Half-sib (HS) mating is the most important breeding method of open-pollinated sexual forage grasses such as tall fescue. Progeny testing procedures of half-sib families can only measure general ...combining ability (GCA) due to the unknown pollen donor. In tall fescue, genetic studies using HS mating and application of molecular markers for paternity identification in polycross progenies are so far limited. In this study, 960 genotypes from sixteen half-sib families of tall fescue derived from polycrosses, along with their corresponding parental genotypes were evaluated for agro-morphological traits in the field during 2016 and 2017. Genetic diversity of the parental genotypes was assessed using twenty EST-SSR markers and then a minimal set of primers was used to identify the most likely male parent in the polycross progenies. Considerable genetic variation was observed among the parental genotypes based on both morphological and molecular assessments. Moderate to high estimates of narrow sense heritability (0.53–0.84) for morphological traits indicated that phenotypic selection can be successful to achieve genetic progress. The EST-SSR markers allowed to identify a unique paternal parent in 177 of the offspring which were divided into 66 full-sib families. The number of genotypes per full-sib family varied from 1 to 11. Marker data used to identify full-sib families, estimate specific combining ability (SCA), and select siblings based on the genetic value of both parents. No clear association was found between GCA of parents and the corresponding SCA for forage yield, indicating that both additive and dominant gene actions may play a role in the genetic control of this trait. The obtained results highlighted the importance of marker-based paternity identification to increase gain from selection in polycross breeding programs of tall fescue.
SUMMARY
Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A ...major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole‐genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7484) biparental F2 population segregating for resistance to Xtg. Using chromosome‐level genome assemblies as references, we were able to define a ~300 kb region highly associated with resistance on pseudo‐chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2‐like disease resistance proteins, cysteine‐rich kinases, and RGA4‐ and RGA5‐like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy of combining BSA studies in very large populations with whole genome deep sequencing and high‐quality genome assemblies to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker‐assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.
Significance Statement
Elucidating the genetic control of phenotypic traits in highly heterozygous, outbreeding plant species is laborious as it requires phenotyping and genotyping of a large number of individuals. Using 7484 individuals of an Italian ryegrass population, bulked segregant analysis, and whole genome deep sequencing of pools, we identified a 300 kb genomic region harboring promising candidate genes for resistance to bacterial wilt, an important target trait in forage grass breeding.
The
species comprises phytopathogenic bacteria that can cause serious damage to cereals and to forage grasses. So far, the genomic resources for
were limited, which hindered further understanding of ...the host-pathogen interactions at the molecular level and the development of disease-resistant cultivars. To this end, we complemented the available complete genome sequence of the
pv.
pathotype strain DSM 18974 by sequencing the genomes of all the other 10
pathotype strains using PacBio long-read technology and assembled complete genome sequences. Phylogeny based on average nucleotide identity (ANI) revealed three distinct clades within the species, which we propose to classify as clades Xt-I, Xt-II, and Xt-III. In addition to 2,181 core
genes, a total of 190, 588, and 168 genes were found to be exclusive to each clade, respectively. Moreover, 29 non-transcription activator-like effector (TALE) and 21 TALE type III effector classes were found, and clade- or strain-specific effectors were identified. Further investigation of these genes could help to identify genes that are critically involved in pathogenicity and/or host adaptation, setting the grounds for the development of new resistant cultivars.
This work is an update and extension of the previously published article "Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly" by Frei
The ...published genome assembly of the doubled haploid perennial ryegrass (
L.) genotype Kyuss (Kyuss v1.0) marked a milestone for forage grass research and breeding. However, order and orientation errors may exist in the pseudo-chromosomes of Kyuss, since barley (
L.), which diverged 30 million years ago from perennial ryegrass, was used as the reference to scaffold Kyuss. To correct for structural errors possibly present in the published Kyuss assembly, we
assembled the genome again and generated 50-fold coverage high-throughput chromosome conformation capture (Hi-C) data to assist pseudo-chromosome construction. The resulting new chromosome-level assembly Kyuss v2.0 showed improved quality with high contiguity (contig N50 = 120 Mb), high completeness (total BUSCO score = 99%), high base-level accuracy (QV = 50), and correct pseudo-chromosome structure (validated by Hi-C contact map). This new assembly will serve as a better reference genome for
spp. and greatly benefit the forage and turf grass research community.
Water limitation is one of the major factors reducing crop productivity worldwide. In order to develop efficient breeding strategies to improve drought tolerance, accurate methods to identify when a ...plant reduces growth as a consequence of water deficit have yet to be established. In perennial ryegrass (
L.), an important forage grass of the Poaceae family, leaf elongation is a key factor determining plant growth and hence forage yield. Although leaf elongation has been shown to be temperature-dependent under non-stress conditions, the impact of water limitation on leaf elongation in perennial ryegrass is poorly understood. We describe a method for quantifying tolerance to water deficit based on leaf elongation in relation to temperature and soil moisture in perennial ryegrass. With decreasing soil moisture, three growth response phases were identified: first, a "normal" phase where growth is mainly determined by temperature, second a "slow" phase where leaf elongation decreases proportionally to soil water potential and third an "arrest" phase where leaf growth terminates. A custom R function was able to quantify the points which demarcate these phases and can be used to describe the response of plants to water deficit. Applied to different perennial ryegrass genotypes, this function revealed significant genotypic variation in the response of leaf growth to temperature and soil moisture. Dynamic phenotyping of leaf elongation can be used as a tool to accurately quantify tolerance to water deficit in perennial ryegrass and to improve this trait by breeding. Moreover, the tools presented here are applicable to study the plant response to other stresses in species with linear, graminoid leaf morphology.
Xanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted ...mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity.
In this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 plant cell wall degradative enzymes.
These results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which could play a crucial role in pathogen adaptation. The large amount of such elements in Xtg compared to other pathovars of the species could, at least partially, explain its high virulence and broad host range. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.
Crown rust, caused by Puccinia coronata f. sp. lolii, is one of the most important diseases of temperate forage grasses, such as ryegrasses (Lolium spp.), affecting yield and nutritional quality. ...Therefore, resistance to crown rust is a major goal in ryegrass breeding programmes. In a two-way pseudo-testcross population consisting of 306 Lolium multiflorum individuals, multisite field evaluations as well as alternative methods based on artificial inoculation with natural inoculate in controlled environments were used to identify QTLs controlling resistance to crown rust. Disease scores obtained from glasshouse and leaf segment test (LST) evaluations were highly correlated with scores from a multisite field assessment (r = 0.66 and 0.79, P < 0.01, respectively) and thus confirmed suitability of these methods for crown rust investigations. Moreover, QTL mapping based on a linkage map consisting of 368 amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers revealed similar results across different phenotyping methods. Two major QTLs were consistently detected on linkage group (LG) 1 and LG 2, explaining up to 56% of total phenotypic variance (V p). Nevertheless, differences between position and magnitude of QTLs were observed among individual field locations and suggested the existence of specific local pathogen populations. The present study not only compared QTL results among crown rust evaluation methods and environments, but also identified molecular markers closely linked to previously undescribed QTLs for crown rust resistance in Italian ryegrass with the potential to be applied in marker-assisted forage crop breeding.
Here, we report the draft genome sequence of the Xanthomonas bromi type strain LMG 947, an important pathogen of bromegrasses (Bromus spp.). Comparative analysis with other Xanthomonas spp. that are ...pathogenic on forage grasses will assist the analysis of host-plant adaptation at the genome level.
Sainfoin (Onobrychis viciifolia Scop.) is a forage legume, which improves animal health and the environmental impact of livestock farming due to its proanthocyanidin content. To identify the impact ...of drought on acetone/water-extractable proanthocyanidin (PA) concentration and composition in the generative and vegetative stages, a rain exclosure experiment was established. Leaves of 120 plants from 5 different sainfoin accessions were sampled repeatedly and analyzed by UPLC-ESI-MS/MS. The results showed distinct differences in response to drought between vegetative and generative plants. Whereas vegetative plants showed a strong response to drought in growth (-56%) and leaf PA concentration (+46%), generative plants showed no response in growth (-2%) or PA concentration (-9%). The PA composition was stable across environments. The five accessions varied in PA concentrations and composition but showed the same pattern of response to the experimental treatments. These results show that the ontogenetic stage at which drought occurs significantly affects the plant's response.
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