Genetic Transformation of Apomictic Grasses: Progress and Constraints

The available methods for plant transformation and expansion beyond its limits remain especially critical for crop improvement. For grass species, this is even more critical, mainly due to drawbacks in in vitro regeneration. Despite the existence of many protocols in grasses to achieve genetic transformation through Agrobacterium or biolistic gene delivery, their efficiencies are genotype-dependent and still very low due to the recalcitrance of these species to in vitro regeneration. Many plant transformation facilities for cereals and other important crops may be found around the world in universities and enterprises, but this is not the case for apomictic species, many of which are C4 grasses. Moreover, apomixis (asexual reproduction by seeds) represents an additional constraint for breeding. However, the transformation of an apomictic clone is an attractive strategy, as the transgene is immediately fixed in a highly adapted genetic background, capable of large-scale clonal propagation. With the exception of some species like Brachiaria brizantha which is planted in approximately 100 M ha in Brazil, apomixis is almost non-present in economically important crops. However, as it is sometimes present in their wild relatives, the main goal is to transfer this trait to crops to fix heterosis. Until now this has been a difficult task, mainly because many aspects of apomixis are unknown. Over the last few years, many candidate genes have been identified and attempts have been made to characterize them functionally in Arabidopsis and rice. However, functional analysis in true apomictic species lags far behind, mainly due to the complexity of its genomes, of the trait itself, and the lack of efficient genetic transformation protocols. In this study, we review the current status of the in vitro culture and genetic transformation methods focusing on apomictic grasses, and the prospects for the application of new tools assayed in other related species, with two aims: to pave the way for discovering the molecular pathways involved in apomixis and to develop new capacities for breeding purposes because many of these grasses are important forage or biofuel resources.

Characteristics of the microspecies aggregations among Alchemilla L. (Rosaceae) coenopopulations on the territory of Mari El Republic

The genus Alchemilla L. is represented by a significant number of apomictic species. This paper deals with the microspecies aggregations among Alchemilla coenopopulations from 5 nature districts on the territory of Mari El Republic. Alchemilla coenopopulations grow in meadow and forest margin communities, in disturbed habitats, while they have close estimates according to the ecological scales of L.G. Ramensky and D.N. Tsyganov. 25 Alchemilla microspecies have been identified. The number of generative rosette shoots on 1 m area for different microspecies within the coenopopulation varies extremely widely. There are from 3 to 14 microspecies in coenopopulations, one or two from them clearly dominant in number. Dominant microspecies are: A. acutiloba Opiz, A. hirsuticaulis H. Lindb., A. micans Buser, A. monticola Opiz, A. schistophylla Juz., A. subcrenata Buser, A. substrigosa Juz. There is A. micans in all coenopopulations, A. acutiloba , A. glabricaulis H. Lindb., A. monticola , A. sarmatica Juz. and A. subcrenata often occur. Microspecies A. breviloba H. Lindb., A. dasycrater Juz., A. devestiens Juz., A. filicaulis Buser, A. heptagona Juz., A. leiophylla Juz., A. lindbergiana Juz., A. plicata Buser, A. semilunaris Alechin are represented by single generative rosette shoots.

Haploidy in Tobacco Induced by PsASGR-BBML Transgenes via Parthenogenesis

Background: Engineering apomixis in sexually reproducing plants has been long desired because of the potential to fix hybrid vigor. Validating the functionality of genes originated from apomictic species that contribute to apomixis upon transfer to sexually reproducing species is an important step. The PsASGR-BABYBOOM-like (PsASGR-BBML) gene from Pennisetum squamulatum confers parthenogenesis in this apomict, and its functionality was demonstrated in several sexually reproducing monocots but not in any dicots. Methods: We introduced the PsASGR-BBML gene regulated by egg cell-specific promoters, either AtDD45 or AtRKD2, into tobacco, and analyzed progeny of the transgenic lines resulting from self-pollination and crossing by flow cytometry. Results: We identified haploid progeny at a frequency lower than 1% in the AtDD45pro lines, while at a frequency of 9.3% for an octoploid (2n = 8x) AtRKD2pro line. Haploid production in the T2 generation, derived from the tetraploid T1 offspring of this original octoploid AtRKD2pro line, was also observed. Pollinated by homozygous transgenic tobacco carrying a DsRed marker gene, 4x progeny of the AtRKD2pro line yielded parthenogenetic embryos identified as DsRed negative. We verified that the DsRed negative seedlings recovered were haploid (2x). Conclusion: The PsASGR-BBML gene regulated by egg cell-specific promoters could enable parthenogenesis in tobacco, a dicotyledon species.

Plant apomixis is rare in Himalayan high-alpine flora

Gametophytic apomixis is a way of asexual plant reproduction by seeds. It should be advantageous under stressful high altitude or latitude environment where short growing seasons, low temperatures, low pollinator activity or unstable weather may hamper sexual reproduction. However, this hypothesis remains largely untested. Here, we assess the reproductive mode in 257 species belonging to 45 families from the world’s broadest alpine belt (2800–6150 m) in NW Himalayas using flow cytometric seed screen. We found only 12 apomictic species, including several members of Poaceae (Festuca, Poa and Stipa), Rosaceae (Potentilla) and Ranunculaceae (Halerpestes, Ranunculus), which are families typical for high apomict frequency. However, several apomictic species were newly discovered, including the first known apomictic species from the family Biebersteiniaceae (Biebersteinia odora), and first apomicts from the genera Stipa (Stipa splendens) and Halerpestes (Halerpestes lancifolia). Apomicts showed no preference for higher elevations, even in these extreme Himalayan alpine habitats. Additional trait-based analyses revealed that apomicts differed from sexuals in comprising more rhizomatous graminoids and forbs, higher soil moisture demands, sharing the syndrome of dominant species with broad geographical and elevation ranges typical for the late-successional habitats. Apomicts differ from non-apomicts in greater ability of clonal propagation and preference for wetter, more productive habitats.

Assembly of the Boechera retrofracta Genome and Evolutionary Analysis of Apomixis-Associated Genes

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of the apomictic species Boechera divaricarpa. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. 27048 protein-coding genes were predicted using a hybrid approach that combines homology-based and de novo methods. Also repeats, tRNA and rRNA genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. Also, a detailed analysis of evolution of the APOLLO apomixis-associated locus was performed. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species such as B. divaricarpa.

A new species and a new range extension in Hieracium sect. Cernua (Asteraceae) from Romania

Hieracium joannei, a new, apomictic species in H. sect. Cernua is described from the Şureanu Mountains, Southern Carpathians, Romania, and illustrated with photos of the holotype and living plants in the locus classicus. Hieracium zanogae (= H. tubulare), previously considered to be endemic to the Retezat Mountains, has been found in the Parâng Mountains. This is the easternmost occurrence of the species, disjoined ca 50 km from the nearest localities in the Retezat Mountains. A key for the species of H. sect. Cernua in Romania is provided.

Genetic gain in apomictic species of the genus Paspalum

ABSTRACT The objective of this work was to provide means to obtain genetic gain in apomictic species of the genus Paspalum by identifying traits that simultaneously show high correlation and heritability. Thus, to determine the amount of phenotypic variation due to genetic and environmental effects and the degree of association between leaf dry matter production and other traits of forage interest that are easy to select and measure will bring agility and economy in the selection of forage species. The experiment was conducted in the years 2009/10 and 2010/11 in the municipalities of Eldorado do Sul and Augusto Pestana, RS, Brazil, in a randomized complete block design with three replicates. The increase in the direct selection pressure on total and leaf dry matter of apomictic accesses of the genus Paspalum is enhanced by a greater contribution of genetic effects than environmental effects on the composition of the phenotypic variance. Efficient genetic gain in leaf production is achieved by indirect selection of total dry matter, which is a trait of high heritability and easy to select and measure.

A new species of the genus Alchemilla (Rosaceae) from Central Russia

A new apomictic species of Alchemilla from several regions of Central Russia is here described as A. tzvelevii. Description and images of the principal morphological features of this species are provided. Alchemilla tzvelevii combines the features of quite distant groups (i.e. A. subsect. Pubescentes and A. subsect. Alchemilla ser. Alchemilla subser. Heptagonae). Its presumed hybridogenous origin is discussed.