Advances in introgression technologies for precision breeding within the Lolium - Festuca complex

The anticipated complexity of multifunctional grasslands with environment‐friendly and sustainable management practices demands better understanding of traits, their interactions, and their genetic control. Intergeneric hybrids between closely related Lolium and Festuca species are being used to broaden the gene pool and provide the plant breeder with options to combine complementary traits aimed at high quality but more robust grass varieties for the future. New techniques in introgression mapping provide opportunities for precision breeding whereby desirable gene combinations transferred from one species into another are selected preferentially, with the exclusion of deleterious alien genes. The close homology between genomes of Lolium and Festuca species allows high levels of chromosome pairing and recombination. Using genomic in situ hybridisation (GISH) on Lolium/Festuca hybrids and their derivatives, recombination between Lolium and Festuca chromosomes is observed at any point along the chromosome. The system provides unlimited access to any combination of Lolium and Festuca DNA sequence. Moreover, genes transferred between homoeologous chromosome sites are expected to function normally at their new locations. Alien chromosome segments may be reduced in size by further recombination events thereby reducing linkage drag. Molecular markers such as AFLPs, SSRs, SNPs, or RFLPs are being targeted to genes of interest to allow their selection through different generations in plant breeding programmes. Relatively simple PCR‐based marker systems are used for specific traits as breeders' toolkits in plant breeding programmes.     

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.     

Chromosome substitutions and recombination in the amphiploid Lolium perenne×Festuca pratensis cv Prior (2n=4x=28)

 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. Received: 16 June 1998 / Accepted: 17 September 1998 RID="1" ID="1" <E5>Present address:</E5> Lithuanian Institute of Agriculture, 5051 Dotnuva-Akademija, Kedainiai, Lithuania RID=" ID=" Communicated by J. W. Snape RID=" ID=" <E5>Correspondence to</E5> P. H. Canter     

Introgression of chromosomes of Festuca arundinacea var. glaucescens into Lolium multiflorum revealed by genomic in situ hybridisation (GISH)

An F₁ hybrid (n=4x=28) between the tetraploid species Festuca arundinacea var. glaucescens (GGG′G′) and a synthetic tetraploid Lolium multiflorum (LmLmLmLm) was backcrossed to diploid L. multiflorum to produce triploid (2n=3x=21) BC₁ hybrids (LmLmG). At metaphase I of meiosis the triploids had a preponderance of ring bivalents and univalents with some linear and frying-pan trivalents. Genomic in situ hybridisation (GISH) differentiated the Festuca chromosomes from Lolium and revealed that the bivalents were exclusively between Lolium homologues, while the univalents were Festuca. Despite the limited amount of homoeologous chiasmata pairing in the triploids, some recombinant chromosomes were recovered in the second backcross when the hybrids were further crossed to diploid L. multiflorum. The progeny from the second backcross was predominantly diploid. Genotypes with recombinant chromosomes and chromosome additions involving an extra Festuca chromosome were identified using GISH. Changes in plant phenotype were related to the presence of Festuca chromatin. Received: 20 September 2000 / Accepted: 05 January 2001     

Introgression-mapping of genes for drought resistance transferred from Festuca arundinacea var. glaucescens into Lolium multiflorum

Procedures for the transfer of genes for drought resistance from Festuca glaucescens (2n=4x=28) into Lolium multiflorum (2n=2x=14) are described. Following the initial hybridisation of a synthetic autotetraploid of L. multiflorum (2n=4x=28) with F. glaucescens, the F₁ hybrid was backcrossed twice onto diploid L. multiflorum (2n=2x=14) to produce a diploid Lolium genotype with a single F. glaucescens introgression located distally on the nucleolar organiser region arm of chromosome 3. The transmission of F. glaucescens-derived amplified fragment length polymorphisms and a sequence-tagged-site (STS) marker was monitored throughout the breeding programme. Those genotypes of a mapping population of backcross 3 that survived combined severe drought and heat stress all contained the F. glaucescens-derived markers. The STS marker provided a prototype for a PCR-based system for high-throughput screening during cultivar development for the presence of the F. glaucescens-derived genes for drought resistance. The frequency of intergeneric recombination between L. multiflorum and F. glaucescens is described. During the initial stages of the breeding programme, preferential intraspecific chromosome pairing between Lolium homologues and Festuca homoeologues dominated with low frequencies of intergeneric chromosome associations. However, these increased in the backcross 1 due to the absence of opportunities for intraspecific chromosome pairing between homoeologous Festuca chromosomes following the loss of half of the Festuca chromosomes. Once transferred to Lolium, F. glaucescens sequences recombined with Lolium at high frequencies, thereby enabling the loss of potentially deleterious gene combinations that might reduce the forage quality of Lolium.     

Genome balance in six successive generations of the allotetraploid Festuca pratensis × Lolium perenne

In the allotetraploid, Festuca pratensis Huds. (2n = 4x = 28) × Lolium perenne L. (2n = 4x = 28) the balance of chromatin, as determined by GISH, changes over successive generations of open pollination in favour of L. perenne. There is extensive recombination between chromosomes of the two parental genomes, as well as substitution of whole Festuca chromosomes by whole Lolium chromosomes. The total number of Lolium chromosomes increased from a mean 14.36 in the F₂ to 16.26 in the F₆, and the total number of Festuca chromosomes decreased correspondingly from a mean of 13.57 to a value of 11.56. The number of recombinant chromosomes and recombination breakpoints per genotype also increased from generation to generation, although the respective values of both characters were higher for Festuca (0.86–8.41 and 1.14–15.22) than for Lolium (0.68–4.59 and 0.68–6.0). The proportion of total genome length contributed by the L. perenne chromatin increased from about 50% in F₂ to 59.5% in F₆. The results are based on the sample of 134 plants studied (26–28 plants per generation), and are discussed in terms of the dominance of Lolium chromosomes over those of Festuca, and possible mechanisms underlying this phenomenon of chromatin substitution.     

Development of intron-flanking EST markers for the Lolium/Festuca complex using rice genomic information

DNA markers able to distinguish species or genera with high specificity are valuable in the identification of introgressed regions in interspecific or intergeneric hybrids. Intergeneric hybridization between the genera of Lolium and Festuca, leading to the reciprocal introgression of chromosomal segments, can produce novel forage grasses with unique combinations of characteristics. To characterize Lolium/Festuca introgressions, novel PCR-based expression sequence tag (EST) markers were developed. These markers were designed around intronic regions which show higher polymorphism than exonic regions. Intronic regions of the grass genes were predicted from the sequenced rice genome. Two hundred and nine primer sets were designed from Lolium/Festuca ESTs that showed high similarity to unique rice genes dispersed uniformly throughout the rice genome. We selected 61 of these primer sets as insertion-deletion (indel)-type markers and 82 primer sets as cleaved amplified polymorphic sequence (CAPS) markers to distinguish between Lolium perenne and Festuca pratensis. Specificity of these markers to each species was evaluated by the genotyping of four cultivars and accessions (32 individuals) of L. perenne and F. pratensis, respectively. Evaluation using specificity indices proposed in this study suggested that many indel-type markers had high species specificity to L. perenne and F. pratensis, including 15 markers completely specific to both species. Forty-nine of the CAPS markers completely distinguish between the two species at bulk level. Chromosome mapping of these markers using a Lolium/Festuca substitution line revealed syntenic relationships between Lolium/Festuca and rice largely consistent with previous reports. This intron-based marker system that shows a high level of polymorphisms between species in combination with high species specificity will consequently be a valuable tool in Festulolium breeding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Parental genome composition and genetic classifications of derivatives from intergeneric crosses of Festuca mairei and Lolium perenne

Intergeneric hybridization between Festuca and Lolium has been a long-term goal of forage and turfgrass breeders to generate improved cultivars by combining stress tolerance of Festuca and rapid establishment of Lolium. However, wide-distance hybridizations usually result in the wild genome being eliminated from the hybrid due to incomplete chromosome pairing and crossovers. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to detect the parental genome composition of F₁ hybrids and backcross, generated from crosses between Festuca mairei St. Yves (Fm) and Lolium perenne L. (Lp). Each of the hybrids exhibited integration of Fm and Lp genomes with varying levels of Fm/Lp genome ratios. However, cluster and principle component analyses of the progeny consistently revealed four groups depending on the amount of genome introgression from both parents. The parental genome composition and classifications of intergeneric progeny would be useful for breeding material selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cytogenetic identification of a novel dwarf wheat line with introgressed <Emphasis Type="Italic">Thinopyrum ponticum</Emphasis> chromatin

Novel dwarfing germplasms and dwarfing genes are valuable for the wheat breeding. A novel semi-dwarf line, 31505-1, with reduced height compared with its common wheat parent, was derived from a cross between common wheat and Thinopyrum ponticum. Cytological studies demonstrated that 31505-1 contained 42 chromosomes and formed 21 bivalents at meiotic metaphase I. Genomic in situ hybridization (GISH) analysis showed that 31505-1 had no large Th. ponticum chromosome fragments. Fluorescence in situ hybridization (FISH) results revealed the absence of a pAs1 hybridization band on 2DL chromosome of 31505-1. Two SSR markers (Xwmc41 and Xcfd168) and two STS markers (Xmag4059 and Xmag3596), which were located on 2D chromosome, amplified unique bands of Th. Ponticum in 31505-1. These revealed presence of an introgressed Th. ponticum segment in 2DL chromosome of dwarf line 31505-1, although the alien segment could not be detected by GISH.