Advances in genomic characterization of Urochloa humidicola: exploring polyploid inheritance and apomixis.

KEY MESSAGE: We present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and species origin, aiding polyploid and tropical forage research. Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one of the main pasture grasses for poorly drained soils in the tropics. However, genetic and genomic resources for this species are lacking due to its genomic complexity, including high heterozygosity, evidence of segmental allopolyploidy, and reproduction by apomixis. These complexities hinder the application of marker-assisted selection (MAS) in breeding programs. Here, we developed the highest-density linkage map currently available for the hexaploid tropical forage grass U. humidicola. This map was constructed using a biparental F1 population generated from a cross between the female parent H031 (CIAT 26146), the only known sexual genotype for the species, and the apomictic male parent H016 (BRS cv. Tupi). The linkage analysis included 4873 single nucleotide polymorphism (SNP) markers with allele dosage information. It allowed mapping of the ASGR locus and apospory phenotype to linkage group 3, in a region syntenic with chromosome 3 of Urochloa ruziziensis and chromosome 1 of Setaria italica. We also identified hexaploid haplotypes for all individuals, assessed the meiotic configuration, and estimated the level of preferential pairing in parents during the meiotic process, which revealed the autopolyploid origin of sexual H031 in contrast to apomictic H016, which presented allopolyploid behavior in preferential pairing analysis. These results provide new information regarding the genetic organization, mode of reproduction, and allopolyploid origin of U. humidicola, potential SNPs markers associated with apomixis for MAS and resources for research on polyploids and tropical forage grasses.

Polyploid SNP Genotyping Using the MassARRAY System.

Molecular marker discovery and genotyping are major challenges in polyploid breeding programs incorporating molecular biology tools. In this context, this work describes a method for single nucleotide polymorphism (SNP) genotyping in polyploid crops using matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry, the MassARRAY System.

Endophytic and rhizospheric bacteria associated with Paspalum atratum and its potential for plant growth promotion with different phosphate sources.

The genus Paspalum belongs to the family Poaceae and has several species that are native to Brazil. The Paspalum Germplasm Bank (GB) of the Brazilian Agricultural Research Corporation comprises approximately 450 accessions from 50 species. Among these accessions, Paspalum atratum (BGP 308) has economic potential for forage purposes. However, the endophytic and rhizospheric microbial communities within this accession and their ability to promote plant growth remain unknown. The present study aimed to isolate the endophytic and rhizospheric bacteria associated with P. atratum and to assess their potential for plant growth improvement, so-called plant growth-promoting bacteria (PGPB). For the in vitro tests, the ability of nitrogen-fixing bacteria (NFB), phosphate solubilization (PS) and indoleacetic acid (IAA) production were evaluated. A total of 116 endophytic and rhizosphere bacteria were obtained from the isolation. In the in vitro tests, 43 (37.00%) of these isolates showed positive NFB, PS, and IAA results. These isolates were identified by 16S rDNA sequencing. The phosphate solubilization index (PSI) ranged from 2 to 3.61, all 43 strains performed biological nitrogen fixation and the IAA production ranged from 12.85 to 431.41 µg ml-1. Eight of these 43 isolates were evaluated in vivo in a greenhouse using P. atratum caryopsis. The pots were filled with soil prepared with three different phosphate sources and one control without phosphate. After growth, the plants were submitted to morphological, bromatological and chemical determination. Data were analyzed using analysis of variance (ANOVA) and principal component analysis (PCA). In the in vivo test, treatments 105 (Pseudomonas sp.) and 458 (Pseudomonas sp.) were the most significant for the crystalline phosphate source, 109 (Bacillus sp.) for the sedimentary phosphate source and, as for the soluble phosphate source most treatments that received bacterial isolates had higher phosphorus content in the dry matter than the uninoculated soluble phosphate control. The 105FCR (crystalline phosphate + Pseudomonas sp.), 109FSE (sedimentary phosphate + Bacillus sp.), and 110 FSE (sedimentary phosphate + Enterobacter sp.) treatments showed the best results for plant growth promotion. This work made it possible to determine the bacterial community associated with P. atratum (BGP308) and to obtain new potential plant growth-promoting strains.

An Overview of the Genetics and Genomics of the Urochloa Species Most Commonly Used in Pastures.

Pastures based on perennial monocotyledonous plants are the principal source of nutrition for ruminant livestock in tropical and subtropical areas across the globe. The Urochloa genus comprises important species used in pastures, and these mainly include Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, and Urochloa ruziziensis. Despite their economic relevance, there is an absence of genomic-level information for these species, and this lack is mainly due to genomic complexity, including polyploidy, high heterozygosity, and genomes with a high repeat content, which hinders advances in molecular approaches to genetic improvement. Next-generation sequencing techniques have enabled the recent release of reference genomes, genetic linkage maps, and transcriptome sequences, and this information helps improve our understanding of the genetic architecture and molecular mechanisms involved in relevant traits, such as the apomictic reproductive mode. However, more concerted research efforts are still needed to characterize germplasm resources and identify molecular markers and genes associated with target traits. In addition, the implementation of genomic selection and gene editing is needed to reduce the breeding time and expenditure. In this review, we highlight the importance and characteristics of the four main species of Urochloa used in pastures and discuss the current findings from genetic and genomic studies and research gaps that should be addressed in future research.

A Semi-Automated SNP-Based Approach for Contaminant Identification in Biparental Polyploid Populations of Tropical Forage Grasses.

Artificial hybridization plays a fundamental role in plant breeding programs since it generates new genotypic combinations that can result in desirable phenotypes. Depending on the species and mode of reproduction, controlled crosses may be challenging, and contaminating individuals can be introduced accidentally. In this context, the identification of such contaminants is important to avoid compromising further selection cycles, as well as genetic and genomic studies. The main objective of this work was to propose an automated multivariate methodology for the detection and classification of putative contaminants, including apomictic clones (ACs), self-fertilized individuals, half-siblings (HSs), and full contaminants (FCs), in biparental polyploid progenies of tropical forage grasses. We established a pipeline to identify contaminants in genotyping-by-sequencing (GBS) data encoded as allele dosages of single nucleotide polymorphism (SNP) markers by integrating principal component analysis (PCA), genotypic analysis (GA) measures based on Mendelian segregation, and clustering analysis (CA). The combination of these methods allowed for the correct identification of all contaminants in all simulated progenies and the detection of putative contaminants in three real progenies of tropical forage grasses, providing an easy and promising methodology for the identification of contaminants in biparental progenies of tetraploid and hexaploid species. The proposed pipeline was made available through the polyCID Shiny app and can be easily coupled with traditional genetic approaches, such as linkage map construction, thereby increasing the efficiency of breeding programs.

Recombinants from the crosses between amphidiploid and cultivated peanut (Arachis hypogaea) for pest-resistance breeding programs.

Peanut is a major oilseed crop worldwide. In the Brazilian peanut production, silvering thrips and red necked peanut worm are the most threatening pests. Resistant varieties are considered an alternative to pest control. Many wild diploid Arachis species have shown resistance to these pests, and these can be used in peanut breeding by obtaining hybrid of A and B genomes and subsequent polyploidization with colchicine, resulting in an AABB amphidiploid. This amphidiploid can be crossed with cultivated peanut (AABB) to provide genes of interest to the cultivar. In this study, the sterile diploid hybrids from A. magna V 13751 and A. kempff-mercadoi V 13250 were treated with colchicine for polyploidization, and the amphidiploids were crossed with A. hypogaea cv. IAC OL 4 to initiate the introgression of the wild genes into the cultivated peanut. The confirmation of the hybridity of the progenies was obtained by: (1) reproductive characterization through viability of pollen, (2) molecular characterization using microsatellite markers and (3) morphological characterization using 61 morphological traits with principal component analysis. The diploid hybrid individual was polyploidized, generating the amphidiploid An 13 (A. magna V 13751 x A. kempff-mercadoi V 13250)4x. Four F1 hybrid plants were obtained from IAC OL 4 × An 13, and 51 F2 seeds were obtained from these F1 plants. Using reproductive, molecular and morphological characterizations, it was possible to distinguish hybrid plants from selfed plants. In the cross between A. hypogaea and the amphidiploid, as the two parents are polyploid, the hybrid progeny and selves had the viability of the pollen grains as high as the parents. This fact turns the use of reproductive characteristics impossible for discriminating, in this case, the hybrid individuals from selfing. The hybrids between A. hypogaea and An 13 will be used in breeding programs seeking pest resistance, being subjected to successive backcrosses until recovering all traits of interest of A. hypogaea, keeping the pest resistance.

Leaf transcriptome of two highly divergent genotypes of Urochloa humidicola (Poaceae), a tropical polyploid forage grass adapted to acidic soils and temporary flooding areas.

BACKGROUND: Urochloa humidicola (Koronivia grass) is a polyploid (6x to 9x) species that is used as forage in the tropics. Facultative apospory apomixis is present in most of the genotypes of this species, although one individual has been described as sexual. Molecular studies have been restricted to molecular marker approaches for genetic diversity estimations and linkage map construction. The objectives of the present study were to describe and compare the leaf transcriptome of two important genotypes that are highly divergent in terms of their phenotypes and reproduction modes: the sexual BH031 and the aposporous apomictic cultivar BRS Tupi. RESULTS: We sequenced the leaf transcriptome of Koronivia grass using an Illumina GAIIx system, which produced 13.09 Gb of data that consisted of 163,575,526 paired-end reads between the two libraries. We de novo-assembled 76,196 transcripts with an average length of 1,152 bp and filtered 35,093 non-redundant unigenes. A similarity search against the non-redundant National Center of Biotechnology Information (NCBI) protein database returned 65 % hits. We annotated 24,133 unigenes in the Phytozome database and 14,082 unigenes in the UniProtKB/Swiss-Prot database, assigned 108,334 gene ontology terms to 17,255 unigenes and identified 5,324 unigenes in 327 known metabolic pathways. Comparisons with other grasses via a reciprocal BLAST search revealed a larger number of orthologous genes for the Panicum species. The unigenes were involved in C4 photosynthesis, lignocellulose biosynthesis and flooding stress responses. A search for functional molecular markers revealed 4,489 microsatellites and 560,298 single nucleotide polymorphisms (SNPs). A quantitative real-time PCR analysis validated the RNA-seq expression analysis and allowed for the identification of transcriptomic differences between the two evaluated genotypes. Moreover, 192 unannotated sequences were classified as containing complete open reading frames, suggesting that the new, potentially exclusive genes should be further investigated. CONCLUSION: The present study represents the first whole-transcriptome sequencing of U. humidicola leaves, providing an important public information source of transcripts and functional molecular markers. The qPCR analysis indicated that the expression of certain transcripts confirmed the differential expression observed in silico, which demonstrated that RNA-seq is useful for identifying differentially expressed and unique genes. These results corroborate the findings from previous studies and suggest a hybrid origin for BH031.