Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac,parthenogenetic embryo development from the egg cell,and endosperm formation either autonomously,or due to fertilization of polar nuclei by the sperm(under pseudogamous form of apomixis).In the latter case,an obstacle to the normal endosperm development is disturbance of maternal(m)-to-paternal(p)genomic ratio 2m:1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms.Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m:2p that provides formation of kernels with plump endosperm.Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions.These kernels had hybrid endosperm and diploid maternal-type embryo or hybrid embryo with different ploidy level(2n,3n,4n).The frequencies of plump kernels on the ear ranged from 0.2-0.3%to 5.7-6.2%counting from the number of ovaries.Maternal-type plants were found in two maize lines,their frequency varying from 10.7 to 37.5%of the progeny plants.In CMS-lines of sorghum pollinated with tetraploid sorghum accessions,the frequency of plump kernels ranged from 0.6 to 14.0%counting from the number of ovaries;the frequency of maternal-type plants varied from 33.0 up to 96.1%.The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins.These data testify to variable modes of seed formation under diploid×tetraploid crosses in maize and sorghum both by amphi-and by apomixis.Therefore,usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.

Identification of Candidate Genes Related to Polyploidy and/or Apomixis in <i>Eragrostis curvula</i>

This work was aimed at identifying genes that show altered expression profiles in response to changes in ploidy and/or reproductive mode (from sexual to apomictic) in the African grass Eragrostis curvula. A differential display analysis was performed on leaf and flower transcriptomes from a series of genetically related euploid plants, including tetraploid apomictic, diploid sexual, and tetraploid sexual plants. More than 100 primer combinations were used to generate 11,864 total markers, yielding 1293 differential bands. Of these bands, 11.84% to 6.74% were related to ploidy and 0.71% to 2.17% to the reproductive mode, depending on the tissue. A small percentage of bands showed similar expressions between the tetraploid apomictic and the diploid sexual plants. Expression-based similarity dendrograms were constructed. Our data suggested that ploidy is more decisive than tissue type in defining the transcriptome structure. Out of 102 fragments sequenced, 50 showed strong homology to known genes. The differentially expressed genes were mapped in silico onto maize chromosomes. Several candidates mapped within the linkage group syntenic to the Tripsacum dactyloides diplospory-governing region. The evidence indicates that expression of genes located around the diplospory-associated region may be strongly influenced by ploidy and may be silenced in the apomictic genotype. These findings are discussed in the context of diplospory molecular control and its connection with ploidy.

Doubled haploid technology and synthetic apomixis:Recent advances and applications in future crop breeding

Doubled haploid(DH)technology and synthetic apomixis approaches can considerably shorten breeding cycles and enhance breeding efficiency.Compared with traditional breeding methods,DH technology offers the advantage of rapidly generating inbred lines,while synthetic apomixis can effectively fix hybrid vigor.In this review,we focus on(i)recent advances in identifying and characterizing genes responsible for haploid induction(Hl),(ii)the molecular mechanisms of Hl,(ili)spontaneous haploid genome doubling,and(iv)crop synthetic apomixis.We also discuss the challenges and potential solutions for future crop breeding programs utilizing DH technology and synthetic apomixis.Finally,we provide our perspectives about how to integrate DH and synthetic apomixis for precision breeding and de novo domestication.

Synthetic apomixis enables stable transgenerational transmission of heterotic phenotypes in hybrid rice

In hybrid plants,heterosis often produces large,vigorous plants with high yields;however,hybrid seeds are generated by costly and laborious crosses of inbred parents.Apomixis,in which a plant produces a clone of itself via asexual reproduction through seeds,may produce another revolution in plant biology.Recently,synthetic apomixis enabled clonal reproduction of F_(1) hybrids through seeds in rice(Oryza sativa),but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear.Here,we propagated clonal plants to the T_(4) generation and investigated their genetic and molecular stability at each generation.By analyzing agronomic traits,as well as the genome,methylome,transcriptome,and allele-specific transcriptome,we showed that the descendant clonal plants remained stable.Unexpectedly,in addition to normal clonal seeds,the plants also produced a few aneuploids that had eliminated large genomic segments in each generation.Despite the identification of rare aneuploids,the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future.

Fixation of hybrid vigor in rice:synthetic apomixis generated by genome editing

Apomixis is an asexual reproduction process in which clonal seeds are formed without meiosis and fertilization.Because of its potential in permanently preserving hybrid vigor,apomixis has attracted a great deal of interests from plant biologists and the seed industry.However,despite of decades of effort,introgression of apomixis traits from wild relatives into major crops has remained unsuccessful.Therefore,synthetic apomixis has been proposed as an alternative to fix hybrid vigor.In this article,I present the development of the MiMe(Mitosis instead of Meiosis),which turns meiosis into mitosis and leads to the production of clonal gametes.Apomixis-like clonal seeds are generated when MiMe plants are crossed to special genome elimination lines,which contain an altered centromere-specific histone 3(CENH3).Furthermore,induction of haploid plants from egg cells can be achieved by either egg cell-specific expression of BABY BOOM1(BBM1),or disruption of MATRILINEAL(MTL)using CRISPR/Cas9 gene-editing technology.Synthetic apomixis is established and clonal seeds are produced by simultaneous engineering MiMe with altering BBM1 expression or MTL disruption.Finally,I discuss how to further improve the apomixis strategy and its applications in crop breeding.

Initial Study on Sichuan Apomixis Rice (SAR-1)

SAR-1 is a new germplasm which was discovered in the breeding material of southern multiplication in spring, 1988. The material showing high sterility of pollens is able to set seeds spontaneously. Under isolation, the seed-setting highest rate may reach 55.33%. The florets emasculated by clipping spikelet and lukewarm water still set seeds at certain rates. The completely sterile florets, after being emasculated and checked under microscope one by one, still set seeds, and the highest setting rate is 41.80%. Cytoembryological research indicates that the egg of SAR-1, without fertilization, divides independently into an embryo, which follows the normal process to maturity. Adventitious embryos originate from ovary wall cells. Therefore, it is deduced that SAR-1 has multiple mechanisms of apomixis, and the unfertilized polar nuclei fuse and develop into endosperm cells. The endosperm provids the embryo with nutrient for development. The automatic formation of the endosperm is an obvious feature of SAR-1.

Morphological characteristics of apomictic embryo in sorghum

The embryo structure of two apomictir lines 296B and SSA-1 has been com-pared in present paper. The apomictic embryo has the same character of lacking (or short) sus-pensor in sorghum,otherwise,the sexual embryo has a long suspensor in the same species. This character as an index of judgement on apomxis was discussed. The same of unsexual embryos in the lack of suspensor suggested that there were some relationships between non-zagotic embryos.

Apomictic symptoms in the aspect of embryology in a sorghum line 296B

The presence of facultative apomixis In line 296B was proved by the embryologicalstudy.The twin embryosacs were observed at florescence.The autonomous development of em-bryo was confirmed by multiple cell proembryo existing with undeveloped polar nuclei In one embryosac.Cell structure and size of apomictic proembryo were different from sexual proembryo.The structural feature of proembryo can be used to distinguish apomictic proembryo with devel-oped endosperm from sexual embryo.The apomictic development in this line is attributed toaposporous type.The frequency of apomixis Is at least 16—21%.The apomictic characters andthe potential for fixing heterosis in line 296B was dlscussed in the present paper.

CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize

Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate(CRISPRa)or inhibit transcription.Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds.We report here a CRISPR/dCas9-based toolkit equippedwith dCas9-VP64 andMS2-p65-HSF1 effectors that may specifically target genes with high activation capability.We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2(ZmBBM2),acting as a fertilization checkpoint,as a means to engineer parthenogenesis.We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells.Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds.Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.

GISH and BAC-FISH study of apomictic Beta M14

Apomixis is a means of asexual reproduction by which plants produce embryos without fertilization and meiosis,therefore the embryo is of clonal and maternal origin.Interspecific hybrids between dip-loid B.vulgaris(2n=2x=18)and tetraploid B.corolliflora(2n=4x=36)were established,and then back-crossed with B.vulgaris.Among their offspring,monosomic addition line M14(2n=2x=18+1)was se-lected because of the apomictic phenotype.We documented chromosome transmission from B.corol-liflora into M14 by using genomic in situ hybridization(GISH).Suppression of cross-hybridization by blocking DNA was not necessary,indicating that the investigated Beta genome contains sufficient species-specific DNA,thus enabling the determination of genomic composition of the hybrids.We analyzed BAC microarrays of B.corolliflora chromosome 9 by using fluorescence-specific mRNA of B.vulgaris and Beta M14.BAC clones 16-M11 and 26-L15 were detected as fluorescence-specifics of BAC DNA of Beta M14.Then both BAC clones 16-M11 and 26-L15 were in situ hybridized to M14 chromo-somes.The two hybridized BAC clones were located close to the telomere region of the long arm of a single chromosome 9,and showed hemizygosity.The results of BAC microarrays showed that these developments of embryo and endosperm have conservative expression patterns,indicating that sexual reproduction and apomixis have an interrelated pathway with common regulatory components and that the induction of a modified sexual reproduction program may enable the manifestation of apomixis in Beta species.It would be sufficient for the expression of apomixes with those apomictic-specific genes on chromosome 9 of B.corolliflora.

Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion

Parthenogenetic reproduction is taxonomically widespread and occurs through various cytological mechanisms, which have different impact on the genetic variation of the offspring. Extatosoma tiaratum is a facultatively parthenogenetic Australian insect （Phasmatodea）, in which females oviposit continuously throughout their adult lifespan irre- spective of mating. Fertilized eggs produce sons and daughters through sexual reproduction and unfertilized eggs produce female offspring via parthenogenesis. Here, we developed novel microsatellite markers for E. tiaratum and characterized them by genotyping indi- viduals from a natural population. We then used the microsatellite markers to infer the cytological mechanism of parthenogenesis in this species. We found evidence suggesting parthenogenesis in E. tiaratum occurs through automixis with terminal fusion, resulting in substantial loss of microsatellite heterozygosity in the offspring. Loss of microsatel- lite heterozygosity may be associated with loss of heterozygosity in fitness related loci. The mechanism of parthenogenetic reproduction can therefore affect fitness outcomes and needs to be considered when comparing costs and benefits of sex versus parthenogenesis.