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.

A joint learning approach for genomic prediction in polyploid grasses.

Poaceae, among the most abundant plant families, includes many economically important polyploid species, such as forage grasses and sugarcane (Saccharum spp.). These species have elevated genomic complexities and limited genetic resources, hindering the application of marker-assisted selection strategies. Currently, the most promising approach for increasing genetic gains in plant breeding is genomic selection. However, due to the polyploidy nature of these polyploid species, more accurate models for incorporating genomic selection into breeding schemes are needed. This study aims to develop a machine learning method by using a joint learning approach to predict complex traits from genotypic data. Biparental populations of sugarcane and two species of forage grasses (Urochloa decumbens, Megathyrsus maximus) were genotyped, and several quantitative traits were measured. High-quality markers were used to predict several traits in different cross-validation scenarios. By combining classification and regression strategies, we developed a predictive system with promising results. Compared with traditional genomic prediction methods, the proposed strategy achieved accuracy improvements exceeding 50%. Our results suggest that the developed methodology could be implemented in breeding programs, helping reduce breeding cycles and increase genetic gains.

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.

Corrigendum: Genetic Mapping With Allele Dosage Information in Tetraploid Urochloa decumbens (Stapf) R. D. Webster Reveals Insights Into Spittlebug (Notozulia entreriana Berg) Resistance.

[This corrects the article DOI: 10.3389/fpls.2019.00092.].

Genetic Mapping With Allele Dosage Information in Tetraploid Urochloa decumbens (Stapf) R. D. Webster Reveals Insights Into Spittlebug (Notozulia entreriana Berg) Resistance.

Urochloa decumbens (Stapf) R. D. Webster is one of the most important African forage grasses in Brazilian beef production. Currently available genetic-genomic resources for this species are restricted mainly due to polyploidy and apomixis. Therefore, crucial genomic-molecular studies such as the construction of genetic maps and the mapping of quantitative trait loci (QTLs) are very challenging and consequently affect the advancement of molecular breeding. The objectives of this work were to (i) construct an integrated U. decumbens genetic map for a full-sibling progeny using GBS-based markers with allele dosage information, (ii) detect QTLs for spittlebug (Notozulia entreriana) resistance, and (iii) seek putative candidate genes involved in defense against biotic stresses. We used the Setaria viridis genome a reference to align GBS reads and selected 4,240 high-quality SNP markers with allele dosage information. Of these markers, 1,000 were distributed throughout nine homologous groups with a cumulative map length of 1,335.09 cM and an average marker density of 1.33 cM. We detected QTLs for resistance to spittlebug, an important pasture insect pest, that explained between 4.66 and 6.24% of the phenotypic variation. These QTLs are in regions containing putative candidate genes related to defense against biotic stresses. Because this is the first genetic map with SNP autotetraploid dosage data and QTL detection in U. decumbens, it will be useful for future evolutionary studies, genome assembly, and other QTL analyses in Urochloa spp. Moreover, the results might facilitate the isolation of spittlebug-related candidate genes and help clarify the mechanism of spittlebug resistance. These approaches will improve selection efficiency and accuracy in U. decumbens molecular breeding and shorten the breeding cycle.

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.

Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

Chromosome numbers and meiotic analysis in the pre-breeding of Brachiaria decumbens (Poaceae).

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.

A differential phenotypic expression of a divergent spindle mutation in interspecific Brachiaria hybrids.

Several mutations are known to alter the normal progression of meiosis and can be correlated with defects in microtubule distribution. The dv mutation affects the spindle organization and chromosomes do not converge into focused poles. Two Brachiaria hybrids presented the phenotypic expressions of dv mutation but exhibited many more details in the second division. Bivalents were distantly positioned and spread over a large metaphase plate and failed to converge into focused poles. Depending on the distance of chromosomes at the poles, telophase I nuclei were elongated or the chromosomes were grouped into various micronuclei of different sizes in each cell. The first cytokinesis occurred. However, when there were micronuclei, a second cytokinesis immediately took place dividing the prophase II meiocytes into three or four cells. In each meiocyte, meiosis progressed to the second division. Slightly elongated nuclei or micronuclei were recorded in telophase II. After a third cytokinesis, hexads or octads were formed. Pollen grains of different sizes were generated. One of these hybrids presented a higher frequency of abnormal cells than when previously analyzed. The fate of these hybrids as genitors or as candidates for cultivars in the Brachiaria breeding program is discussed.

Desynapsis and precocious cytokinesis in Brachiaria humidicola (Poaceae) compromise meiotic division.

The forage grass species Brachiaria humidicola is native to African savannas. Owing to its good adaptation to poorly drained and infertile acid soils, it has achieved wide utilization for pastures in Brazilian farms. Among the 55 accessions of B. humidicola analysed from the Embrapa Beef Cattle collection, one (H022), presented desynapsis and an abnormal pattern of cytokinesis in the first meiotic division. Among 28 inflorescences analysed in this accession, 12 were affected by the anomaly. In affected meiocytes, the first cytokinesis occurred in metaphase I and was generally perpendicular to a wide-metaphase plate, dividing the genome into two parts with an equal or unequal number of chromosomes. The normal cytokinesis after telophase I did not occur, and the meiocytes entered metaphase II, progressing to the end of meiosis with the occurrence of the second cytokinesis. As the first cytokinesis occurred precociously, whereas the second was normal, tetrads were formed but with unbalanced chromosome numbers in microspores. Abnormal cytokinesis occurred only in those meiocytes that underwent desynapsis after diakinesis. The implications of this abnormality in the Brachiaria breeding programme are discussed.

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae).

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.

Multiple spindles and cellularization during microsporogenesis in an artificially induced tetraploid accession of Brachiaria ruziziensis (Gramineae).

The genus Brachiaria is characterized by a majority of polyploid accessions--mainly tetraploid--and apomictic reproduction. Sexuality is found among diploids. To overcome incompatibility barriers, accessions with the same ploidy level are necessarily used in hybridization. Thus, sexual diploid accessions were tetraploidized to be used as female genitors. This paper reports microsporogenesis in an artificially induced tetraploid accession of Brachiaria ruziziensis. Chromosome pairing at diakinesis ranged from univalents to tetravalents, with predominance of bivalents. Irregular chromosome segregation was frequent in both meiotic divisions. During the first division, multiple spindles showing different arrangements were recorded. The spindle position determined the plane of first cytokinesis and the number of chromosomes determined the size of the cell. Meiotic products were characterized by polyads with spores of different sizes. Pollen sterility was estimated at 61.38%. The limitations of using this accession in the breeding program are discussed.

Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae).

The genus Brachiaria (Trin.) Griseb. has achieved considerable importance to cattle production systems, as a result of the good production and adaptation of a few cultivars to poor and acid soils of the Brazilian savannas. Many of its species and accessions are polyploid and apomictic, which limits direct hybridization. To assist the breeding program, cytogenetic characterization has been undertaken on the accessions of Brachiaria collection at the Embrapa Beef Cattle Research Center. In this study, chromosome number and meiotic behavior are reported for the Brachiaria nigropedata (Ficalho & Hiern) Stapf collection. The 20 available accessions are tetraploid (2n = 4x = 36). Chromosomes paired preferentially as bivalents, but quadrivalents were found in high frequencies in some cells. Meiotic behavior was, in general, irregular, and varied among accessions. Most accessions presented more than 20% of abnormal tetrads. The most common meiotic abnormalities were those related to irregular chromosome segregation due to polyploidy, leading to micronuclei formation in the tetrad stage. A low frequency of other meiotic abnormalities such as the absence of cytokinesis, chromosome stickiness, cell fusion, anaphase bridges, and chromosome transfer among microsporocytes were also recorded in some accessions. Limitations of these accessions for use in hybridization programs are discussed.

Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae).

The genus Brachiaria (Trin.) Griseb. has achieved considerable importance to cattle production systems, as a result of the good production and adaptation of a few cultivars to poor and acid soils of the Brazilian savannas. Many of its species and accessions are polyploid and apomictic, which limits direct hybridization. To assist the breeding program, cytogenetic characterization has been undertaken on the accessions of Brachiaria collection at the Embrapa Beef Cattle Research Center. In this study, chromosome number and meiotic behavior are reported for the Brachiaria nigropedata (Ficalho & Hiern) Stapf collection. The 20 available accessions are tetraploid (2n = 4x = 36). Chromosomes paired preferentially as bivalents, but quadrivalents were found in high frequencies in some cells. Meiotic behavior was, in general, irregular, and varied among accessions. Most accessions presented more than 20

of abnormal tetrads. The most common meiotic abnormalities were those related to irregular chromosome segregation due to polyploidy, leading to micronuclei formation in the tetrad stage. A low frequency of other meiotic abnormalities such as the absence of cytokinesis, chromosome stickiness, cell fusion, anaphase bridges, and chromosome transfer among microsporocytes were also recorded in some accessions. Limitations of these accessions for use in hybridization programs are discussed.

Asynchronous meiosis in an interspecific hybrid of Brachiaria ruziziensis and B. brizantha.

Male meiosis is generally synchronous in higher plants. The regulation of the cell cycle is still not well understood, and a powerful tool for gaining an understanding of this regulation is the development of mutations that affect cell-cycle synchrony. We report here asynchronous microsporogenesis in an interspecific hybrid between two important tropical grasses. In young spikelets of the interspecific hybrid 49.10% of anther meiocytes entered meiosis, exhibiting typical phases of the first and second divisions, while the other 50.90% showed distinctive features of early prophase. In older spikelets, anthers containing mature pollen grains also displayed meiocytes still undergoing meiosis. At this time, the latter cells were enclosed by the exine wall. Despite asynchrony, all cells completed meiosis. Old anthers contained only pollen grains that appeared to be in the same stage of development. Pollen fertility was estimated to be 52.76% in dehiscent anthers. An independent genetic control for meiosis synchrony and meiotic stages is suggested.

A mutation in the spindle checkpoint arresting meiosis II in Brachiaria ruziziensis.

Cytological characterization of BRA005568 accession of Brachiaria ruziziensis (2n = 2x = 18) showed a totally unexpected high frequency of abnormal meiotic products, from triads to hexads, and also tetrads with micro nuclei or microcytes. Meiosis I had a low frequency of abnormalities, mainly related to the chiasma terminalization process. In meiosis II, however, frequency of abnormalities increased exceptionally. Early prophase II was normal with the chromosome set enclosed by the nuclear envelope. However, in late prophase II, owing to the breakdown of the nuclear envelope, the chromosomes were scattered in the cytoplasm. Some chromosomes did not reach the metaphase II plate and remained scattered. The behavior of sister cells was inconsistent. While in one cell the chromosomes were totally aligned at the metaphase II plate, in the other they could be found completely scattered, leading to an asynchronous cell division. Cells with scattered chromosomes were unable to progress in meiosis. Thus, anaphase II failed to occur and sister chromatids were not released. Cells with non-aligned chromosomes in the metaphase II plate did not receive the "go ahead" sign to initiate anaphase II. Consequently, the scattered chromosomes produced telophase II nuclei of different sizes in situ. The asynchronous behavior led to the formation of a wide range of meiotic products. Results suggest that the present accession contains a mutation affecting the spindle checkpoint that arrests the second meiotic division.