Genome dosage alteration caused by chromosome pyramiding and shuffling effects on karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids.

KEY MESSAGE: We developed an array of Zea-Tripsacum tri-hybrid allopolyploids with multiple ploidies. We unveiled that changes in genome dosage due to the chromosomes pyramiding and shuffling of three species effects karyotypic heterogeneity, reproductive diversity, and phenotypic variation in Zea-Tripsacum allopolyploids. Polyploidy, or whole genome duplication, has played a major role in evolution and speciation. The genomic consequences of polyploidy have been extensively studied in many plants; however, the extent of chromosomal variation, genome dosage, phenotypic diversity, and heterosis in allopolyploids derived from multiple species remains largely unknown. To address this question, we synthesized an allohexaploid involving Zea mays, Tripsacum dactyloides, and Z. perennis by chromosomal pyramiding. Subsequently, an allooctoploid and an allopentaploid were obtained by hybridization of the allohexaploid with Z. perennis. Moreover, we constructed three populations with different ploidy by chromosomal shuffling (allopentaploid × Z. perennis, allohexaploid × Z. perennis, and allooctoploid × Z. perennis). We have observed 3 types of sexual reproductive modes and 2 types of asexual reproduction modes in the tri-species hybrids, including 2n gamete fusion (2n + n), haploid gamete fusion (n + n), polyspermy fertilization (n + n + n) or 2n gamete fusion (n + 2n), haploid gametophyte apomixis, and asexual reproduction. The tri-hybrids library presents extremely rich karyotype heterogeneity. Chromosomal compensation appears to exist between maize and Z. perennis. A rise in the ploidy of the trihybrids was linked to a higher frequency of chromosomal translocation. Variation in the degree of phenotypic diversity observed in different segregating populations suggested that genome dosage effects phenotypic manifestation. These findings not only broaden our understanding of the mechanisms of polyploid formation and reproductive diversity but also provide a novel insight into genome pyramiding and shuffling driven genome dosage effects and phenotypic diversity.

Multispecies polyploidization, chromosome shuffling, and genome extraction in Zea/Tripsacum hybrids.

By hybridization and special sexual reproduction, we sequentially aggregated Zea mays, Zea perennis, and Tripsacum dactyloides in an allohexaploid, backcrossed it with maize, derived self-fertile allotetraploids of maize and Z. perennis by natural genome extraction, extended their first six selfed generations, and finally constructed amphitetraploid maize using nascent allotetraploids as a genetic bridge. Transgenerational chromosome inheritance, subgenome stability, chromosome pairings and rearrangements, and their impacts on an organism's fitness were investigated by fertility phenotyping and molecular cytogenetic techniques genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). Results showed that diversified sexual reproductive methods produced highly differentiated progenies (2n = 35-84) with varying proportions of subgenomic chromosomes, of which one individual (2n = 54, MMMPT) overcame self-incompatibility barriers and produced a self-fertile nascent near-allotetraploid by preferentially eliminating Tripsacum chromosomes. Nascent near-allotetraploid progenies showed persistent chromosome changes, intergenomic translocations, and rDNA variations for at least up to the first six selfed generations; however, the mean chromosome number preferably maintained at the near-tetraploid level (2n = 40) with full integrity of 45S rDNA pairs, and a trend of decreasing variations by advancing generations with an average of 25.53, 14.14, and 0.37 for maize, Z. perennis, and T. dactyloides chromosomes, respectively. The mechanisms for three genome stabilities and karyotype evolution for formatting new polyploid species were discussed.

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.

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.