Bridging genomics and genetic diversity : association between sequence polymorphism and trait variation in a spring barley collection

Association analysis has become common praxis in plant genetics for high-resolution mapping of quantitative trait loci (QTL), validating candidate genes, and identifying important alleles for crop improvement. In the present study the feasibility of association mapping in barley is investigated by associating DNA polymorphisms in selected candidate genes with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. (1) As a starting point a worldwide collection of spring barley (Hordeum vulgare L.) accessions has been established to serve as an association platform for the present and possible further studies. This collection of 224 accessions, sampled from the IPK genebank, consists of 109 European, 45 West Asian and North African, 40 East Asian and 30 American entries. Forty-five EST derived polymorphic SSRs were used to determine the genetic structure. The markers were equally distributed over all seven chromosomes. Phenotypic data were assessed in field experiments performed at three locations in 2004 and 2005 in Germany. (2) Seven candidate genes were considered. Fragments of these genes were amplified and sequenced in the established collection. Single nucleotide polymorphisms (SNPs), haplotype variants, and linkage disequilibrium (LD) were investigated. (3) One gene was additionally analysed in 42 bread wheat (Triticum aestivum L.) accessions in order to compare barley and wheat for nucleotide diversity and LD. (4) Association analysis between SNPs and haplotype variants of the selected candidate genes and the phenotypic variation in thousand-grain weight, crude protein content, starch content, plant height, and flowering time was used to identify candidate genes influencing the variation of these traits in spring barley. A mixed model association-mapping method was employed for this purpose. In the established collection, significant genotypic variation was observed for all traits under study. Genotype×environment interaction variances were much smaller than the genotypic variances and heritability coefficients exceeded 0.9. Statistical analyses of population stratification revealed two major subgroups, mainly comprising two-rowed and six-rowed accessions, respectively. Within the sequenced fragments (13kb) of the seven candidate genes, 216 polymorphic sites and 93 haplotypes were detected demonstrating a moderate to high level of nucleotide and haplotype diversity in the germplasm collection. Most haplotypes (74.2%) occurred at a low frequency (95%) in the coding sequences, the distribution of polymorphisms was also similar in the two species, and both map to a syntenic position on chromosome 3. However, the genes were different in both collections with respect to LD and Tajima?s D statistic. In the barley collection only a moderate level of LD was observed whereas in wheat, LD was absolute between polymorphic sites located in the first intron while it decayed by distance between the former sites and those located downstream the first intron. Differences in Tajima?s D values indicate a lower selection pressure on the gene in barley than in wheat. In conclusion, the established association platform represents an excellent resource for marker-trait association studies. The germplasm collection displays a wide range of genotypic and phenotypic diversity providing phenotypic data for economically important traits and comprehensive information about the nucleotide and haplotype polymorphism of seven candidate genes. Association results demonstrate that the candidate gene-based approach of association mapping is an appropriate tool for characterising gene loci that have a significant impact on plant development and grain quality in spring barley.Die Assoziationsanalyse ist in der Pflanzengenetik eine aktuelle Methode zur hochauflösenden Kartierung quantitativer Merkmale (?quantitative trait loci?, QTL), Validierung von Kandidatengenen und Identifizierung merkmalsrelevanter Allele für die Züchtung. In der vorliegenden Arbeit soll durch die Verknüpfung von Sequenzpolymorphismus in ausgewählten Kandidatengenen und der Variation in den Merkmalen Kornqualität, Pflanzenhöhe und Blühzeitpunkt die Eignung der Assoziationskartierung zur Ermittlung von funktionalen und diagnostischen Markern in Genen, die die Ausprägung dieser Merkmale beeinflussen, geprüft werden. (1) Für diese und mögliche weitere Assoziationsstudien wurde im Rahmen der Arbeit eine Kollektion ausgewählter Sommergerstenakzessionen (Hordeum vulgare L.) der Gaterslebener Genbank zusammengestellt. Die Kollektion umfasst 224 Akzessionen, von denen 109 aus Europa, 45 aus Westasien und Nordafrika, 40 aus Ostasien und 30 aus Amerika stammen. Ein Set von 45 EST(?expressed sequence tag?)-abgeleiteten SSRs wurde zur Bestimmung der genetischen Struktur der Kollektion herangezogen. Die phänotypischen Daten beruhen auf Feldversuchen, die in den Jahren 2004 und 2005 an drei Standorten in Deutschland durchgeführt wurden. (2) Sieben Kandidatengene wurden ausgewählt. Fragmente dieser Kandidatengene wurden amplifiziert und innerhalb der etablierten Kollektion sequenziert. Nukleotiddiversität und Linkage Disequilibrium (LD) wurden untersucht. (3) Ein Gen wurde zusätzlich in einer Kollektion von 42 Brotweizenakzessionen (Triticum aestivum L.) hinsichtlich Nukleotiddiversität und LD untersucht. (4) Die Assoziation zwischen den Merkmalen Rohproteingehalt, Stärkegehalt, Tausendkorngewicht, Blühzeitpunkt und Pflanzenhöhe und Haplotypen bzw. Einzelnukleotidaustauschen (?single nucleotide polymorphisms?, SNPs) der sieben untersuchten Kandidatengene wurde in der oben beschriebenen Kollektion unter Verwendung eines gemischten Modells getestet. In der etablierten Kollektion konnte eine signifikante genotypische Variation für alle untersuchten Merkmale nachgewiesen werden. Die Varianz der Genotyp×Umwelt Interaktion war wesentlich geringer im Vergleich zur genotypischen Varianz, und die Heritabilitätskoeffizienten lagen über 0,9. Statistische Analysen zur Ermittlung der Populationsstruktur ergaben zwei Untergruppen, welche die Akzessionen im Wesentlichen in zweizeilige und sechszeilige Ährentypen unterteilten. Innerhalb der sequenzierten Fragmente (13kb) der sieben Kandidatengene konnten in der Kollektion 216 SNP-Marker und 93 Haplotypen detektiert werden. Die Mehrzahl der Haplotypen (74,2%) war jedoch nur mit einer geringen Frequenz (95%) innerhalb der kodierenden Sequenz, ergab eine syntenische Kartenpositionen auf Chromosom 3 und die Verteilung der Polymorphismen war in beiden Kollektionen ähnlich. Im Hinblick auf das LD und Tajima?s D verhielten sich beide Kollektionen an diesem Locus jedoch unterschiedlich. In der Gerstenkollektion wurde ein mittleres LD beobachtet. Bei Weizen trat zwischen den im ersten Intron lokalisierten Polymorphismen absolutes LD auf, während es zwischen den strangabwärts gelegenen Polymorphismen mit zunehmender Distanz stark zurückging. Diese Unterschiede sind vermutlich auf den, im Vergleich zum orthologen Weizengen, geringeren Selektionsdruck im Gerstengen zurückzuführen. Mit der beschriebenen Kollektion wurde eine Ressource geschaffen, die sich hervorragend als Plattform zur Assoziationskartierung bei Sommergerste eignet. Die phänotypischen und genotypischen Daten der Kollektion repräsentieren ein breites Diversitätsspektrum. Die vorliegende Arbeit zeigt, dass die Kandidatengen-basierte Assoziationskartierung eine effektive Methode zur Identifizierung von Genen darstellt, die an der Ausprägung quantitativer Merkmale wie Blühzeitpunkt, Pflanzenhöhe und Kornqualität beteiligt sind

High levels of nucleotide diversity and fast decline of linkage disequilibrium in rye (Secale cereale L.) genes involved in frost response

<p>Abstract</p> <p>Background</p> <p>Rye (<it>Secale cereale </it>L.) is the most frost tolerant cereal species. As an outcrossing species, rye exhibits high levels of intraspecific diversity, which makes it well-suited for allele mining in genes involved in the frost responsive network. For investigating genetic diversity and the extent of linkage disequilibrium (LD) we analyzed eleven candidate genes and 37 microsatellite markers in 201 lines from five Eastern and Middle European rye populations.</p> <p>Results</p> <p>A total of 147 single nucleotide polymorphisms (SNPs) and nine insertion-deletion polymorphisms were found within 7,639 bp of DNA sequence from eleven candidate genes, resulting in an average SNP frequency of 1 SNP/52 bp. Nucleotide and haplotype diversity of candidate genes were high with average values <it>π </it>= 5.6 × 10^-3and <it>Hd </it>= 0.59, respectively. According to an analysis of molecular variance (AMOVA), most of the genetic variation was found between individuals within populations. Haplotype frequencies varied markedly between the candidate genes. <it>ScCbf14</it>, <it>ScVrn1</it>, and <it>ScDhn1 </it>were dominated by a single haplotype, while the other 8 genes (<it>ScCbf2</it>, <it>ScCbf6</it>, <it>ScCbf9b</it>, <it>ScCbf11</it>, <it>ScCbf12</it>, <it>ScCbf15</it>, <it>ScIce2</it>, and <it>ScDhn3</it>) had a more balanced haplotype frequency distribution. Intra-genic LD decayed rapidly, within approximately 520 bp on average. Genome-wide LD based on microsatellites was low.</p> <p>Conclusions</p> <p>The Middle European population did not differ substantially from the four Eastern European populations in terms of haplotype frequencies or in the level of nucleotide diversity. The low LD in rye compared to self-pollinating species promises a high resolution in genome-wide association mapping. SNPs discovered in the promoters or coding regions, which attribute to non-synonymous substitutions, are suitable candidates for association mapping.</p

DNA polymorphisms and haplotype patterns of transcription factors involved in barley endosperm development are associated with key agronomic traits

<p>Abstract</p> <p>Background</p> <p>Association mapping is receiving considerable attention in plant genetics for its potential to fine map quantitative trait loci (QTL), validate candidate genes, and identify alleles of interest. In the present study association mapping in barley (<it>Hordeum vulgare </it>L.) is investigated by associating DNA polymorphisms with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. We focused on the four loci <it>BLZ1</it>, <it>BLZ2</it>, <it>BPBF </it>and <it>HvGAMYB </it>that play a role in the regulation of B-hordein expression, the major fraction of the barley storage protein. The association was tested in a collection of 224 spring barley accessions using a two-stage mixed model approach.</p> <p>Results</p> <p>Within the sequenced fragments of four candidate genes we observed different levels of nucleotide diversity. The effect of selection on the candidate genes was tested by Tajima's D which revealed significant values for <it>BLZ1</it>, <it>BLZ2</it>, and <it>BPBF </it>in the subset of two-rowed barleys. Pair-wise LD estimates between the detected SNPs within each candidate gene revealed different intra-genic linkage patterns. On the basis of a more extensive examination of genomic regions surrounding the four candidate genes we found a sharp decrease of LD (<it>r</it>²<0.2 within 1 cM) in all but one flanking regions.</p> <p>Significant marker-trait associations between SNP sites within <it>BLZ1 </it>and flowering time, <it>BPBF </it>and crude protein content and <it>BPBF </it>and starch content were detected. Most haplotypes occurred at frequencies <0.05 and therefore were rejected from the association analysis. Based on haplotype information, <it>BPBF </it>was associated to crude protein content and starch content, <it>BLZ2 </it>showed association to thousand-grain weight and <it>BLZ1 </it>was found to be associated with flowering time and plant height.</p> <p>Conclusions</p> <p>Differences in nucleotide diversity and LD pattern within the candidate genes <it>BLZ1</it>, <it>BLZ2</it>, <it>BPBF</it>, and <it>HvGAMYB </it>reflect the impact of selection on the nucleotide sequence of the four candidate loci.</p> <p>Despite significant associations, the analysed candidate genes only explained a minor part of the total genetic variation although they are known to be important factors influencing the expression of seed quality traits. Therefore, we assume that grain quality as well as plant height and flowering time are influenced by many factors each contributing a small part to the expression of the phenotype. A genome-wide association analysis could provide a more comprehensive picture of loci involved in the regulation of grain quality, thousand grain weight and the other agronomic traits that were analyzed in this study. However, despite available high-throughput genotyping arrays the marker density along the barely genome is still insufficient to cover all associations in a whole genome scan. Therefore, the candidate gene-based approach will further play an important role in barley association studies.</p

Genome-wide prediction methods for detecting genetic effects of donor chromosome segments in introgression populations

BACKGROUND:Introgression populations are used to make the genetic variation of unadapted germplasm or wild relatives of crops available for plant breeding. They consist of introgression lines that carry small chromosome segments from an exotic donor in the genetic background of an elite line. The goal of our study was to investigate the detection of favorable donor chromosome segments in introgression lines with statistical methods developed for genome-wide prediction. RESULTS:Computer simulations showed that genome-wide prediction employing heteroscedastic marker variances had a greater power and a lower false positive rate compared with homoscedastic marker variances when the phenotypic difference between the donor and recipient lines was controlled by few genes. The simulations helped to interpret the analyses of glycosinolate and linolenic acid content in a rapeseed introgression population and plant height in a rye introgression population. These analyses support the superiority of genome-wide prediction approaches that use heteroscedastic marker variances. CONCLUSIONS:We conclude that genome-wide prediction methods in combination with permutation tests can be employed for analysis of introgression populations. They are particularly useful when introgression lines carry several donor segments or when the donor segments of different introgression lines are overlapping

From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

<p>Abstract</p> <p>Background</p> <p>The improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (<it>Secale cereale </it>L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology.</p> <p>Results</p> <p>More than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool.</p> <p>Conclusions</p> <p>The established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye.</p

High level of conservation between genes coding for the GAMYB transcription factor in barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.) collections

The transcription factor GAMYB is involved in gibberellin signalling in cereal aleurone cells and in plant developmental processes. Nucleotide diversity of HvGAMYB and TaGAMYB was investigated in 155 barley (Hordeum vulgare) and 42 wheat (Triticum aestivum) accessions, respectively. Polymorphisms defined 18 haplotypes in the barley collection and 1, 7 and 3 haplotypes for the A, B, and D genomes of wheat, respectively. We found that (1) Hv- and TaGAMYB genes have identical structures. (2) Both genes show a high level of nucleotide identity (>95%) in the coding sequences and the distribution of polymorphisms is similar in both collections. At the protein level the functional domain is identical in both species. (3) GAMYB genes map to a syntenic position on chromosome 3. GAMYB genes are different in both collections with respect to the Tajima D statistic and linkage disequilibrium (LD). A moderate level of LD was observed in the barley collection. In wheat, LD is absolute between polymorphic sites, mostly located in the first intron, while it decays within the gene. Differences in Tajima D values might be due to a lower selection pressure on HvGAMYB, compared to its wheat orthologue. Altogether our results provide evidence that there have been only few evolutionary changes in Hv- and TaGAMYB. This confirms the close relationship between these species and also highlights the functional importance of this transcription factor