Michael W. Humphreys

Discriminating the ancestral progenitors of hexaploid Festuca arundinacea using genomic in situ hybridization

The phylogeny of Festuca arundinacea Schreb. (2n = 6x = 42) was determined using GISH. Total genomic DNA of putative ancestral species was labelled with rhodamine and hybridized to chromosome preparations of hybrids involving these species and F. arundinacea. The degree of hybridization to chromosomes known to be homologous to the probe DNA was compared with that found simultaneously on chromosomes of the genome of F. arundinacea. It was concluded that the tetraploid species Festuca arundinacea var. glaucescens contributed two genomes and the diploid species Festuca pratensis one, to create the allohexaploid species F. arundinacea.

Identification of parental and recombined chromosomes in hybrid derivatives of Lolium multiflorum × Festuca pratensis by genomic in situ hybridization

Genomic in situ hybridization (GISH) was used to identify Festuca chromatin in mitotic chromosomes of Lolium multiflorum (Lm) × Festuca pratensis (Fp) hybrids and hybrid derivatives. In two inverse autoallotriploids LmLmFp and LmFpFp, in situ hybridization was able to discriminate between the Lolium and Festuca chromosomes. In a third triploid hybrid produced by crossing an amphiploid of L. multiflorum × F. pratensis (2n=4x=28) with L. multiflorum (2n=2x=14), the technique identified chromosomes with interspecific recombination. Also, in an introgressed line of L. multiflorum which was homozygous for the recessive sid (senescence induced degradation) allele from F. pratensis, a pair of chromosome segments carrying the sid gene could be identified, indicating the suitability of GISH in showing the presence and location of introgressed genes. By screening backcross progeny for the presence of critical alien segments and the absence of other segments the reconstitution of the genome of the recipient species can be accelerated.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.

Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses

Abstract Simple-sequence repeats (SSRs) comprising three tetranucleotide repeat sequences with two-base ’anchors’, namely 5´-(AGAC)4GC, 5´-AC(GACA)4 and 5´-(GACA)4GT, were used in PCR reactions as primers to develop inter-SSR DNA fingerprints of the outbreeding grass species Lolium multiflorum, L. perenne, Festuca pratensis and F. arundinacea. Each species was represented by DNA samples from 3 to 6 varieties. In all four species distinctive species-specific DNA profiles were produced that were common across a number of varieties despite their diverse origin. While the fingerprints of the two ryegrasses, L. multiflorum and L. perenne, were the most similar, a number of inter-SSR DNA markers were generated that enabled them to be distinguished from each other. Some slight variations were found between varieties, which provided putative variety-specific markers for cultivar identification. In addition, variations in the DNA profiles of the genotypes of L. multiflorum and F. pratensis were examined, and the results showed that variety-specific fingerprints are integrated patterns made up from the profiles of individual genotypes. Amongst the primers used, AC(GACA)4 generated the best distinction between Lolium and Festuca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequences, ranging in size between 550 bp and 1,600 bp, were cloned: three clones for F. pratensis, one clone for L. multiflorum and one clone for F. arundinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridization confirmed the presence of this fragment in F. arundinacea (which contains one genome of F. pratensis), but no homology was found with L. multiflorum. However, a F. arundinacea clone amplified with (GACA)4GT, pFa 104H1350, was found to be unique to the F. arundinacea genome.

Designing grasses with a future - combining the attributes of Lolium and Festuca

Lolium species (considered the ideal grasses for European agriculture) are not sufficiently robust to meet many of the environmental challenges that face extensive agriculture in less favoured areas. Fortunately, adaptations to abiotic and biotic stresses exist amongst Festuca species related closely to Lolium. The complex of species has an enormous wealth of genetic variability and potentiality for genetic exchange, thus offering unique opportunities for the production of versatile hybrid varieties with new combinations of useful characters suited to modern grassland farming. The attributes of Lolium and Festuca can be combined into a single genotype by amphiploidy or alternatively, a limited number of characters can be selectively introgressed from Festucainto Lolium or vice versa. Androgenesis of the interspecific hybrids can generate genotypes combining characters that may not be recovered by sexual backcrossing. Genomic in situ hybridization(GISH) can differentially ‘paint’ the chromosomes of Lolium and Festuca and identify Lolium-Festuca recombinant chromosomes. GISH is valuable in the analysis of amphiploids, introgressions and androgenic genotypes and can be used to physically map introgressed traits. Introgression mapping is a powerful new approach to the mapping of traits and arises from a fusion of physical and genetic mapping. For example, in a diploidLolium introgression genotype with only one introgressed Festucasegment, the gene(s) for any Festucaderived trait expressed by the plant must be located within the segment. Using GISH and molecular markers, a dense but highly localised map of the Festuca segment is made in isolation of the Loliumgenome – this may simplify QTL analysis.

GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum

The first backcross breeding programme for the transfer of freezing-tolerance genes from winter hardy Festuca pratensis to winter-sensitive Lolium multiflorum is described. A partly fertile, triploid F1 hybrid F. pratensis (2n=2x=14) × L. multiflorum (2n=4x=28) was employed initially, and after two backcrosses to L. multiflorum (2x) a total of 242 backcross two (BC2) plants were generated. Genomic in situ hybridisation (GISH) was performed on 61 BC2 plants selected for their good growth and winter survival characters in the spring following one Polish winter (2000–2001). Among the winter survivors, diploid chromosome numbers were present in 80% of plants. An appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased freezing tolerance without compromise to the good growth and plant vigour found in Lolium. Among all the diploids, a total of 20 individuals were identified, each with a single F. pratensis chromosome segment. Another diploid plant contained 13 Lolium chromosomes and a large metacentric F. pratensis chromosome, identified as chromosome 4, with two large distal Lolium introgressions on each chromosome arm. Three of the diploid BC2, including the genotype with Festuca chromosome 4 DNA sequences, were found to have freezing tolerance in excess of that of L. multiflorum, and in one case in excess of the F. pratensis used as control. A detailed cytological analysis combining GISH and fluorescence in situ hybridisation analyses with rDNA probes revealed that the other two freezing-tolerant genotypes carried a Festuca chromosome segment at the same terminal location on the non-satellite arm of Lolium chromosome 2.

Chromosome substitutions and recombination in the amphiploid lolium perenne×festuca pratensis cv prior (2n=4x=28)

Abstract The synthetic amphiploid cv Prior was created in the early 1970s at the Welsh Plant Breeding Station by crossing colchicine-induced autotetraploids of Lolium perenne (2n=14) and Festuca pratensis (2n=14). Meiosis in the early generations was characterized as stable, with frequent bivalent formation. In situ hybridization of a L. perenne total genomic DNA probe to mitotic chromosome spreads of 12 plants, from two extant populations of Prior, demonstrates extensive recombination between the two genomes. Recombination events occur along the whole length of chromosome arms but with a higher frequency in the medial portion. The species origins of chromosomes were assigned by the presence or absence of a fluorescent probe at the centromere. There has been a substitution of Festuca-origin chromosomes by those of Lolium-origin, resulting in a mean of 17.9 (15–21) Lolium and 9.7 (7–13) Festuca chromosomes per genotype. Mean chromatin length per genotype comprised 62.1% Lolium and 37.9% Festuca. On average 9.3 Lolium (51.1% of those present) and 3.5 Festuca (37.8%) chromosomes had no recombined segments. For chromosomes which did show recombination, fewer alien segments were observed in Lolium than in Festuca chromosomes. Festuca chromosomes in genotypes selected for drought resistance had undergone more recombination than in genotypes from an unselected population, though this difference was not statistically significant for the small sample examined.

The controlled introgression of Festuca arundinacea genes into Lolium multiflorum

SummaryUsing phosphoglucoisomerase (PGI/2) as a genetic marker, it has been shown to be possible to transfer genes from Festuca arundinacea into diploid Lolium multiflorum using the pentaploid hybrid L. multiflorum (4x) x F. arundinacea (6x). The pentaploid hybrid was sufficiently fertile to be used in reciprocal crosses with diploid. L. multiflorum. When used as the male parent, only two backcross generations were then required to reconstitute the diploid genotype. Intergeneric recombinants including a F. arundinacea PGI/2 allele were found among the diploid BC2. Cytological data indicates that although the majority of chromosome associations involve only homologous Lolium chromosomes, associations involving Lolium and Festuca chromosomes also occur.Interpollinating the pentaploid hybrids prior to commencing a backcrossing programme increases the number of cycles of recombination and improves the chance of recovering intergeneric recombinants. The crossing programme described is proposed to be an effective method of introducing F. arundinacea genes into L. multiflorum.

Genetic Improvement of Forage Species to Reduce the Environmental Impact of Temperate Livestock Grazing Systems

Abberton, M. T., Marshall, A. H., Humphreys, M. W., Macduff, J. H., Collins, R. P., Marley, C. L. (2008). Genetic improvement of forage species to reduce the environmental impact of temperate livestock grazing systems. Advances in Agronomy, 98, 311-355.

Global agricultural intensification during climate change: a role for genomics

Summary Agriculture is now facing the ‘perfect storm’ of climate change, increasing costs of fertilizer and rising food demands from a larger and wealthier human population. These factors point to a global food deficit unless the efficiency and resilience of crop production is increased. The intensification of agriculture has focused on improving production under optimized conditions, with significant agronomic inputs. Furthermore, the intensive cultivation of a limited number of crops has drastically narrowed the number of plant species humans rely on. A new agricultural paradigm is required, reducing dependence on high inputs and increasing crop diversity, yield stability and environmental resilience. Genomics offers unprecedented opportunities to increase crop yield, quality and stability of production through advanced breeding strategies, enhancing the resilience of major crops to climate variability, and increasing the productivity and range of minor crops to diversify the food supply. Here we review the state of the art of genomic‐assisted breeding for the most important staples that feed the world, and how to use and adapt such genomic tools to accelerate development of both major and minor crops with desired traits that enhance adaptation to, or mitigate the effects of climate change.