) markers aided both the introgression of Sub1 and the subsequent recovery of the recurrent parental background. Introgression of Sub1 converted 'Swarna' to a submergence-tolerant variety within three backcross generations (2-3 years).

Table 12.1. Recent marker systems developed and applied to marker assisted selection (MAS).

Acronym

Description

RFLPs

Restriction fragment length polymorphisms

RAPDs

Random amplified length polymorphisms

STS

Sequence tagged site

AFLPs

Amplified fragment length polymorphisms

SSRs

Simple sequence repeats or 'microsatellites'

SNPs

Single nucleotide polymorphisms

marker-assisted pyramiding MAS may be used to pyramid multiple monogenic traits, or several QTLs for a single target trait, with complex inheritance such as drought tolerance. Root architecture is a secondary trait intrinsically linked to drought tolerance.

Example 11: The effect of QTLs for root architecture on yield has been reported under varying moisture regimes in rice and maize (reviewed by Collins et al., 2008). After the identification of four major root architecture QTLs in rice, MAS aided the introgression of all alleles for increased root length from 'Azucena' into 'Kalinga III', an upland variety (Steele et al., 2006, 2007).

early-generation mas MAS is often simpler than phenotypic screening selection and can be carried out at early stages of development and on single plants, rather than plant families or plots. Using MAS to select in 'offseason' nurseries enables cost-effective production of more generations per year. DNA extraction is the largest cost in MAS and is often the primary rate-limiting factor for scaling up the whole process (Xu and Crouch, 2008).

Recent development of non-destructive single seed-based DNA extraction and geno-typing systems is enhancing MAS efficiency significantly and is being applied to the International Maize and Wheat Improvement Center's (CIMMYT) maize molecular breeding programmes (Gao et al., 2008).

metabolite-assisted breeding Genomic-based technologies such as metabolic profiling are now used in addition to MAS. The rapid development of high-throughput tools for metabolite profiling makes DNA sequence-based profiling cost competitive (Kopka et al., 2004). Metabolite profiling will assist in the selection of components of yield and stress tolerance (Fernie and Schauer, 2009).

Example 12: Important metabolic traits include carotenoid content of tomato, protein content of maize and starch content of both potato and rice (Fernie and Schauer, 2009). High-throughput metabolomic screening of large tomato breeding populations for carotenoid metabolites has used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Profiling of lines from two tomato populations (Solanum pennellii introgression lines and saturated mutants) identified germplasm likely to assist breeding of fruit containing high levels of nutriceuticals (Fraser et al., 2007).

Combined MAS

Genetic gain can be improved when pheno-typic selection is combined with MAS. Even where relationships between gene information and phenotypic variation are well defined, a lack of appropriate computational tools has hampered incorporation into breeding programmes (Xu and Crouch, 2008). However, new simulation and decision-support software are enabling the integration of genomics into breeding programmes, increasing the scale, efficiency and impact of MAS. Combining screening technologies and computational modelling should shorten the introduction of new varieties by between 3 and 5 years.

Example 13: The genetics and breeding simulation tool, QuLiNE/QuCiM, has been used by wheat breeders to predict cross performance and compare selection strategies (Wang et al., 2003, 2007; Kuchel et al., 2005).

Analysis of diversity and population dynamics

Applying molecular marker technologies to large breeding programmes has advanced genetic mapping; many QTLs controlling a range of abiotic stresses have been identified.

SSRs, amplified fragment length polymorphisms (AFLPs) and random amplified length polymorphisms (RAPDs) have been used to assess genetic diversity in synthetic wheat derivatives (Zhang et al., 2005) and landraces (Strelchenko et al., 2004), important sources of abiotic stress tolerance.

Genotypic variation is used to improve stress tolerance in elite germplasm. Superior genotypes can be developed by the molecular measurement of genetic similarity or genetic distance between parents (Korzun, 2003).

Rapidly identifying genotypes using DNA-based molecular marker technologies is helping breeders to select elite genotypes without extensive field-based testing (Reynolds et al., 2009).

Abiotic stress tolerance diversity in wild relatives and breeding populations is also used to validate candidate genes.

Management of germplasm resources is a major problem for many crop improvement programmes. Diversity surveys help with the compilation of smaller genotype-based reference sets reflecting the allelic diversity present in the larger germplasm reserves.

Example 16: The analysis of 3000 chickpea accessions with 48 SSR markers revealed extensive allelic diversity: 78% of all alleles were captured in a reference set of 300 accessions (Upadhyaya et al, 2008).

High-throughput technologies (genotyping, phenotyping)

It typically takes 12 years to release a commercial cereal variety from the time of the initial cross, and perennials may take longer. The increasing rate of climate change requires accelerated breeding, now being assisted by high-throughput genotyping and phenotyping technologies.

Phenotyping

For many traits, phenotyping is the limiting component. Extensive studies of the genetic control of drought tolerance have not yet resulted in the deployment of markers for specific loci and alleles in breeding programmes. It is difficult to reproduce seasonal differences in combination with different genetic backgrounds, and validating marker-trait associations has been problematic. However, phenotyping is often more reliable for some factors affecting root health, notably tolerance to root disease, pests and nutrient deficiencies. Reliable phenotyping leads to more reliable mapping, usually linked to higher heritability, from which markers can be readily developed and deployed.

High-throughput phenotyping facilities using robotics and image analysis are being constructed at many research sites (APPF, 2009) but it will be several years before their impact can be measured. Similar facilities are already widely used by industry (CropDesign, 2009) to accurately and objectively measure plant characteristics under a range of stresses.

Example 15: Collaboration between CIMMYT, Cornell University and the Chinese, Kenyan, Thai and Zimbabwean governments is identifying key regulators in drought response pheno-types from 350 tropical maize lines. Metabolites such as sucrose, glucose, starch, ABA and the ABA glucose ester of leaves and reproductive organs are being assessed under both water-stressed and well-watered conditions, alongside yield components and secondary traits. The genotypic component of the association test involves haplotyping about 130 ABA and carbohydrate synthesis pathway candidate genes and drought-tolerance response genes involved from maize and other plant species (Ribaut et al., 2009). One- and two-dimensional gas chro-matography/mass spectrometry (GC-MS) has been used to survey 70 rice cultivars for important nutritional metabolites (Kusano et al., 2007; Oryzabase, 2009).

Example 14: In common bean (Phaseolus vulgaris L.), ultrametric genetic distances between progeny and a target parent were used in combination with nine indexed QTL-linked markers, weighted according to the amount of phenotypic variance they explained, to select high-yielding lines that retained important QTLs in a desirable genetic background (Tar'an et al., 2003). Critical for this methodology was a bioinformatic platform capable of compiling and comparing complex molecular fingerprints and delivering predictions of genetic distance and variance.

Phenotyping systems focusing on clusters of mega-environments and high-throughput field-based phenotyping criteria have been used by CIMMYT and the International Rice Research Institute (IRRI). When combined with sampling and data acquisition systems, phenomics-based protocols for breeding programmes can be developed. In natural or controlled environments, drought-tolerance breeding programmes (CIMMYT, 2009a; IRRI, 2009) are incorporating techniques such as remote sensing of plant water status, canopy chlorophyll content and canopy temperature.

Genotyping

PCR-based assays have allowed extensive automation of genotyping, but high marker development costs and low levels of polymorphisms in breeding material have inhibited the use of MAS in many breeding programmes. Cheap, fast-screening using single nucleotide polymorphisms (SNPs) has led to the development of a large SNP detection industry largely servicing medical geno-typing but also applicable to crop plants. Next Generation Sequencing Technologies, such as Solexa and 454/FLX, have dramatically reduced sequencing costs and SNP discovery is now possible in species where other marker systems are poorly developed such as cowpea, chickpea, pigeonpea and groundnut (Varshney et al., 2009).

Private companies are using high-throughput technologies for transgene testing in several model systems.

Example 17: Mendel Biotechnology has over-expressed 1700 transcription factors in Arabidopsis and identified transcription factors related to biomass production, seed yield and a 'stay-green' phenotype under drought stress (Gutterson, 2005). With Monsanto, these genes have been introduced into important cereal crops (Monsanto, 2009a). CropDesign has tested 1400 constructs in rice and identified genes that enhance seed yield and biomass (e.g. SYT1 and STZ) (CropDesign, 2009).

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