Plant Science

Plant genomes are pronouncedly enriched in repeat elements such as transposons. These repeats are epigenetically regulated by DNA methylation. Whole genome high-depth sequencing after bisulfite treatment remains an expensive and laborious method to reliably profile the DNA methylome, especially when considering large genomes such as in crops. Here, we present a simple reproducible Southern hybridisation–based assay to obtain incontrovertible methylation patterns from targeted regions in the rice genome. By employing minor but key modifications, we reliably detected transposon copy number variations over multiple generations. This method can be regarded as a gold standard for validation of epigenetic variations at target loci, and the consequent proliferation of transposons, or segregation in several plant replicates and genotypes.

Graphical abstract:

Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high throughput genotyping. We present here a pipeline for developing a reservoir of SNP and SSR markers for Mangifera indica L. as an example for fruit tree crops having no genomic information available. Our pipeline includes de novo assembly of reference transcriptome with MIRA and CAP3 based on reads produced by 454-GS FLX technology; Polymorphic loci discovery by alignment of Illumina resequencing to the transcriptome reference; Identifying a subset of loci that are polymorphic in the entire germplasm collection for downstream diversity analysis by genotyping with Fluidigm technology.

The genus Lavandula comprises of several economically important lavender species that are mainly cultivated worldwide for essential oil production. Identification of lavender species and their cultivars has been a huge bottleneck in lavender industries due to lack of appropriate identification mechanisms. Recent advances in modern technologies would help to address these identification issues through development of potential molecular markers, including simple sequence repeats (SSRs). SSRs can be developed from specific species, and can be potentially used for related species, which lack the source sequences to develop species-specific SSRs. Here, we describe the guidelines and steps of identifying and analyzing SSRs from expressed sequence tag (EST) sequences of lavender species. We also detail the validation procedures of selected EST-SSRs in distinguishing source (donor) species as well as related species.

Detection of natural hybrids is of great significance for plant taxonomy, reproductive biology, and population genetic studies. Compared with methods depending on morphological characters, molecular markers provide reliable and much more accurate results. This protocol describes approaches employing microsatellite (SSR) markers to identify inter-specific hybrids in Mussaenda (Rubiaceae).

Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.

Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected on a capillary electrophoresis system. This method allows the cost-effective genotyping of several SNPs in a multiplexed manner at an overall low cost in a short period of time. Additionally, this method could be efficiently combined with the simultaneous detection of SSRs at the same electrophoresis run resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

Transposable elements represent a major part of any eukaryotic genomes. Notably in plants they can account for more than 80% of the whole genomic sequence (such as in maize). Due to their mobility across the genome, they can act as mutagens but can also be considered as an important source of genetic diversity. It has been shown that they may be activated following various stresses, and it has been assumed that they may contribute to genome evolution and adaptation. Molecular methods have thus been proposed to allow identification of new transposition events, or more generally to tag transposable element insertion site polymorphisms. Sequence-Specific Amplification Polymorphism (SSAP) is a high throughput method derived from AFLP, which has been first tested on the barley genome (Waugh et al., 1997). Its efficiency in tagging TEs in comparison to AFLP is based on the use of specific primers anchored in the TE sequences of interest, requiring the TEs under survey to be previously characterized. SSAP can thus be used to identify any genomic reorganization in the vicinity of TE insertion sites, and still represents an efficient approach to analyse evolutionary dynamics of TEs.

Transposable elements (TEs) are repetitive sequences, capable of inducing genetic mutations through their transpositional activity, or by non-homologous or illegitimate recombination. Because of their similarity and often high copy numbers, examining the effects of mutations caused by TEs in different samples (tissues, individuals, species, etc.) can be difficult. Thus, high throughput methods have been developed for genotyping TEs in un-sequenced genomes. A common method is termed Transposon Display (or transposon SSAP), which utilizes restriction enzymes and PCR amplification to produce chimeric DNA molecules that include genomic and TE DNA. The advent of second generation sequencing technologies, such as 454-pyrosequencing, have dramatically improved the resolution of this assay, allowing the simultaneous sequencing of all PCR products, representing all amplified TE sites in a specific genome.

In this protocol, determination of seed paternity by microsatellite markers in Nicotiana attenuata is described. However, this does not include a protocol for the novel marker selection/identification, but rather exploits the markers generated for a closely related species N. tabacum (Bindler et al., 2007). This is a high-throughput protocol optimized and streamlined for one skilled person to process 384 (96 x 4) seeds in 5 days, from DNA isolation (from seedlings) to paternity assessment by microsatellite genotype data.