MIME-Mitosis instead of meiosis and its application in crop apomixis / 生物工程学报

Apomixis has been widely concerned because of its great potential in heterosis fixation. Artificial apomixis is an important direction of current apomixis research. Mitosis instead of Meiosis (MIME) produces diploid gametes that is identical with the maternal genetic composition and is a key step in the artificial creation of apomixes. This paper reviews the occurrence of MIME and its application in crop apomixis and the problems encountered, in an aim to provide reference for expanding the application of MIME in crop apomixis.

Development of EST-SSR markers in Cenchrus ciliaris and their applicability in studying the genetic diversity and cross-species transferability

Most of the grasses of the genus Cenchrus (20–25 species) and Pennisetum (80–140 species) are distributed throughout the tropical and subtropical regions of the world and reproduce both by sexual and apomictic modes. However, the relationships among the Cenchrus–Pennisetum species are not very clear yet. Molecular markers like expressed sequence tag-simple sequence repeats (EST-SSRs) have been reported to be a better choice for resolving the phylogenetic relationships and to estimate the genetic diversity. The present study describes the identification of EST-SSR markers based on the transcriptome data of Cenchrus ciliaris inflorescence and illustrates the genetic diversity and phylogenetic relationships among these species. Of the 378 primer pairs used across 33 accessions of 21 Cenchrus, Pennisetum, and related grass (Bothriochloa, Dichanthium and Panicum) species, 116 EST-SSR markers were found to be polymorphic with an average polymorphism information content (PIC) of 0.49. Fifty-one EST-SSR loci and 520 alleles showed that where the PIC value is >0.5 there the GAG repeat motif was highly polymorphic. Two EST-SSR markers, CcSSR_80 and CcSSR_102, are polymorphic among the Cenchrus species, while they are absent in Pennisetum and the allied species. Five SSR markers (CcSSR_75, CcSSR_85, CcSSR_87, CcSSR_88 and CcSSR_114) showed 100% cross-transferability among the 21 Cenchrus–Pennisetum species. Species-specific alleles could also be detected for seven species of Cenchrus, Pennisetum and Panicum across 10 SSR markers. Assay of polymorphism across these agamic complexes showed that the three SSR markers (CcSSR_26, CcSSR_97 and CcSSR_109) were associated with Cenchrus–Pennisetum complex, and one (CcSSR_47) with Bothriochloa–Dichanthium complex. Markers with high discriminating power, namely CcSSR_4, CcSSR_38, CcSSR_48, CcSSR_66, CcSSR_67 and CcSSR_70, can be used to estimate the allelic sequence divergence across the sexual and apomictic lineages. Genetic diversity analysis using neighbour-joining (NJ) and principal co-ordinate analysis (PCoA) based approaches showed six and five clusters for the 33 accessions, respectively, having congruence in the pattern of clustering. These accessions were grouped according to their mode of reproduction. Cenchrus and Pennisetum species were grouped separately within the same clade, implying monophyletic group within a ‘bristle clade’. Thus, this study showed high discrimination power of microsatellite (EST-SSR) markers to resolve the phylogenetic relationships.

A brief note on genes that trigger components of apomixis

Apomixis or asexual reproduction through seeds occurs in about 400 species of flowering plants producing geneticallyuniform progeny. During apomixis, meiosis is bypassed and embryos develop by parthenogenesis. However, the endospermcould form either autonomously without fertilization or sexually, depending on the plant species. Most probably, aheterochronic expression of sexually expressed genes is one of the reason that causes apomixis. A better understanding ofthe genetic components regulating apomixis is important for developmental and evolutionary studies and also for engineering apomixis traits into crop plants that may realize a possibility to propagate hybrid vigor in a range of subsequentgenerations.

Forage characters of different Paspalum species in Rio Grande do Sul: a meta-analysis / Caracteres forrageiros de diferentes espécies do gênero Paspalum no Rio Grande do Sul: uma meta-análise

ABSTRACT: The objective of this study was to evaluate, through meta-analysis, the forage characteristics of various species of the genus Paspalum and to use them to select the best ecotypes that can be used in artificial hybridization as parents and hybrids for pasture production and natural pasture recovery systems. Data were obtained from studies conducted by the Department of Forage Plants and Agrometeorology of the Universidade Federal do Rio Grande do Sul. Database comprised tests conducted with ecotypes and/or hybrids of Paspalum spp. in plots for evaluating total dry mass production, leaf dry mass production, and stem dry mass production by means of cuts. Total dry mass production, which included leaves and stems, differed between the ecotypes and hybrids. Hybrid H12 was the most divergent of all evaluated accessions. The greatest genetic divergence occurred due to dry mass production. Hybrids showed high total dry mass production, comprised mainly of leaves. Hybrid H12 and the accession of Paspalum lepton 28E were identified as the most dissimilar based on the generalized Mahalanobis distance using Tocher’s method. Total dry mass production is the characteristic that most contributed to the detection of genetic variability.

RESUMO: O objetivo deste trabalho foi avaliar, por meio de meta-análise, a variabilidade dos caracteres forrageiros de espécies do gênero Paspalum e utilizá-los para selecionar os melhores ecótipos para serem utilizados em hibridações artificiais como genitores e híbridos para serem empregados em sistemas de produção a pasto e recuperação de pastagens naturais. Os dados foram obtidos a partir ensaios, do Departamento de Plantas Forrageiras e Agrometeorologia da Universidade Federal do Rio Grande do Sul. A base de dados foi composta por ensaios conduzidos com ecótipos e/ou híbridos do gênero Paspalum, em parcelas, avaliando por meio de cortes a produção de massa seca total, de folhas e colmos. Houve diferença entre ecótipos/híbridos para produção de matéria seca total, de folhas e colmos. O híbrido H12 foi o mais divergente dos acessos avaliados. A maior divergência genética ocorreu devido à produção de massa seca. Os híbridos apresentam elevada produção de massa seca total, sendo esta composta principalmente por folhas. O método de Tocher utilizando a distância generalizada de Mahalanobis identifica o híbrido H12 e o acesso de Paspalum lepton 28E como os mais dissimilares. A produção de massa seca total é o caractere que mais contribui para a detecção da variabilidade genética.

Forage performance of Paspalum hybrids from an interspecific cross / Desempenho forrageiro de híbridos de Paspalum obtidos por meio de cruzamentos interespecíficos

ABSTRACT: The goal of this study was to assess the variability of Paspalum plicatulum x P. guenoarum interspecific hybrids in terms of agronomic performance and their tolerance to cold conditions, as well as to estimate the correlation of different phenotypic characters associated with forage production. Twenty hybrids plants were used, besides one access P. guenoarum , one of P. plicatulum and the cultivar 'Pensacola' ( P. notatum ). Assessments were performed on individual plants, in a completely randomized design with five repetitions. Paspalum genotypes presented variability in total dry matter, leaf dry mass, leaf: stem ratio, and tolerance to cold. We suggest selection of the hybrids 08Q01 and 08Q44 for new stages within a forage improvement program. Estimates of phenotypic correlation may aid in the selection of genotypes with better agronomic traits.

RESUMO: O objetivo deste estudo foi avaliar a variabilidade de híbridos interespecíficos de Paspalum plicatulum x P. guenoarum por meio do desempenho agronômico e tolerância ao frio, bem como estimar as correlações fenotípicas de diferentes caracteres ligados à produção de forragem. Foram utilizados 20 híbridos, um acesso de P. guenoarum , um de P. plicatulum e a cultivar 'Pensacola' ( P. notatum ). As avaliações foram realizadas em plantas individuais, em delineamento completamente casualizado, com cinco repetições. Os genótipos de Paspalum apresentam variabilidade para massa seca total, massa seca de folhas e relação folha: colmo e na tolerância ao frio. É possível selecionar os híbridos 08Q01 e 08Q44 para novas etapas dentro de um programa de melhoramento de forrageiras. As estimativas de correlações fenotípicas podem auxiliar na seleção de genótipos com caracteres agronômicos superiores.

Establishment of a Simple Plant Regeneration System Using Callus from Apomictic and Sexual Seeds of Guinea Grass (Panicum maximum).

Aims: For analysis of apomixis genes, as the first step, an efficient and simple plant regeneration system has been established using callus from apomictic and sexual seeds of guinea grass (Panicum maximum). Study Design: The best basic medium for callus formation from matured seeds of guinea grass was selected, and then, the best combinations of growth regulators on different media were selected for plant regeneration by indirect organogenesis. Methodology: Guinea grass accessions of sexual and apomict of seeds were used for culture. Seeds sterilized were cultured in Murashige and Skoog [10] medium (MS) and in that proposed by Chu et al. [12] (N6D) for callus formation. The best medium and, the effects of L-proline and growth regulators’ type and concentrations on callus formation and plant regeneration in 3 accessions were examined, respectively. After the plantlets rooting in MS hormone-free medium, the complete plants were planted in pots for hardening. Results: N6D medium has given better rates for callus formation, that is, 97.1% in sexual N68/96- 8-o-5, and 91.7% and 84.6% in apomict N68/96-8-o-11 and ‘Natsukaze’, respectively. MS medium with 0.2 mg/l of Kinetin has given the best rate or plant regeneration among the used 4 kinds of the media. For each material, the best results were obtained on MS with 0.2 mg/l of Kinetin for N68/96- 8-o-5, and MS with 2.0 mg/l of L-proline and 0.2 mg/l of Kinetin for ‘Natsukaze’. After hardening of the regenerated plants in soil, 100% of surviving rates were obtained, and showed normal growth comparing with the plants-derived from seeds. Conclusion: We have established, as the first case, an efficient and simple plant regeneration system by using callus from apomictic and sexual seeds of guinea grass for analysis of apomixis genes, consisting of analysis of the best media, L-proline usages and phytohormone combinations for callus formation, plant regeneration and hardening in different stages, respectively.

Establishment of Efficient System for Isolation and Manipulation of Single Protoplasts Containing Aposporous Embryo Sac Initial Cell in Guinea Grass (Panicum maximum).

Aims: In order to do the molecular analysis of mechanism of aposporous embryo sac initial cell (AIC) appearance, as the first step, we attempted to establish the system of isolating and manipulating single cells containing AIC using different methods in guinea grass (Panicum maximum). Study Design: At first, single protoplasts were isolated from the ovaries staged in different developmental stages using different pre-treatments and different concentrations of enzyme solutions on ovaries; Based on it, the single protoplasts containing AIC were manipulated with ultra particle electronic syringe picopipet (UPESP) machine set onto microscope. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2012 and December 2013. Methodology: The ovary of facultatively apomictic guinea grass (Panicum maximum) was used as materials in this study for single protoplast isolation. The ovaries were classified and collected from young buds and flowers staged in different developmental stages, according to the colors of stigma and the ovary length. And then, the ovaries were pre-treated with needle into different shapes, and treated with different kinds of enzyme solution with different concentrations of mannitol to increase efficiency of protoplast isolation. In final, the single protoplasts containing AIC isolated from different stages of ovaries were manipulated by handle control. Results: 1) The ovaries in different stages of before AIC appearance, AIC appearance, and AIC-derived embryo sac formation were collected successfully and respectively, indicating that the stigma colors and length of ovaries are proportionate to stages of ovary mature. And single protoplasts containing AIC were isolated from the ovaries staged in white to yellow color. 2) The ovaries be flooded in enzyme solution, were pre-treated with needle in 4 types, that is, (1) Cut in micropylar end; (2) Cut in chalazal end; (3) Cut in middle part; (4) after 1hr of (3) treatment, cut in micropylar end. As a result, the efficiency of protoplast isolation of (3) and (4) was 1-2 hrs shorter than that of (1) and (2). 12%, 11%, 10% and 9% were proper enzyme concentrations for obtaining perfect shingle protoplasts from the ovaries with white, yellow, peach and purple colors, respectively. 3) The single protoplasts containing AIC were collected and manipulated with UPESP in the performance of controlled aspiration and spit. Conclusion: In this study, in order to do molecular analysis of the mechanism of AIC appearance, we focus on that, the key points were to isolate AIC single protoplasts from apomictic guinea grass using different methods, and then to establish the method of controlling a single protoplast using UPESP machine. These results obtained in this study will be a useful tool for molecular analysis of AIC, and provide important information for clarification of apomixis reproductive mode.

Establishment of Agrobacterium-Mediated Transformation System in Sweet Potato (Ipomoea batatas) by Culture of Leaf Segments for Functional Analysis of ASG-1, an Apomixis- Specific Gene.

Aims: In order to do the functional analysis of apomixis-specific gene (ASG-1), which was isolated from apomictic guineagrass, the sweet potato was used to establish an Agrobacterium-mediated transformation system. Study Design: At first, plant regeneration was achieved from the culture of leaf segments of sweet potato. Based on it, a binary vector pSMA35H2-NG for transformation of ASG-1 was used for establishment of a suitable procedure for plant regeneration of transformants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2009 and December 2012. Methodology: The leaf segments were used for somatic embryogenesis and plantlets regeneration. For the preliminary transformation, a GUS gene set in pSMA35H2-NG was introduced into the Agrobacterium strain GV3101/PMP9, and the Agrobacterium was used to infect the callus derived from leaf segments of sweet potato “Miyazakibeni” and the callus derived from seeds of rice “Nipponbare”. For the plasmid construction, the GUS was replaced by ASG-1, named as pSMA35H2/ASG1. The resultant plasmid was mobilized into Agrobacterium strain GV3101/PMP9 for transformation. For detection of ASG-1, DNAs of the transgenic plantlets were used for PCR, using the primers designed according to ASG-1 and hygromycin, respectively. Results: 1) When the leaf segments were sterilized with sodium hypochlorite solution of 0.3% and 0.4% for 15 min, 100% of surviving rates was achieved. And the segments cultured on Murashige and Skoog (1962) gave 100% of callus formation rates. 2) When the calli were placed onto Komamine and Nomura (1998) medium for differentiation, somatic embryogenesis was obtained with white color and grain-like tissue, and plantlets with multiple shoot-like tissues were obtained from the somatic embryo. 3) For the preliminary transformation, the calli showed GUS blue spots gradually on the surface. 4) When the pSMA35H2/ASG1 was used to the transformation of the embryogenic calli, the plantlets were developed through multiple shoots. 5) The specific bands of ASG-1 and hygromycin were observed from the PCR products of the plantlets’ DNAs, respectively. Conclusion: Overall the above results, the procedure using the binary vector pSMA35H2/ASG1 containing ASG-1 revealed, as the first case, that Agrobacteriummediated transformation system in sweet potato was established using the culture of leaf segments in this study.

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.

The synthesis of a new cassava-derived species, Manihot vieiri Nassar

A new species was synthesized artificially by chromosome doubling in a hybrid. The ensuing polyploid type exhibits an apomictic nature and maintains its morphological characteristics in the progeny. It showed a frequency of multiembryonic sacs of 29% in the ovules examined, whereas sacs were absent in the diploid type.

Molecular analysis of apomixis in cassava

Cassava is the main staple for more than 800 million people in the tropics. It is propagated vegetatively by stem cuttings, which maintains superior genotypes but favors disease accumulation and spread. In this report, we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixes using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environmental effect.

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.

Breeding cassava for apomixis

Apomixis genes have been successfully transferred to cassava (Manihot esculenta) by hybridizing it with the wild species, M. glaziovii. The interspecific hybrid of cassava and M. glaziovii was exposed to open pollination during three subsequent generations. Seven sibs and the maternal progenitor of the fourth generation were genotyped using five microsatellite loci previously developed for cassava. All sibs were identical with each other and with their maternal progenitor. Sibs from M. glaziovii proved to be identical when examined by the same microsatellite loci. This evidence leads to the conclusion that apomixis does occur in wild-cassava relatives and apparently has played an important role in Manihot speciation. This is the first report of nearly 100% apomixis.