Pattern analysis of the diversity of morphological plant attributes and herbage yield in a world collection of white clover (Trifolium repens L.) germplasm characterised in a summer moisture stress environment of Australia

Information on the variation available for different plant attributes has enabled germplasm collections to be effectively utilised in plant breeding. A world sourced collection of white clover germplasm has been developed at the White Clover Resource Centre at Glen Innes, New South Wales. This collection of 439 accessions was characterised under field conditions as a preliminary study of the genotypic variation for morphological attributes; stolon density, stolon branching, number of nodes. number of rooted nodes, stolon thickness, internode length, leaf length, plant height and plant spread, together with seasonal herbage yield. Characterisation was conducted on different batches of germplasm (subsets of accessions taken from the complete collection) over a period of five years. Inclusion of two check cultivars, Haifa and Huia, in each batch enabled adjustment of the characterisation data for year effects and attribute-by-year interaction effects. The component of variance for seasonal herbage yield among batches was large relative to that for accessions. Accession-by-experiment and accession-by-season interactions for herbage yield were not detected. Accession mean repeatability for herbage yield across seasons was intermediate (0.453). The components of genotypic variance among accessions for all attributes, except plant height, were larger than their respective standard errors. The estimates of accession mean repeatability for the attributes ranged from low (0.277 for plant height) to intermediate (0.544 for internode length). Multivariate techniques of clustering and ordination were used to investigate the diversity present among the accessions in the collection. Both cluster analysis and principal component analysis suggested that seven groups of accessions existed. It was also proposed from the pattern analysis results that accessions from a group characterised by large leaves, tall plants and thick stolons could be crossed with accessions from a group that had above average stolon density and stolon branching. This material could produce breeding populations to be used in recurrent selection for the development of white clover cultivars for dryland summer moisture stress environments in Australia. The germplasm collection was also found to be deficient in genotypes with high stolon density, high number of branches high number of rooted nodes and large leaves. This warrants addition of new germplasm accessions possessing these characteristics to the present germplasm collection.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

Comparative productivity of irrigated short-term ryegrass (Lolium multiflorum) pasture receiving nitrogen, grown alone or in a mixture with white (Trifolium repens) and Persian (T. resupinatum) clovers

Dairy farms in subtropical Australia use irrigated, annually sown short-term ryegrass (Lolium multiflorum) or mixtures of short-term ryegrass and white (Trifolium repens) and Persian (shaftal) (T. resupinatum) clover during the winter-spring period in all-year-round milk production systems. A series of small plot cutting experiments was conducted in 3 dairying regions (tropical upland, north Queensland, and subtropical southeast Queensland and northern New South Wales) to determine the most effective rate and frequency of application of nitrogen (N) fertiliser. The experiments were not grazed, nor was harvested material returned to the plots, after sampling. Rates up to 100 kg N/ha.month (as urea or calcium ammonium nitrate) and up to 200 kg N/ha every 2 months (as urea) were applied to pure stands of ryegrass in 1991. In 1993 and 1994, urea, at rates up to 150 kg N/ha.month and to 200 kg N/ha every 2 months, was applied to pure stands of ryegrass; urea, at rates up to 50 kg N/ha.month, was also applied to ryegrass-clover mixtures. The results indicate that applications of 50-85 kg N/ha.month can be recommended for short-term ryegrass pastures throughout the subtropics and tropical uplands of eastern Australia, irrespective of soil type. At this rate, dry matter yields will reach about 90% of their potential, forage nitrogen concentration will be increased, there is minimal risk to stock from nitrate poisoning and there will be no substantial increase in soil N. The rate of N for ryegrass-clover pastures is slightly higher than for pure ryegrass but, at these rates, the clover component will be suppressed. However, increased ryegrass yields and higher forage nitrogen concentrations will compensate for the reduced clover component. At application rates up to 100 kg N/ha.month, build-up of NO3--N and NH4+-N in soil was generally restricted to the surface layers (0-20 cm) of the soil, but there was a substantial increase throughout the soil profile at 150 kg N/ha.month. The build-up of NO3--N and NH4+-N was greater and was found at lower rates on the lighter soil compared with heavy clays. Generally, most of the soil N was in the NO3--N form and most was in the top 20 cm.

Análisis comparativo del crecimiento y la partición de asimilatos de plantulas de Senecio madagascariensis, Crepis capillaris, Trifolium repens y Lolium perenne

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Análisis y evaluación de pérdidas en la cosecha de trébol blanco (Trifolium repens L.) de dos sistemas de recolección : el tradicional y recolección directa

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Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.

Protein precipitation behaviour of condensed tannins from Lotus pedunculatus and Trifolium repens with different mean degrees of polymerization

The precipitation of bovine serum albumin (BSA), lysozyme (LYS) and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited high purity profiles by 1D/2D NMR and purities >90% (determined by thiolysis). At pH 6.5, large CTs with a mean degree of polymerization (mDP) of ~18 exhibited similar protein precipitation behaviors and were significantly more effective than medium CTs (mDP ~9). Medium CTs exhibited similar capacities to precipitate ALF or BSA, but showed small but significant differences in their capacity to precipitate LYS. All CTs precipitated ALF more effectively than BSA or LYS. Aggregation of CT-protein complexes likely aided precipitation of ALF and BSA, but not LYS. This study, one of the first to use CTs of confirmed high purity, demonstrates that mDP of CTs influences protein precipitation efficacy.

Caracterização morfológica e molecular da coleção básica de trevo-branco (Trifolium repens L.)

O objetivo deste trabalhofoi avaliarcaracterísticas moleculares e morfológicasde 79 acessos pertencentes à coleção básica de trevo-branco obtida do Departamento de Agricultura dos Estados Unidos(USDA), a fim de caracterizar a variabilidade genética existente.

Efeito de sistemas de pastejo sobre o desempenho e o comportamento de suínos em crescimento e terminação, mantidos em pastagem de trevo-branco (Trifolium repens L.)

O presente trabalho foi conduzido com o objetivo de avaliar o efeito de diferentes sistemas de pastejo (contínuo, alternado e rotativo) sobre o desempenho e o comportamento de suínos, assim como, sobre a disponibilidade e a composição química do trevo-branco (Trifolium repens L.). Foram utilizados 48 suínos machos, castrados, com peso médio inicial de 27 kg, sendo doze mantidos em sistema confinado, os quais foram utilizados como tratamento testemunha. Os animais foram distribuídos em um delineamento de blocos casualizados, com três tratamentos e três repetições. Não foram observadas diferenças significativas (P>0,05) entre os sistemas de pastejo nas características de desempenho e de disponibilidade. No entanto, foram observadas diferenças significativas (P<0,05) da composição química da pastagem entre os sistemas de pastejo estudados. Verificou-se que os suínos em sistemas de pastejo contínuo, alternado e rotativo consumiram de 13% a 17% a menos de ração (P<0,05) e apresentaram menores ganhos de peso (P<0,05) e espessura de toucinho (P<0,05) do que os suínos criados em confinamento. Não foram observadas diferenças significativas (P>0,05) na conversão alimentar entre os suínos mantidos em confinamento e sobre pastagem. Deitar, beber água, ingerir ração, pastejar e caminhar foram as atividades que apresentaram maiores freqüências de ocorrência, independente dos sistemas de pastejo estudados. Houve efeito significativo dos sistemas de pastejo sobre a atividade de ingerir ração (P=0,0186) e interação de sistema x dia de observação para as atividades de deitar (P=0,0345) e pastejar (P=0,0678).

Avaliação do potencial de produção de sementes de acessos de trevo branco (Trifolium repens L)

A produção de sementes de qualidade é um dos pontos chave na difusão do trevo branco em nosso meio. Esta leguminosa é uma espécie de boa produção de sementes, entretanto, como as demais espécies de Trifolium, é conhecida por flutuações no rendimento de sementes. Com o objetivo de avaliar o potencial de produção de sementes de 27 acessos da coleção básica de trevo branco conduziu-se o presente trabalho na região da Depressão Central do RS, EEA/UFRGS, Eldorado do Sul, no período de outubro de 2003 a março de 2004. As variáveis estudadas através de amostragens semanais realizadas entre 06/11 e 10/03 foram: número de inflorescências por planta, número de flores por inflorescência, número de inflorescências maduras, número de legumes maduros por inflorescência, peso de 1000 sementes e rendimento de sementes por planta. Foram separados três grupos distintos quanto à produção de sementes. O grupo superior com o acesso 53 (Peru), outro intermediário com os acessos 2 (Israel), 3 (EUA), 7 (Etiópia), 13 (Espanha), 19 (Costa Rica), 20 (Uruguai), 28 (Austrália), 33 (Irlanda), 50 (África do Sul), 58 (Índia), 64 (Suíça), 68 (Jamaica) e 75 (Uruguai) e um grupo inferior com os acessos 15 (Nova Zelândia), 22 (Hungria), 23 (Bélgica), 29 (França), 31 (EUA), 38 (Irã), 54 (Nova Zelândia), 59 (Líbano), 79 (Nova Zelândia) e 80 (EUA). O número de inflorescências, número de inflorescências maduras e peso de 1000 sementes apresentaram correlação positiva e altamente significativa em relação ao rendimento de sementes. O acesso 20 apresentou superioridade no número de inflorescências por planta e número de inflorescências maduras por planta, no entanto, o acesso 53 foi o de maior potencial de produção de sementes, em função de sua superioridade no peso de 1000 sementes. Os acessos 27 (Canadá), 65 (Dinamarca) e 73 (Polônia) não produziram sementes nas condições locais.

Identification and expression of different dehydrin subclasses involved in the drought response of Trifolium repens

Reverse transcribed RNAs coding for YnKn, YnSKn, SKn, and KS dehydrin types in drought-stressed white clover (Trifolium repens) were identified and characterized. The nucleotide analyses revealed the complex nature of dehydrin-coding sequences, often featured with alternative start and stop codons within the open reading frames, which could be a prerequisite for high variability among the transcripts originating from a single gene. For some dehydrin sequences, the existence of natural antisense transcripts was predicted. The differential distribution of dehydrin homologues in roots and leaves from a single white clover stolon under normal and drought conditions was evaluated by semi-quantitative RT-PCR and immunoblots with antibodies against the conserved K-, Y- and S-segments. The data suggest that different dehydrin classes have distinct roles in the drought stress response and vegetative development, demonstrating some specific characteristic features. Substantial levels of YSK-type proteins with different molecular weights were immunodetected in the non-stressed developing leaves. The acidic SK2 and KS dehydrin transcripts exhibited some developmental gradient in leaves. A strong increase of YK transcripts was documented in the fully expanded leaves and roots of drought-stressed individuals. The immunodetected drought-induced signals imply that Y- and K-segment containing dehydrins could be the major inducible Late Embryogenesis Abundant class 2 proteins (LEA 2) that accumulate predominantly under drought.

Natural antisense transcripts of Trifolium repens dehydrins

The recently described complex nature of some dehydrin-coding sequences in Trifolium repens could explain the considerable variability among transcripts originating from a single gene.1 For some of the sequences the existence of natural antisense transcripts (NAT s), which could form sense-antisense (SAS) pairs, was predicted. The present study demonstrates that cis-natural antisense transcripts of 2 dehydrin types (YnKn and YnSKn) accumulate in white clover plants subjected to treatments with polyethylene glycol (PEG), abscisic acid (ABA), and high salt concentration. The isolated YnKn cis-NAT s mapped to sequence site enriched in alternative start codons. Some of the sense-antisense pairs exhibited inverse expression with differing profiles which depended on the applied stress. A natural antisense transcript coding for an ABC F family protein (a trans-NAT) which shares short sequence homology with YnSKn dehydrin was identified in plants subjected to salt stress. Forthcoming experiments will evaluate the impact of NAT s on transcript abundances, elucidating the role of transcriptional and post-transcriptional interferences in the regulation of dehydrin levels under various abiotic stresses.

Transgenerational effects of land use on offspring performance and growth in Trifolium repens

entral European grasslands vary widely in productivity and in mowing and grazing regimes. The resulting differences in competition and heterogeneity among grasslands might have direct effects on plants, but might also affect the growth and morphology of their offspring through maternal effects or adaptive evolution. To test for such transgenerational effects, we grew plants of the clonal herb Trifolium repens from seeds collected in 58 grassland sites differing in productivity and mowing and grazing intensities in different treatments: without competition, with homogeneous competition, and with heterogeneous competition. In the competition-free treatment, T. repens from more productive, less frequently mown, and less intensively grazed sites produced more vegetative offspring, but this was not the case in the other treatments. When grown among or in close proximity to competitors, T. repens plants did not show preferential growth towards open spaces (i.e., no horizontal foraging), but did show strong vertical foraging by petiole elongation. In the homogeneous competition treatment, petiole length increased with the productivity of the parental site, but this was not the case in the heterogeneous competition treatment. Moreover, petiole length increased with mowing frequency and grazing intensity of the parental site in all but the homogeneous competition treatment. In summary, although the expression of differences between plants from sites with different productivities and land-use intensities depended on the experimental treatment, our findings imply that there are transgenerational effects of land use on the morphology and performance of T. repens.

Caracterização isoenzimática de oito espécies do gênero Trifolium L., ocorrentes no Rio Grande do Sul

O presente estudo tem como objetivos: contribuir para o desenvolvimento de oito espécies do gênero Trifolium L., Trifolium incamatum L., Trifolium polymorphum Poir, Trifolium pratense L., Trifolium repens L., trifolium resupinatum L., Trifolium riograndense Burkart, Trifolium subterraneum L. e Trifolium vesiculosum Savi, através da caracterização da variabilidade isoenzimática presente em quatro sistemas enzimáticos, Fosfoglicoisomerase (PGI), Esterase (EST), Malato desidrogenase (MDH) e Superóxido dismutase (SOD) e fornecer subsídios para futuros trabalhos de melhoramento de plantas forrageiras.