Development of Apomictic 56-Chromosomal Maize-Tripsacum Hybrids: A Potential Breakthrough in Heterosis Fixation.

Maize (Zea mays L.) is one of the most demanded grain crops in the world. Currently, production has exceeded one billion tons and is increasing by 3-5% annually. Such growth is due to the genetic potential of the crop and the use of heterosis F1 hybrids in production. However, the need to produce first-generation seed annually poses significant challenges and is an economically costly technology. A solution to this problem may be the transfer of the asexual (apomictic) mode of reproduction to maize from its wild relative, eastern gamagrass (Tripsacum dactyloides L.). In this work, we report the production of 56-chromosome apomictic hybrids of maize (Zea mays L.) with eastern gamagrass (T. dactyloides L.) with restored anther fertility. The mode of reproduction of the plant was confirmed by counting chromosomes and sequencing the nuclear gene (Pox3) and chloroplast tRNA-Leu (trnL) gene. These apomictic hybrids had karyotypes of 2n = 56 = [(10Zm(573MB) + 36Td) + 10Zm(611CB)] and 2n = 56 = [(10Zm(611CB) + 36Td) + 10Zm(611CB)]. The resulting hybrids can be widely used as a fodder crop.

Interactions with fungi vary among Tripsacum dactyloides genotypes from across a precipitation gradient.

Plant-associated microbes, specifically fungal endophytes, augment the ability of many grasses to adapt to extreme environmental conditions. Tripsacum dactyloides (Eastern gamagrass) is a perennial, drought-tolerant grass native to the tallgrass prairies of the central USA. The extent to which the microbiome of T. dactyloides contributes to its drought tolerance is unknown. Ninety-seven genotypes of T. dactyloides were collected from native populations across an east-west precipitation gradient in Kansas, Oklahoma and Texas, and then grown together in a common garden for over 20 years. Root and leaf samples were visually examined for fungal density. Because fungal endophytes confer drought-tolerant capabilities to their host plants, we expected to find higher densities of fungal endophytes in plants from western, drier regions, compared to plants from eastern, wetter regions. Results confirmed a negative correlation between endophyte densities in roots and precipitation at the genotype's original location (r = -0.21 P = 0.04). Our analyses reveal that the host genotype's origin along the precipitation gradient predicts the absolute abundance of symbionts in the root, but not the relative abundances of particular organisms or the overall community composition. Overall, these results demonstrate that genetic variation for plant-microbe interactions can reflect historical environment, and reinforce the importance of considering plant genotype in conservation and restoration work in tallgrass prairie ecosystems.

Integrated single-molecule real-time sequencing and RNA sequencing reveal the molecular mechanisms of salt tolerance in a novel synthesized polyploid genetic bridge between maize and its wild relatives.

BACKGROUND: Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear. RESULTS: Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance. CONCLUSIONS: Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.

The complete chloroplast genome of Tripsacum laxum (Gramineae).

Tripsacum laxum (Guatemalan grass) is a perennial fodder grasses, which is commonly growing in large parts of Africa for a source of livestock feed. It has a high economic value as a forage. In this study, we obtained a complete chloroplast genome of T. laxum by Illumina sequencing. The results showed a circular genome of 140,556 bp, including the large single copy region (LSC, 82,939 bp), the small single-copy region (SSC, 12,573 bp), and a pair of 22,522 bp inverted repeat regions (IRs). The circular genome contained 120 genes, including 74 protein-coding genes, eight ribosomal RNA genes and 38 tRNA genes. Evolutionary relationship analysis indicates that T. laxum is more closely related to previously reported T. dactyloides.

Megabase-scale presence-absence variation with Tripsacum origin was under selection during maize domestication and adaptation.

BACKGROUND: Structural variants (SVs) significantly drive genome diversity and environmental adaptation for diverse species. Unlike the prevalent small SVs (< kilobase-scale) in higher eukaryotes, large-size SVs rarely exist in the genome, but they function as one of the key evolutionary forces for speciation and adaptation. RESULTS: In this study, we discover and characterize several megabase-scale presence-absence variations (PAVs) in the maize genome. Surprisingly, we identify a 3.2 Mb PAV fragment that shows high integrity and is present as complete presence or absence in the natural diversity panel. This PAV is embedded within the nucleolus organizer region (NOR), where the suppressed recombination is found to maintain the PAV against the evolutionary variation. Interestingly, by analyzing the sequence of this PAV, we not only reveal the domestication trace from teosinte to modern maize, but also the footprints of its origin from Tripsacum, shedding light on a previously unknown contribution from Tripsacum to the speciation of Zea species. The functional consequence of the Tripsacum segment migration is also investigated, and environmental fitness conferred by the PAV may explain the whole segment as a selection target during maize domestication and improvement. CONCLUSIONS: These findings provide a novel perspective that Tripsacum contributes to Zea speciation, and also instantiate a strategy for evolutionary and functional analysis of the "fossil" structure variations during genome evolution and speciation.

Evolutionary Dynamics of Transposable Elements Following a Shared Polyploidization Event in the Tribe Andropogoneae.

Both polyploidization and transposable element (TE) activity are known to be major drivers of plant genome evolution. Here, we utilize the Zea-Tripsacum clade to investigate TE activity and accumulation after a shared polyploidization event. Comparisons of TE evolutionary dynamics in various Zea and Tripsacum species, in addition to two closely related diploid species, Urelytrum digitatum and Sorghum bicolor, revealed variation in repeat content among all taxa included in the study. The repeat composition of Urelytrum is more similar to that of Zea and Tripsacum compared to Sorghum, despite the similarity in genome size with the latter. Although LTR-retrotransposons were abundant in all species, we observed an expansion of the copia superfamily, specifically in Z. mays and T. dactyloides, species that have adapted to more temperate environments. Additional analyses of the genomic distribution of these retroelements provided evidence of biased insertions near genes involved in various biological processes including plant development, defense, and macromolecule biosynthesis. Specifically, copia insertions in Zea and T. dactyloides were significantly enriched near genes involved in abiotic stress response, suggesting independent evolution post Zea-Tripsacum divergence. The lack of copia insertions near the orthologous genes in S. bicolor suggests that duplicate gene copies generated during polyploidization may offer novel neutral sites for TEs to insert, thereby providing an avenue for subfunctionalization via TE insertional mutagenesis.

Analysis of the genitor origin of an intergeneric hybrid clone between Zea and Tripsacum for forage production by McGISH.

In this study, the chromosome number and composition of a novel perennial forage crop, 'Yucao No. 6' (Yu6), was revealed by chromosome spread and McGISH (multicolor genomic in situ hybridization) techniques to clarify its genitor origin. Cytogenetic analysis showed that Yu6, which has 56 chromosomes, is an aneuploid representing 12, 17 and 27 chromosomes from Zea mays ssp. mays L. (Zm, 2n = 2x = 20), Tripsacum dactyloides L. (Td, 2n = 4x = 72), and Z. perennis (Hitchc.) Reeves & Mangelsd. (Zp, 2n = 4x = 40), respectively. This finding indicates that Yu6 is the product of a reduced egg (n = 36 = 12Zm + 17Td + 7Zp) of MTP (a near-allohexaploid hybrid, 2n = 74 = 20Zm + 34Td + 20Zp) fertilized by a haploid sperm nucleus (n = 20Zp) of Z. perennis. Moreover, 3 translocated chromosomes consisting of the maize-genome chromosome with the segment of Z. perennis were observed. These results suggest that it is practical to develop perennial forage maize by remodeling the chromosomal architecture of MTP offspring with Z. perennis as a pollen parent. Finally, the overview of forage breeding in the Zea and Tripsacum genera was discussed.

Tripsazea, a Novel Trihybrid of Zea mays, Tripsacum dactyloides, and Zeaperennis.

A trispecific hybrid, MTP (hereafter called tripsazea), was developed from intergeneric crosses involving tetraploid Zea mays (2n = 4x = 40, genome: MMMM), tetraploid Tripsacum dactyloides (2n = 4x = 72, TTTT), and tetraploid Zperennis (2n = 4x = 40, PPPP). On crossing maize-Tripsacum (2n = 4x = 56, MMTT) with Zperennis, 37 progenies with varying chromosome numbers (36-74) were obtained, and a special one (i.e., tripsazea) possessing 2n = 74 chromosomes was generated. Tripsazea is perennial and expresses phenotypic characteristics affected by its progenitor parent. Flow cytometry analysis of tripsazea and its parents showed that tripsazea underwent DNA sequence elimination during allohexaploidization. Of all the chromosomes in diakinesis I, 18.42% participated in heterogenetic pairing, including 16.43% between the M- and P-genomes, 1.59% between the M- and T-genomes, and 0.39% in T- and P-genome pairing. Tripsazea is male sterile and partly female fertile. In comparison with previously synthesized trihybrids containing maize, Tripsacum and teosinte, tripsazea has a higher chromosome number, higher seed setting rate, and vegetative propagation ability of stand and stem. However, few trihybrids possess these valuable traits at the same time. The potential of tripsazea is discussed with respect to the deployment of the genetic bridge for maize improvement and forage breeding.

Characterization of the complete chloroplast genome of the Eastern gamagrass, Tripsacum dactyloides.

Tripsacum dactyloides, known as eastern gamagrass, is used as a donor of valuable traits. It grows naturally in the same region where maize is commercially cultured in the USA and has the ability to hybridize to maize. The wild genotype of eastern gamagrass is threaten by the gene flow from the transgenic maize. The circular genome is 141,050 bp in length and contains 120 genes, including 73 protein-coding genes (PCG), 39 transfer RNA genes (tRNA) and eight ribosomal RNA genes (rRNA). The overall nucleotide composition is: 30.8% A, 19.2% C, 19.3% G, 30.7% T, with a total G + C content of 38.5%. The phylogenetic tree was constructed to explore the taxonomic status of Tripsacum dactyloides, which contributes to phylogenetic studies and further conservation strategies for this species.

Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

Estudio citológico en poblaciones diploides y poliploides del género tripsacum / CYTOLOGICAL STUDY IN DIPLOID AND POLIPOID POPULATIONS OF THE Tripsacum GENUS / ESTUDO CITOLÓGICO EM POPULAÇÕES DIPLÓIDES E POLIPLÓIDES DO GÊNERO Tripsacum

El género Tripsacum (Poaceae) tiene afinidad filogenética con el maíz y se distribuye ampliamente en América. El número básico de cromosomas es x=18 y existen poblaciones naturales desde diploides (2n=2x=36) hasta hexaploides (2n=6x=108). Las plantas de este género se reproducen de manera sexual y asexual y, en poliploides, ocurre la apomixis. Se realizó un estudio citológico en plantas con diferente nivel de ploidía. En espigas se hizo análisis de la meiosis, y de semillas se obtuvieron plántulas a las cuales se determinó el número cromosómico en ápices radicales y por cuantificación de ADN mediante citometría de flujo. Se encontraron poblaciones constituidas únicamente por triploides o tetraploides, y otras que contienen una mezcla de diploides hasta hexaploides. En diacinesis, los cromosomas de los diploides se asocian en bivalentes; en los triploides se forman univalentes, bivalentes y trivalentes; mientras que en tetraploides en univalentes, bivalentes, trivalentes y cuadrivalentes. En profase II, los diploides forman, en alta frecuencia, grupos cromosómicos segregantes de 18; los triploides desde 21 hasta 33; y los tetraploides desde 32 hasta 38. El número de cromosomas en progenies de plantas diploides fue 2x=36 y 3x=54; de triploides, 3x=54, 4x=72, 5x=90 y 6x=108; y de tetraploides, 4x=72, 5x=90 y 6x=108. Los resultados indican que los diploides tienen un origen sexual y que los triploides y tetraploides posiblemente sean apomícticos facultativos.

The Tripsacum (Poaceae) genus has phylogenetic affinity with maize and is widely distributed in America. The basic number of chromosomes is x=18, and there are natural populations ranging from diploids (2n=2x=36) to hexaploids (2n=6x=108). The plants of this genus reproduce sexually and asexually and, in polyploids, apomixis occurs. A cytological study was conducted in plants with different ploidy level. Analysis of meiosis was made in tassels, and seedlings were obtained whose chromosomal number was determined in root tips and by DNA quantification using flow cytometry. Populations made up only of triploids or tetraploids were found, and others containing a mixture from diploids to hexaploids. During diakinesis, diploid chromosomes associate in bivalents; in triploids, univalents, bivalents, and trivalents are formed; and in tetraploids, univalents, bivalents, trivalents, and quadrivalents are formed. In prophase II, the diploids form with high frequencies segregating chromosomal groups of 18; the triploids, groups from 21 to 33; and tetraploids, from 32 to 38. The chromosomal number in progenies of diploid plants was 2x=36 and 3x=54; of triploids 3x=54, 4x=72, 5x=90, and 6x=108; and from tetraploids, 4x=72, 5x=90, and 6x=108. Results indicate that the diploid individuals have sexual origin and the triploids and tetraploids may be apomictic.

O gênero Tripsacum (Poaceae) tem afinidade filogenética com o milho e se distribui amplamente na América. O número básico de cromossomas é x=18 e existem populações naturais desde diplóides (2n=2x=36) até hexaplóides (2n=6x=108). As plantas deste gênero se reproduzem de maneira sexual e asexual e, em poliplóides, ocorre a apomixia. Realizou-se um estudo citológico em plantas com diferente nível de ploidia. Foi realizada análise em espigas da meiose e das sementes foram obtidas plântulas nas quais se determinou o número cromossômico em ápices radicais e por quantificação de DNA mediante citometria de fluxo. Encontraram-se populações constituídas unicamente por triplóides ou tetraplóides, e outras que contêm uma mistura de diplóides até hexaplóides. Em diacinese, os cromossomas dos diplóides se associam em bivalentes; nos triplóides se formam monovalentes, bivalentes e trivalentes; enquanto que em tetraplóides em monovalentes, bivalentes, trivalentes e tetravalentes. Na profase II, os diplóides formam, em alta frequência, grupos cromossômicos segregantes de 18; os triplóides desde 21 até 33; e os tetraplóides desde 32 até 38. O número de cromossomas em progênies de plantas diplóides foi 2x=36 e 3x=54; de triplóides, 3x=54, 4x=72, 5x=90 e 6x=108; e de tetraplóides, 4x=72, 5x=90 e 6x=108. Os resultados indicam que os diplóides têm uma origem sexual e que os triplóides e tetraplóides possívelmente sejam apomícticos facultativos.

Novel sources of resistance to Striga hermonthica in Tripsacum dactyloides, a wild relative of maize.

• The parasitic weed Striga hermonthica lowers cereal yield in small-holder farms in Africa. Complete resistance in maize to S. hermonthica infection has not been identified. A valuable source of resistance to S. hermonthica may lie in the genetic potential of wild germplasm. • The susceptibility of a wild relative of maize, Tripsacum dactyloides and a Zea mays-T. dactyloides hybrid to S. hermonthica infection was determined. Striga hermonthica development was arrested after attachment to T. dactyloides. Vascular continuity was established between parasite and host but there was poor primary haustorial tissue differentiation on T. dactyloides compared with Z. mays. Partial resistance was inherited in the hybrid. • Striga hermonthica attached to Z. mays was manipulated such that different secondary haustoria could attach to different hosts. Secondary haustoria formation was inhibited on T. dactyloides, moreover, subsequent haustoria formation on Z. mays was also impaired. • Results suggest that T. dactyloides produces a signal that inhibits haustorial development: this signal may be mobile within the parasite haustorial root system.

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.).

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 µM) was applied to 27 microtillers for 3-5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually.