Identification of Stipules reduced, a leaf morphology gene in pea (Pisum sativum).

Pea (Pisum sativum) is one of relatively few genetically amenable plant species with compound leaves. Pea leaves have a variety of specialized organs: leaflets, tendrils, pulvini and stipules, which enable the identification of mutations that transform or affect distinct parts of the leaf. Characterization of these mutations offers insights into the development and evolution of novel leaf traits. The previously characterized morphological gene Cochleata, conferring stipule identity, was known to interact with Stipules reduced (St), which conditions stipule size in pea, but the St gene remained unknown. Here we analysed Fast Neutron irradiated pea mutants by restriction site associated DNA sequencing. We identified St as a gene encoding a C2H2 zinc finger transcription factor that is regulated by Cochleata. St regulates both cell division and cell expansion in the stipule. Our approach shows how systematic genome-wide screens can be used successfully for the analysis of traits in species for which whole genome sequences are not available.

Implementation of Genomic Prediction in Lolium perenne (L.) Breeding Populations.

Perennial ryegrass (Lolium perenne L.) is one of the most widely grown forage grasses in temperate agriculture. In order to maintain and increase its usage as forage in livestock agriculture, there is a continued need for improvement in biomass yield, quality, disease resistance, and seed yield. Genetic gain for traits such as biomass yield has been relatively modest. This has been attributed to its long breeding cycle, and the necessity to use population based breeding methods. Thanks to recent advances in genotyping techniques there is increasing interest in genomic selection from which genomically estimated breeding values are derived. In this paper we compare the classical RRBLUP model with state-of-the-art machine learning techniques that should yield themselves easily to use in GS and demonstrate their application to predicting quantitative traits in a breeding population of L. perenne. Prediction accuracies varied from 0 to 0.59 depending on trait, prediction model and composition of the training population. The BLUP model produced the highest prediction accuracies for most traits and training populations. Forage quality traits had the highest accuracies compared to yield related traits. There appeared to be no clear pattern to the effect of the training population composition on the prediction accuracies. The heritability of the forage quality traits was generally higher than for the yield related traits, and could partly explain the difference in accuracy. Some population structure was evident in the breeding populations, and probably contributed to the varying effects of training population on the predictions. The average linkage disequilibrium between adjacent markers ranged from 0.121 to 0.215. Higher marker density and larger training population closely related with the test population are likely to improve the prediction accuracy.

Fructan synthesis, accumulation and polymer traits. II. Fructan pools in populations of perennial ryegrass (Lolium perenne L.) with variation for water-soluble carbohydrate and candidate genes were not correlated with biosynthetic activity and demonstrated constraints to polymer chain extension.

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism, and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favoring high sink/low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.

Temporal changes in population genetic diversity and structure in red and white clover grown in three contrasting environments in northern Europe.

UNLABELLED: BACKGOUND AND AIMS: Extending the cultivation of forage legume species into regions where they are close to the margin of their natural distribution requires knowledge of population responses to environmental stresses. This study was conducted at three north European sites (Iceland, Sweden and the UK) using AFLP markers to analyse changes in genetic structure over time in two population types of red and white clover (Trifolium pratense and T. repens, respectively): (1) standard commercial varieties; (2) wide genetic base (WGB) composite populations constructed from many commercial varieties plus unselected material obtained from germplasm collections. METHODS: At each site populations were grown in field plots, then randomly sampled after 3-5 years to obtain survivor populations. AFLP markers were used to calculate genetic differentiation within and between original and survivor populations. KEY RESULTS: No consistent changes in average genetic diversity were observed between original and survivor populations. In both species the original varieties were always genetically distinct from each other. Significant genetic shift was observed in the white clover 'Ramona' grown in Sweden. The WGB original populations were more genetically similar. However, genetic differentiation occurred between original and survivor WGB germplasm in both species, particularly in Sweden. Regression of climatic data with genetic differentiation showed that low autumn temperature was the best predictor. Within the set of cold sites the highest level of genetic shift in populations was observed in Sweden. CONCLUSIONS: The results suggest that changes in population structure can occur within a short time span in forage legumes, resulting in the rapid formation of distinct survivor populations in environmentally challenging sites.

Allelic variation in the perennial ryegrass FLOWERING LOCUS T gene is associated with changes in flowering time across a range of populations.

The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5' and 3' noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for identical protein products and the third contained two amino acid substitutions. Association analysis using either a mixed model with a relationship matrix to correct for population structure and relatedness or structured association with further correction using genomic control indicated significant associations between LpFT3 and variation in flowering time. These associations were corroborated in a validation population segregating for the same major alleles. The most "diagnostic" region of genomic variation was situated 5' of the coding sequence. Analysis of this region identified that the interhaplotype variation was closely associated with sequence motifs that were apparently conserved in the 5' region of orthologs of LpFT3 from other plant species. These may represent cis-regulatory elements involved in influencing the expression of this gene.

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen.

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.

Comparative analyses between lolium/festuca introgression lines and rice reveal the major fraction of functionally annotated gene models is located in recombination-poor/very recombination-poor regions of the genome.

Publication of the rice genome sequence has allowed an in-depth analysis of genome organization in a model monocot plant species. This has provided a powerful tool for genome analysis in large-genome unsequenced agriculturally important monocot species such as wheat, barley, rye, Lolium, etc. Previous data have indicated that the majority of genes in large-genome monocots are located toward the ends of chromosomes in gene-rich regions that undergo high frequencies of recombination. Here we demonstrate that a substantial component of the coding sequences in monocots is localized proximally in regions of very low and even negligible recombination frequencies. The implications of our findings are that during domestication of monocot plant species selection has concentrated on genes located in the terminal regions of chromosomes within areas of high recombination frequency. Thus a large proportion of the genetic variation available for selection of superior plant genotypes has not been exploited. In addition our findings raise the possibility of the evolutionary development of large supergene complexes that confer a selective advantage to the individual.

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus x canescens L.) for copper in vitro.

Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) M) incubated for 21 days on aseptic tissue culture media WPM containing 1 microM Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(-2) M). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.