Martine Jean

An Improved Genotyping by Sequencing (GBS) Approach Offering Increased Versatility and Efficiency of SNP Discovery and Genotyping

Highly parallel SNP genotyping platforms have been developed for some important crop species, but these platforms typically carry a high cost per sample for first-time or small-scale users. In contrast, recently developed genotyping by sequencing (GBS) approaches offer a highly cost effective alternative for simultaneous SNP discovery and genotyping. In the present investigation, we have explored the use of GBS in soybean. In addition to developing a novel analysis pipeline to call SNPs and indels from the resulting sequence reads, we have devised a modified library preparation protocol to alter the degree of complexity reduction. We used a set of eight diverse soybean genotypes to conduct a pilot scale test of the protocol and pipeline. Using ApeKI for GBS library preparation and sequencing on an Illumina GAIIx machine, we obtained 5.5 M reads and these were processed using our pipeline. A total of 10,120 high quality SNPs were obtained and the distribution of these SNPs mirrored closely the distribution of gene-rich regions in the soybean genome. A total of 39.5% of the SNPs were present in genic regions and 52.5% of these were located in the coding sequence. Validation of over 400 genotypes at a set of randomly selected SNPs using Sanger sequencing showed a 98% success rate. We then explored the use of selective primers to achieve a greater complexity reduction during GBS library preparation. The number of SNP calls could be increased by almost 40% and their depth of coverage was more than doubled, thus opening the door to an increase in the throughput and a significant decrease in the per sample cost. The approach to obtain high quality SNPs developed here will be helpful for marker assisted genomics as well as assessment of available genetic resources for effective utilisation in a wide number of species.

Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana

Abstract DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability. Using degenerate primers, we have cloned the first plant homologue of the E. coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1. AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4. Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins. As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants. One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated. Surprisingly, although the insertion occurred only ≈80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines. RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site. Finally, no increase in microsatellite instability – a phenotype often associated with mutations in mismatch repair genes – was observed in plants homozygous for this insertion.

The Arabidopsis DNA mismatch repair gene PMS1 restricts somatic recombination between homeologous sequences

The eukaryotic DNA mismatch repair (MMR) system contributes to maintaining the fidelity of genetic information by correcting replication errors and preventing illegitimate recombination events. This study aimed to examine the function(s) of the Arabidopsis thaliana PMS1 gene (AtPMS1), one of three homologs of the bacterial MutL gene in plants. Two independent mutant alleles (Atpms1-1 and Atpms1-2) were obtained and one of these (Atpms1-1) was studied in detail. The mutant exhibited a reduction in seed set and a bias against the transmission of the mutant allele. Somatic recombination, both homologous and homeologous, was examined using a set of reporter constructs. Homologous recombination remained unchanged in the mutant while homeologous recombination was between 1.7- and 4.8-fold higher than in the wild type. This increase in homeologous recombination frequency was not correlated with the degree of sequence divergence. In RNAi lines, a range of increases in homeologous recombination were observed with two lines showing a 3.3-fold and a 3.6-fold increase. These results indicate that the AtPMS1 gene contributes to an antirecombination activity aimed at restricting recombination between diverged sequences.

Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids.

Genotyping-by-Sequencing on Pooled Samples and its Use in Measuring Segregation Bias during the Course of Androgenesis in Barley

Estimation of allelic frequencies is often required in breeding but genotyping many individuals at many loci can be expensive. We have developed a genotyping‐by‐sequencing (GBS) approach for estimating allelic frequencies on pooled samples (Pool‐GBS) and used it to examine segregation distortion in doubled haploid (DH) populations of barley (Hordeum vulgare L.). In the first phase, we genotyped each line individually and exploited these data to explore a strategy to call single nucleotide polymorphisms (SNPs) on pooled reads. We measured both the number of SNPs called and the variance of the estimated allelic frequencies at various depths of coverage on a subset of reads containing 5 to 25 million reads. We show that allelic frequencies could be cost‐effectively and accurately estimated at a depth of 50 reads per SNP using 15 million reads. This Pool‐GBS approach yielded 1984 SNPs whose allelic frequency estimates were highly reproducible (CV = 10.4%) and correlated (r = 0.9167) with the “true” frequency derived from analysis of individual lines. In a second phase, we used Pool‐GBS to investigate segregation bias throughout androgenesis from microspores to a population of regenerated plants. No strong bias was detected among the microspores resulting from the meiotic divisions, whereas significant biases could be shown to arise during embryo formation and plant regeneration. In summary, this methodology provides an approach to estimate allelic frequencies more efficiently and on materials that are unsuitable for individual analysis. In addition, it allowed us to shed light on the process of androgenesis in barley.

A new method for studying population genetics of cyst nematodes based on Pool-Seq and genomewide allele frequency analysis

Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time‐consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping‐by‐sequencing and Pool‐Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well‐characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time‐ and cost‐efficient and could be applied to other cyst nematode species.

Population isolation shapes plant genetics, phenotype and germination in naturally patchy ecosystems

Aims Habitat connectivity is important in conservation since isolation can diminish the potential of a population for adaptation and increase its risk of extinction. However, conservation of naturally patchy ecosystems such as peatlands has mainly focused on preserving specific sites with exceptional characteristics, neglecting the potential interconnectivity between patches. In order to better understand plant dynamics within a peatland network, we assessed the effect of population isolation on genetic distinctiveness, phenotypic variations and germination rates using the peatland-obligate whitefringed orchid (Platanthera blephariglottis). Methods Fifteen phenotypic traits were measured for 24 individuals per population (20 distinct populations, Quebec, Canada) and germination rates of nearly 20 000 seeds were assessed. Genetic distinctiveness was quantified for 26 populations using single nucleotide polymorphism markers obtained via a pooled genotyping-by-sequencing approach. Geographic isolation was measured as the distance to the nearest population and as the number of populations occurring in concentric buffer zones (within a radius of 2, 5 and 10 km) around the studied populations. Important Findings All phenotypic traits showed significant differences among populations. Genetic results also indicated a pattern of isolation-bydistance, which suggests that seed and/or pollen exchange is restricted geographically. Finally, all phenotypic traits, as well as a reduced germination rate, were correlated with either geographic isolation or genetic distance. We conclude that geographic isolation likely restricts gene flow, which in turn may affect germination. Consequently, it is imperative that conservation programs take into account the patchy nature of such ecosystems, rather than targeting a few specific sites with exceptional character for preservation.