An SSR-based genetic linkage map for perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

A simple sequence repeat (SSR)-based linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross reference population. A total of 309 unique perennial ryegrass SSR (LPSSR) primer pairs showing efficient amplification were evaluated for genetic polymorphism, with 31% detecting segregating alleles. Ninety-three loci have been assigned to positions on seven linkage groups. The majority of the mapped loci are derived from cloned sequences containing (CA)_n-type dinucleotide SSR arrays. A small number (7%) of primer pairs amplified fragments that mapped to more than one locus. The SSR locus data has been integrated with selected data for RFLP, AFLP and other loci mapped in the same population to produce a composite map containing 258 loci. The SSR loci cover 54% of the genetic map and show significant clustering around putative centromeric regions. BLASTN and BLASTX analysis of the sequences flanking mapped SSRs indicated that a majority (84%) are derived from non-genic sequences, with a small proportion corresponding to either known repetitive DNA sequence families or predicted genes. The mapped LPSSR loci provide the basis for linkage group assignment across multiple mapping populations.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Genetic characterisation of seed yield and fertility traits in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Seed yield is a trait of major interest for the key grassland species Lolium perenne L. An F2 mapping population of perennial ryegrass (VrnA), recently characterised for vernalisation response, was assessed in a glasshouse for traits related to seed yield based on a lattice design with four replications over 2 years. The traits heading date, plant height, length of panicles, number of panicles per plant, seed yield per panicle, flag leaf length, flag leaf width and seed yield per plant revealed repeatabilities ranging from 41 to 76% and a considerable amount of genetic variation in the VrnA population. Path analysis partitioned the direct and indirect effects of seed yield components on seed yield per plant. Seed yield per panicle showed the highest effect on total seed yield. The adjusted mean values of each trait and a genetic linkage map consisting of 97 anonymous and 85 gene associated DNA markers were used for quantitative trait loci (QTL) analysis. Of particular interest were two QTL on linkage group (LG) 1 and LG 2, explaining 41 and 18%, respectively, of the observed phenotypic variation for the trait seed yield per panicle. Both QTL co-located with two major QTL for total seed yield per plant possibly representing the S and Z loci of the gametophytic self incompatibility (SI) system of perennial ryegrass. The diversity of SI alleles in mapping parents and the degree of heterozygosity at SI loci in the full sib progeny determines the interference of self incompatibility with seed production.     

Quantitative Trait Locus (QTL) meta-analysis and comparative genomics for candidate gene prediction in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Background

In crop species, QTL analysis is commonly used for identification of factors contributing to variation of agronomically important traits. As an important pasture species, a large number of QTLs have been reported for perennial ryegrass based on analysis of biparental mapping populations. Further characterisation of those QTLs is, however, essential for utilisation in varietal improvement programs.

Results

A bibliographic survey of perennial ryegrass trait-dissection studies identified a total of 560 QTLs from previously published papers, of which 189, 270 and 101 were classified as morphology-, physiology- and resistance/tolerance-related loci, respectively. The collected dataset permitted a subsequent meta-QTL study and implementation of a cross-species candidate gene identification approach. A meta-QTL analysis based on use of the BioMercator software was performed to identify two consensus regions for pathogen resistance traits. Genes that are candidates for causal polymorphism underpinning perennial ryegrass QTLs were identified through in silico comparative mapping using rice databases, and 7 genes were assigned to the p150/112 reference map. Markers linked to the Lp DGL1, Lp Ph1 and Lp PIPK1 genes were located close to plant size, leaf extension time and heading date-related QTLs, respectively, suggesting that these genes may be functionally associated with important agronomic traits in perennial ryegrass.

Conclusions

Functional markers are valuable for QTL meta-analysis and comparative genomics. Enrichment of such genetic markers may permit further detailed characterisation of QTLs. The outcomes of QTL meta-analysis and comparative genomics studies may be useful for accelerated development of novel perennial ryegrass cultivars with desirable traits.

     

Regeneration from leaf-base explants of <Emphasis Type="Italic">Lolium perenne</Emphasis> L. and <Emphasis Type="Italic">Lolium multiflorum</Emphasis> L.

The regenerability of Lolium perenne and Lolium multiflorum was investigated using leaf-base sections excised from plantlets grown in vitro. Young leaf bases were cut into three sections each 2 mm in length, the lowest 0–2 mm section adjacent to, but not including, the apical meristem. Callus development followed by embryogenesis occurred only in the lowest 2 mm leaf-base sections of L. perenne cv. Limes, whereas L. multiflorum leaf bases showed an embryogenic response along the entire 6 mm section. Rooted, green plantlets were obtained from 100% of the 0–2 mm sections of L. perenne cv. Limes and from 91%, 65% and 26% of leaf-base sections at 0–2, 2–4 and 4–6 mm respectively from L. multiflorum. Microprojectile bombardment of leaf-base explants resulted in transient expression of the -glucuronidase gene.     

Inheritance patterns of the response to in vitro doubled haploid induction in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

The ability to produce doubled haploid (DH) plants has found broad application in research and breeding. For major crop species such as maize (Zea mays L.) and barley (Hordeum vulgare L.), routine large-scale production of DHs has enabled the acceleration of breeding processes, for example through efficient generation of homozygous lines. However, in forage crops such as perennial ryegrass (Lolium perenne L.), low and genotype-specific responses to in vitro anther culture (AC) still limit wide-spread use of DHs. Here, we report the responses of nine bi-parental populations, segregating for microspore embryogenesis and plant regeneration capacity, to an effective AC protocol. Genotypes of exceptionally high androgenic ability, producing over 200 green plants per 100 anthers cultured, could be selected. Continuous and distinctly shaped distributions for the evaluated traits were indicative of quantitative polygenic control and the presence of different alleles in each population. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield (ρ?=?0.20), suggested that different genes influence these traits. The populations evaluated here provide a rich source of alleles needed for the introgression of high levels of androgenic capacity into recalcitrant material. Moreover, this germplasm is ideally suited for use in future genotyping and mapping studies so that the genetic control of androgenic capacity in perennial ryegrass can be elucidated. Ultimately, our results will help to realize the potential of DH induction in one of the world’s most important forage crop species.     

Molecular breeding of transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered fructan biosynthesis through the expression of fructosyltransferases

Perennial ryegrass is the most important forage grass used in temperate agriculture. Transgenic perennial ryegrass events with altered fructan biosynthesis have the potential not only to increase animal production by improving digestibility of the grasses, but also to increase pasture intake by the animal due to lower neutral detergent fibre (NDF) concentrations. Transgenic perennial ryegrass plants were shown to have increased (P?<?0.05) in fructan concentrations of leaf blades in transgenic T₀ events and have thus an increase in water-soluble carbohydrate and in vivo dry matter digestibility concentrations as well as a decrease in the NDF concentration within the plant in spring and summer. These changes in nutritive value have led to an increase in metabolisable energy of up to 1.7 MJ ME·kg DM^?1 in selected T₀ events compared to FLp418-20 during spring and summer, with no differences in autumn or winter. The field evaluation of these events and the further development of these events using molecular breeding technologies are described in this paper.     

Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Perennial ryegrass is an obligate outbreeding pasture grass of the Poaceae family, with a two-locus (S and Z) gametophytic self-incompatibility (SI) mechanism. This system has provided a major obstacle to targeted varietal development, and enhanced knowledge is expected to support more efficient breeding strategies. Comparative genetics and physical mapping approaches have been developed to permit molecular cloning of the SI genes. SI gene-linked genetic markers based on heterologous cDNA restriction fragment length polymorphisms (RFLPs) and homologous genomic DNA-derived simple sequence repeats (SSRs) were converted to single nucleotide polymorphism (SNP) format for efficient genotyping. Genetic mapping identified the location of SI loci and demonstrated macrosynteny between related grass species. S- and Z-linked bacterial artificial chromosome (BAC) clones were sequenced using massively parallel pyrosequencing technology to provide the first physical mapping data for Poaceae SI loci. The sequence assembly process suggested a lower prevalence of middle repetitive sequences in the Z locus region and hence precedence for positional cloning strategy. In silico mapping using data from rice, Brachypodium distachyon and Sorghum revealed high sequence conservation in the vicinity of the Z locus region between SI and self-compatible (SC) grass species. Physical mapping identified a total of nine genes encoded in the Z locus region. Expression profiling and nucleotide diversity assessment identified two Z-linked genes, LpTC116908 and LpDUF247, as plausible candidates for the male and female determinants of the S-Z SI system.     

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered expression of fructosyltransferases

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+?(gene of interest positive) and GOI? (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+?and GOI? for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI? compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI?. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.     

Genetic dissection of a genomic region for a quantitative trait locus, <Emphasis Type="Italic">Hd3</Emphasis>, into two loci, <Emphasis Type="Italic">Hd3a</Emphasis> and <Emphasis Type="Italic">Hd3b</Emphasis>, controlling heading date in rice

The rice photoperiod sensitivity gene Hd3 was originally detected as a heading date-related quantitative trait locus localized on chromosome 6 of rice. High-resolution linkage mapping of Hd3 was performed using a large segregating population derived from advanced backcross progeny between a japonica variety, Nipponbare, and an indica variety, Kasalath. To determine the genotype of Hd3, we employed progeny testing under natural field and short-day conditions. As a result, two tightly linked loci, Hd3a and Hd3b, were identified in the Hd3 region. Nearly-isogenic lines for Hd3a and Hd3b were selected from progeny using marker-assisted selection. The inheritance mode of both Hd3a and Hd3b was found to be additive. Analysis of daylength response in nearly-isogenic lines of Hd3a and Hd3b showed that the Kasalath allele at Hd3a promotes heading under short-day conditions while that at Hd3b causes late heading under long-day and natural field conditions.     

Use of a gnotobiotic plant assay for assessing root colonization and mineral phosphate solubilization by <Emphasis Type="Italic">Paraburkholderia bryophila</Emphasis> Ha185 in association with perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Aims

The mechanisms by which rhizosphere bacteria increase the availability of mineral P precipitates for plant use are understudied. However, Paraburkholderia bryophila Ha185 is known to solubilize inorganic phosphate in vitro via a novel process. Therefore, this study aimed to demonstrate P solubilization by Ha185 in association with roots of perennial ryegrass (Lolium perenne L.).

Methods

We developed a gnotobiotic plant assay to assess P solubilization by Ha185 on ryegrass roots under various nutrient conditions. A green fluorescent protein (GFP)-tagged derivative of Ha185 was used in conjunction with fluorescent microscopy and confocal microscopy to visualize colonization of ryegrass roots.

Results

Ha185 solubilized mineral P (hydroxyapatite) in association with ryegrass roots and increased ryegrass growth by 20% under P-limited conditions. The GFP-tagged Ha185 strain colonized the rhizoplane and penetrated the primary root of ryegrass, possibly through “crack entry” at the point of lateral root emergence, but also by entering the epidermal cells via root hairs.

Conclusions

Ha185 supported ryegrass growth under P-limited conditions, indicating this strain may improve availability of soil P for uptake by ryegrass. Tools developed in this study have broad application in the study of rhizobacteria-plant interactions.

     

Detection of favorable alleles for plant height and crown rust tolerance in three connected populations of perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Plant height, which is an estimator of vegetative yield, and crown rust tolerance are major criteria for perennial ryegrass breeding. Genetic improvement has been achieved through phenotypic selection but it should be speeded up using marker-assisted selection, especially in this heterozygous species suffering from inbreeding depression. Using connected multiparental populations should increase the diversity studied and could substantially increase the power of quantitative trait loci (QTL) detection. The objective of this study was to detect the best alleles for plant height and rust tolerance among three connected populations derived from elite material by comparing an analysis per parent and a multipopulation connected analysis. For the studied traits, 17 QTL were detected with the analysis per parent while the additive and dominance models of the multipopulation connected analysis made it possible to detect 33 and 21 QTL, respectively. Favorable alleles have been detected in all parents. Only a few dominance effects were detected and they generally had lower values than the additive effects. The additive model of the multipopulation connected analysis was the most powerful as it made it possible to detect most of the QTL identified in the other analyses and 11 additional QTL. Using this model, plant growth QTL and rust tolerance QTL explained up to 19 and 38.6% of phenotypic variance, respectively. This example involving three connected populations is promising for an application on polycross progenies, traditionally used in breeding programs. Indeed, polycross progenies actually are a set of several connected populations.     

Allelopathic interference of alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) genotypes to annual ryegrass (<Emphasis Type="Italic">Lolium rigidum</Emphasis>)

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.     

Discovery and genetic mapping of single nucleotide polymorphisms in candidate genes for pathogen defence response in perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.     

Molecular genetics of the<Emphasis Type="Italic"> Alhambra</Emphasis> (<Emphasis Type="Italic"> Drosophila AF10</Emphasis>) complex locus of<Emphasis Type="Italic"> Drosophila</Emphasis>

The Alhambra ( Alh) gene is the Drosophila homologue of the human AF10 gene. AF10 has been identified as a fusion partner of MLL, a human homologue of the fly gene trithorax, in infant leukemias. The endogenous function of human AF10 is not known, but may be vital to its role in acute leukemia. This prompted us to analyse Alh function. We describe here the genetic organisation of the Alh locus in D. melanogaster. We show that an independent lethal complementation group encoding a muscle protein ( Mlp84B) is located within an Alh intron. We have already shown that the leucine zipper (LZ) domain of ALH activates several Polycomb group-responsive elements. We further demonstrate that the LZ domain on its own bears the Alh vital function, since it is necessary and sufficient for rescue of Alh mutant lethality. Finally, we demonstrate that, in contrast to a previous report, Alh does not affect position-effect variegation.Communicated by G. Reuter     

Assessment of genetic diversity of <Emphasis Type="Italic">Saltol</Emphasis> QTL among the rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Eight Saltol quantitative trait locus (QTL) linked simple sequence repeat (SSR) markers of rice (Oryza sativa L.) were used to study the polymorphism of this QTL in 142 diverse rice genotypes that comprised salt tolerant as well as sensitive genotypes. The SSR profiles of the eight markers generated 99 alleles including 20rare alleles and 16 null alleles. RM8094 showed the highest number (13) of alleles followed by RM3412 (12), RM562 (11), RM493 (9) and RM1287 (8) while as, RM10764 and RM10745 showed the lowest number (6) of alleles. Based on the highest number of alleles and PIC value (0.991), we identified RM8094 as suitable marker for discerning salt tolerant genotypes from the sensitive ones. Based upon the haplotype analysis using FL478 as a reference (salt tolerant genotypes containing Saltol QTL), we short listed 68 rice genotypes that may have at least one allele of FL478 haplotype. Further study may confirm that some of these genotypes might have Saltol QTL and can be used as alternative donors in salt tolerant rice breeding programmes.     

Identification and characterization of a major QTL responsible for erect panicle trait in <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Panicle erectness (PE) is one of the most important traits for high-yielding japonica cultivars. Although several cultivars with PE trait have been developed and released for commercial production in China, there is little information on the inheritance of PE traits in rice. In the present study, 69 widely cultivated japonica cultivars and a double haploid (DH) population derived from a cross between a PE cultivar (Wuyunjing 8) and a drooping panicle cultivar (Nongken 57) were utilized to elucidate the mechanisms of PE formation and to map PE associated genes. Our data suggested that panicle length (PL) and plant height (PH) significantly affected panicle curvature (PC), with shorter PL and PH resulting in smaller PC and consequently more erect. A putative major gene was identified on chromosome 9 by molecular markers and bulk segregant analysis in DH population. In order to finely map the major gene, all simple sequence repeats (SSR) markers on chromosome 9 as well as 100 newly developed sequence-tagged site (STS) markers were used to construct a linkage group for quantitative trait locus (QTL) mapping. A major QTL, qPE9-1, between STS marker H90 and SSR marker RM5652, was detected, and accounted for 41.72% of PC variation with pleiotropic effect on PH and PL. another QTL, qPE9-2, was also found to be adjacent to qPE9-1. In addition, we found that H90, the nearest marker to qPE9-1, used for genotyping 38 cultivars with extremely erect and drooping panicles, segregated in agreement with PC, suggesting the H90 product was possibly part of the qPE9-1 gene or closely related to it. These data demonstrated that H90 could be used for marker-aided selection for the PE trait in breeding and in the cloning of qPE9-1.     

Micro-colinearity between rice, <Emphasis Type="Italic">Brachypodium</Emphasis>, and <Emphasis Type="Italic">Triticum monococcum</Emphasis> at the wheat domestication locus <Emphasis Type="Italic">Q</Emphasis>

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.