Advances in next generation technologies have driven the costs of DNA sequencing down to the point that genotyping-by-sequencing (GBS) is now feasible for high diversity, large genome species. Here, we report a procedure for constructing GBS libraries based on reducing genome complexity with restriction enzymes (REs). This approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. By using methylation-sensitive REs, repetitive regions of genomes can be avoided and lower copy regions targeted with two to three fold higher efficiency. This tremendously simplifies computationally challenging alignment problems in species with high levels of genetic diversity. The GBS procedure is demonstrated with maize (IBM) and barley (Oregon Wolfe Barley) recombinant inbred populations where roughly 200,000 and 25,000 sequence tags were mapped, respectively. An advantage in species like barley that lack a complete genome sequence is that a reference map need only be developed around the restriction sites, and this can be done in the process of sample genotyping. In such cases, the consensus of the read clusters across the sequence tagged sites becomes the reference. Alternatively, for kinship analyses in the absence of a reference genome, the sequence tags can simply be treated as dominant markers. Future application of GBS to breeding, conservation, and global species and population surveys may allow plant breeders to conduct genomic selection on a novel germplasm or species without first having to develop any prior molecular tools, or conservation biologists to determine population structure without prior knowledge of the genome or diversity in the species.

/pdf/a-robust-simple-genotyping-by-sequencing-gbs-approach-for-2orahkct9n.pdf

A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species

DNA markers have enormous potential to improve the efficiency and precision of conventional plant breeding via marker-assisted selection (MAS). The large number of quantitative trait loci (QTLs) mapping studies for diverse crops species have provided an abundance of DNA marker–trait associations. In this review, we present an overview of the advantages of MAS and its most widely used applications in plant breeding, providing examples from cereal crops. We also consider reasons why MAS has had only a small impact on plant breeding so far and suggest ways in which the potential of MAS can be realized. Finally, we discuss reasons why the greater adoption of MAS in the future is inevitable, although the extent of its use will depend on available resources, especially for orphan crops, and may be delayed in less-developed countries. Achieving a substantial impact on crop improvement by MAS represents the great challenge for agricultural scientists in the next few decades.

/pdf/marker-assisted-selection-an-approach-for-precision-plant-1be57ryhno.pdf

Marker-assisted selection: an approach for precision plant breeding in the twenty-first century

We developed a simple marker technique called sequence-related amplified polymorphism (SRAP) aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (”filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. For the first five cycles the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by autoradiography. We tested the marker technique in a series of recombinant inbred and doubled-haploid lines of Brassica oleracea L. After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty percent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in nine major linkage groups, not differing in this regard to AFLP markers. We successfully tagged the glucosinolate desaturation gene BoGLS-ALK with these markers. SRAPs were also easily amplified in other crops such as potato, rice, lettuce, Chinese cabbage (Brassica rapa L.), rapeseed (Brassica napus L.), garlic, apple, citrus, and celery. We also amplified cDNA isolated from different tissues of Chinese cabbage, allowing the fingerprinting of these sequences.

Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica

Drought tolerance improvement in crop plants: An integrated view from breeding to genomics

There is tremendous interest in using association mapping to identify genes responsible for quantitative variation of complex traits with agricultural and evolutionary importance. Recent advances in genomic technology, impetus to exploit natural diversity, and development of robust statistical analysis methods make association mapping appealing and affordable to plant research programs. Association mapping identifi es quantitative trait loci (QTLs) by examining the marker-trait associations that can be attributed to the strength of linkage disequilibrium between markers and functional polymorphisms across a set of diverse germplasm. General understanding of association mapping has increased signifi cantly since its debut in plants. We have seen a more concerted effort in assembling various association-mapping populations and initiating experiments through either candidategene or genome-wide approaches in different plant species. In this review, we describe the current status of association mapping in plants and outline opportunities and challenges in complex trait dissection and genomics-assisted crop improvement. L arge-scale genome-wide association analyses of major human diseases have yielded very promising results, corroborating fi ndings of previous candidategene association studies and identifying novel disease loci that were previously unknown (Th e Wellcome Trust Case Control Consortium, 2007). Th e same strategy is being exploited in many plant species thanks to the dramatic reduction in costs of genomic technologies. In contrast to the widely used linkage analysis traditional mapping research in plants, association mapping searches for functional variation in a much broader germplasm context. Association mapping enables researchers to use modern genomic technologies to exploit natural diversity, the wealth of which is known to plant geneticists and breeders but has been utilized only on a small scale before the genomics era. Owing to the ease of producing large numbers of progenies from controlled crosses and conducting replicated trials with immortal individuals (inbreds and recombinant inbred lines, RILs), association mapping in plants may prove to be more promising than in human or animal genetics. In the current review,

/pdf/status-and-prospects-of-association-mapping-in-plants-h5kcg1ymdj.pdf

Status and Prospects of Association Mapping in Plants

Breeding by design.

AFLPTM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring.

QTL analysis of pepper fruit characters was performed in an F3 population derived from a cross between two Capsicum annuum genotypes, the bell-type cultivar Maor and the Indian small-fruited line Perennial. RFLP, AFLP®1, RAPD and morphological markers (a total of 177) were used to construct a comparative pepper-tomato genetic map for this cross, and 14 quantitatively inherited traits were evaluated in 180 F3 families. A total of 55 QTL were identified by interval analysis using LOD 3.0 as the threshold for QTL detection. QTL for several traits including fruit diameter and weight, pericarp thickness and pedicel diameter were often located in similar chromosomal regions, thus reflecting high genetic correlations among these traits. A major QTL that accounts for more than 60% of the phenotypic variation for fruit shape (ratio of fruit length to fruit diameter) was detected in chromosome 3. This chromosome also contained QTL for most of the traits scored in the population. Markers in linkage groups 2, 3, 8 and 10 were associated with QTL for multiple traits, thereby suggesting their importance as loci that control developmental processes in pepper. Several QTL in pepper appeared to correspond to positions in tomato for loci controlling the same traits, suggesting the hypothesis that these QTL may be orthologous in the two species.

QTL mapping of fruit-related traits in pepper (Capsicum annuum)

Two independent F2 populations of Lycopersicon esculentum×L. pennellii which have previously been investigated in RFLP mapping studies were used for construction of a highly saturated integrated AFLP map. This map spanned 1482 cM and contained 67 RFLP markers, 1078 AFLP markers obtained with 22 EcoRI+MseI primer combinations and 97 AFLP markers obtained with five PstI+MseI primer combinations, 231 AFLP markers being common to both populations. The EcoRI+MseI AFLP markers were not evenly distributed over the chromosomes. Around the centromeric region, 848 EcoRI+ MseI AFLP markers were clustered and covered a genetic distance of 199 cM, corresponding to one EcoRI+ MseI AFLP marker per 0.23 cM; on the distal parts 1283 cM were covered by 230 EcoRI+MseI AFLP markers, corresponding to one marker per 5.6 cM. The PstI/MseI AFLP markers showed a more even distribution with 16 PstI/MseI AFLP markers covering a genetic distance of 199 cM around the centromeric regions and 81 PstI/MseI AFLP markers covering a genetic distance of 1283 cM on the more distal parts, corresponding to one marker per 12 and 16 cM respectively. In both populations a large number of loci showed a significant skewed segregation, but only chromosome 10 loci showed skewness that was similar for both populations. This ultra-dense molecular-marker map provides good perspectives for genetic and breeding purposes and map-based cloning.

An integrated high-density RFLP-AFLP map of tomato based on two Lycopersicon esculentum x L. pennellii F2 populations

Although DNA microarrays are currently the standard tool for genome-wide expression analysis, their application is limited to organisms for which the complete genome sequence or large collections of known transcript sequences are available. Here, we describe a protocol for cDNA-AFLP, an AFLP-based transcript profiling method that allows genome-wide expression analysis in any species without the need for prior sequence knowledge. In essence, the cDNA-AFLP method involves reverse transcription of mRNA into double-stranded cDNA, followed by restriction digestion, ligation of specific adapters and fractionation of this mixture of cDNA fragments into smaller subsets by selective PCR amplification. The resulting cDNA-AFLP fragments are separated on high-resolution gels, and visualization of cDNA-AFLP fingerprints is described using either a conventional autoradiography platform or an automated LI-COR system. Observed differences in band intensities between samples provide a good measure of the relative differences in the gene expression levels. Identification of differentially expressed genes can be accomplished by purifying cDNA-AFLP fragments from sequence gels and subsequent sequencing. This method has found widespread use as an attractive technology for gene discovery on the basis of fragment detection and for temporal quantitative gene expression analysis. The protocol can be completed in 3-4 d.

J. Peleman

Papers

Breeding by design.

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring.

QTL mapping of fruit-related traits in pepper (Capsicum annuum)

An integrated high-density RFLP-AFLP map of tomato based on two Lycopersicon esculentum x L. pennellii F2 populations

AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis