Quality characteristics of backcross hybrids between Trifolium repens and Trifolium ambiguum

Two generations of novel backcross hybrids between white clover (Trifolium repens L.) and the related species Trifolium ambiguum,with white clover as the recurrent parent, were analysed for forage quality characteristics. Water soluble carbohydrate (WSC) levels, nitrogen concentrations (%N), and dry matter digestibility (DMD) were recorded for the hybrids and parental species grown in deep soil bins with and without a companion grass. Half the experimental material was subjected to moisture stress by being left unwatered through the four-week period when measurements were made. Four such four -week cycles of drought were imposed. The first and second generation backcrosses had the highest values for WSC, but were lowest for N%. There was no difference between the different legume lines (parents and backcrosses) for DMD. No effects of either moisture stress or presence of a grass companion on quality traits were observed. The WSC and %N of the grass component differed significantly depending on which legume line it was grown with. The importance of these results for the potential use of these hybrids in agriculture is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rhizobium specificity of Trifolium ambiguum M. Bieb × T. repens L. hybrid clovers

Growth of full-sib families of an F₁ interspecific hybrid between Trifolium ambiguum M.Bieb and T. repens L, and two generations of backcross hybrids (BC₁F₂ and BC₂F₁) with T. repens as the recurrent parent, were compared to their parental species. Plants were grown in a N-free medium and inoculated with Rhizobium leguminosarum biovar trifolii rhizobia effective on T. ambiguum or T. repens. Hybridisation produced progeny that nodulated with rhizobia from either T. ambiguum or T. repens, but plant growth varied. Mean weights of T. repens and hybrids, particularly F₁ and BC₁F₂, were higher when inoculated with a mixture of strains isolated from field grown T. repens than with the New Zealand inoculant strain for T. repens. When inoculated with the mix of rhizobia from T. repens, mean weights were 242, 189, 132, 125, and 100 mg/plant for T. repens, BC₂F₁, BC₁F₂, F₁ and T. ambiguum, respectively. However, although the mean weight of BC₂F₁ lines was significantly less than T. repens, there was considerable variation in individual full-sib families indicating the potential to select within BC₂F₁ hybrids for high plant growth/symbiotic nitrogen fixation. The weight of T. ambiguumplants inoculated with the New Zealand inoculant strain for hexaploid T. ambiguum was similar to T. repens inoculated with the mix of rhizobia from T. repens (253 and 242 mg/plant, respectively). Mean fresh weights of F₁ hybrid plants were similar when inoculated with rhizobia for T. ambiguum or T. repens (125 and 130 mg/plant, respectively). However, weight of T. repens, BC₁F₂ and BC₂F₁ hybrids inoculated with rhizobia for T. ambiguum were all less than 90 mg/plant. There was a significant relationship between plant fresh weight and ethylene production. The results indicate that measuring weights of inoculated plants growing in N-free media is a rapid initial method of screening a range of plant germplasm for plant growth/symbiotic nitrogen fixation rates. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen Fixation in Hybrids of White Clover (Trifolium repens L.) and Caucasian Clover (Trifolium ambiguum M. Bieb)

Hybrids between white clover (Trifolium repens L.) and Caucasian clover (or Kura clover, Trifolium ambiguum M. Bieb) are a potential route for the improvement of drought tolerance and persistence in white clover. However, to be agronomically viable they must show no significant reduction in their potential for nitrogen fixation relative to white clover. A comparative study of growth rate and nitrogen fixation was carried out in flowing solution culture without a supply of mineral nitrogen to the plants. The two parental species and two generations of backcross hybrids, with white clover as the recurrent parent, were assessed. The growth rate and N content of T. ambiguum were significantly lower than those of the other lines. However, dry matter production, nodule biomass per plant and rates of fixation were similar in second‐generation backcross plants and white clover. The results suggest that the agronomic potential of this novel germplasm is not compromised by limitations with respect to nitrogen fixation.     

Introgression as a Route to Improved Drought Tolerance in White Clover (Trifolium repens L.)

Backcross hybrids between the important forage legume white clover Trifolium repens , which is stoloniferous, and the related rhizomatous species T. ambiguum have been produced using T. repens as the recurrent parent. The drought tolerance of parents and two generations of backcross plants, possessing both stolons and rhizomes, was studied in deep soil bins in a glasshouse both as monocultures and in mixtures with perennial ryegrass. Soil moisture content, leaf relative water content, and leaf water potential were measured on plants subjected to drought cycles of 4 weeks duration and those watered normally and maintained at field capacity. Six cycles of drought, carried out over 2 years, showed that T. ambiguum and the first and second generation backcross hybrids maintained a greater leaf relative water content and higher leaf water potential than T. repens at comparable levels of soil moisture. The dry matter yield of T. repens was significantly reduced over each drought cycle compared to the watered treatments, while the yield of T. ambiguum increased over the 4-week cycle in both treatments. The yield of the backcross generations was intermediate between those of the two parental species. The implications of these results for white clover germplasm improvement programmes are discussed.     

Evidence of functional unreduced gametes in Trifolium repens L.

The current research reports the first evidence of functional 2n gametes in megaspores of Trifolium repens L. Reciprocal backcrossing of a colchicine doubled hexaploid (2n = 6x = 48) T. repens × T. nigrescens (6x H-6909-5) F₁ hybrid to T. repens produced four seeds from 1700 pollinations. The backcross involving 6x H-6909-5 as the female parent and T. repens as the male resulted in only one seed which was, as expected, a pentaploid (2n = 5x = 40) presumably carrying four genomes of T. repens and one genome of T. nigrescens. On the other hand, those backcrosses which utilised T. repens as the female parent and 6x H-6909-5 as the male parent, resulted in 3 viable seeds. These were unexpectedly all heptaploid (2n = 7x = 56) with presumably six genomes of T. repens and one genome of T. nigrescens. The occurrence of heptaploids from a T. repens × 6x H-6909-5 (4x–6x) cross can only be explained by the union of n (= 3x = 24) pollen from 6x H-6909-5 with 2n (= 4x = 32) eggs from T. repens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Self-compatibility and heterosis in white clover (Trifolium repens L.)

Inbred lines of white clover (Trifolium repens L.) have been produced utilising the rare self-fertility (Sf) allele. Twenty-two lines of four distinct groups have been maintained through five generations of selfing by single seed descent. Fourteen lines were used in crosses to produce F¹s. Both parents and hybrids were analysed for a range of morphological characters. Positive heterosis for dry matter production was observed in half the hybrids. No other trait showed significant heterosis. The degree of heterosis appears to be related to the extent of variation in morphological characters between the parental lines, some combinations of lines yielding heterotic hybrids in all cases, others in none. The majority of the F¹ hybrids are superior in terms of dry matter production to eight control varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of divergent selection on correlations between morphological traits in white clover (Trifolium repens L.)

The effects of two generations of divergent selection for stolon diameter and stolon amount on the pattern of phenotypic correlations between important agronomic traits in white clover (Trifolium repens L.) were studied. The effects of selection were: the proportions of the variances of traits under direct selection were considerably lower within selection lines than overall and this was associated with a loss of correlation between traits. Unselected traits showed higher proportions of variances remaining within the selection lines and shifts from non-significant to significant correlations from the plants overall to selection lines. The significance of these results and their relevance to strategies of germplasm improvement is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparing mating designs to restore seed production of interspecific hybrids between Trifolium repens (white clover) and Trifolium uniflorum

Interspecific hybrids between Trifolium uniflorum and cultivated white clover (Trifolium repens) have highly useful characteristics for temperate pastoral systems derived from both parent species. However, the early hybrids (F₁ and BC₁) also have unacceptably poor seed production for commerce. This study analysed the basis for the poor seed production and investigated breeding strategies for overcoming the problem. The BC₁F₁ generation produced lower‐than‐expected numbers of heads per plant and seeds per floret. Backcrossing of selected hybrids to white clover corrected these deficiencies and created new variation. Seed numbers were also returned to near target levels by recurrent selection within the BC₁ generation. Thus, it was possible to retain a theoretical average of 25% of T. uniflorum genome and still achieve high seed production per plant. The BC₁F₂ and BC₂F₁ generations produced high seed numbers per plant, along with reasonable variation. Both of these second‐generation hybrid forms have high reproductive potential and should be the focus for the selection of the desired combinations of agronomic and seed production traits.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

Relationships between genetic distance measured by RAPD-PCR and heterosis in inbred lines of white clover (Trifolium repens L.)

The RAPD-PCR technique has been used to generate DNA amplification profiles from a set of advanced generation inbred lines of white clover ( Trifolium repens L.). The genetic distances between lines have been estimated and have been compared with the extent of positive heterosis for dry matter yield in hybrid crosses. The analysis clearly separated the four major groups (H, J, R and S), with little within group variation. Heterosis, previously shown to be positively correlated with the extent of phenotypic variation between parental lines, was greatest between lines from groups showing the least genetic distance. The lack of correlation between heterosis and genetic divergence is discussed in the context of results from other systems. Additionally, the data allow a comparison of the extent of phenotypic variation for a trait under selective pressure with the degree of variation at the DNA level detected with the RAPD technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Development of breeding populations from interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative of T. repens (white clover) with adaptation to dry, saline coastal habitats. Transfer of drought and salt‐tolerant adaptive traits to white clover could be potentially valuable if interspecific hybridization can be achieved efficiently and leads to fertile hybrid populations. To achieve hybridization, 4x plants of T. occidentale were generated. Efficient techniques for generation of 4x plants and their identification using dry pollen shape are described. Interspecific 4x F₁ plants were achieved without embryo rescue. F₂ populations and first backcross hybrids to white clover were also efficiently achieved. Although male and female fertility were lower than in white clover, they were adequate to produce large amounts of seed from small numbers of inflorescences. Thus, early generation interspecific hybrid prebreeding populations can be readily developed, opening the way for transfer of traits from T. occidentale to white clover.     

Characterization of novel inbred lines of white clover (Trifolium repens L.). I. Dynamics of plant growth and nodule development in flowing solution culture

Growth, dry matter partitioning between shoots and roots, and extent of nodulation were characterised in four novel self-fertile highly inbred lines (referred to as A, B, C and D) of white clover (Trifolium repens L.) thought to differ fundamentally in their nitrogen relations and therefore regarded as candidate material for genetic mapping. Plants were inoculated with a mixture of three strains of Rhizobium and grown in flowing nutrient solutions without N for 18 d. Half the plants were then supplied with 20 μM NO₃ ^- during a 36 d treatment period, the remaining plants acting as ‘controls’ solely dependent upon N₂ fixation for acquisition of N. Total dry matter production and shoot:root ratios were similar in all lines supplied with NO₃ ^- and in control plants of lines B and C. Growth was severely reduced in control plants of line D and to a lesser extent in control line A. These effects were attributed to a failure to develop effective N₂ fixation capacity after apparently normal infection. Mean nodule size, nodule numbers and nodule d.w. per plant were extremely low in line D compared with the other lines, irrespective of whether NO₃ ^- was supplied. Lines A, B and C differed in the severity with which NO₃ ^- decreased mean d.w. per nodule and total nodule d.w. per plant relative to corresponding control plants, with line C being least sensitive. Nitrate also decreased the total number of nodules per plant relative to control plants after day 14 in all lines except C, and most severely in line B. This revised version was published online in August 2006 with corrections to the Cover Date.     

影响白三叶草高频率植株再生因素的研究

以白三叶草子叶和下胚轴为外植体,进行直接再生系统中不同基本培养基成分和激素浓度的研究发现：白三叶草下胚轴顶端细胞的分化能力最强,分化率在65％以上,子叶次之,下胚轴其他部位几乎不分化。高维生素和微量元素可有效提高子叶的再生频率15％,而对下胚轴顶端细胞的分化影响不大。在6-BA 3mg／L NAA0．08ms／L的浓度下,下胚轴上段的再生频率最高,达80％以上。     

铜尾矿对白车轴草种子萌发和幼苗生长的影响

四川彭州铜尾矿库尾沙中含有铜、镍、铅、锌、镉等重金属元素，除镍和锌元素外，其它几种重金属元素含量均已超过国家标准，对土壤和水源造成污染。该尾矿沙能够延迟白车轴草（Trifolium repens L.）种子的萌发，使胚根长度和小苗重量降低，但对最终发芽率无影响。盆栽改良试验表明，铜尾矿沙能够降低白车轴草的根系活力、叶绿素含量和生物量，增加无污染土壤的比例可以使其伤害降低。     

Evaluation of the effects of resistance to stem nematode (Ditylenchus dipsaci) in white clover (Trifolium repens L.) under sheep grazing and cutting

Two field experiments were carried out to analyze the performance of white clover varieties differing in their resistance to stem nematode. Varieties were compared under grazing and cutting regimes in mixed swards with or without the addition of nematode and dry matter yields of white clover and perennial ryegrass recorded over a 3-year period. The resistant variety did not show a yield advantage in the absence of nematode but did so in the presence of nematode in the first year and for several of the sampling dates in the second year. White clover yields under grazing were significantly less than under cutting in both experiments. The hypothesis that the additional stress of grazing would increase the benefits of resistance relative to a cutting management was not supported.     

低温处理对白三叶叶绿素、可溶性糖和丙二醛等的影响

为探索低温胁迫对白三叶的叶绿素、可溶性糖和丙二醛等生理指标的影响,为白三叶的抗寒性育种和资源的合理利用提供基础数据,试验以新品系08-2和海法白三叶(Trifolium repens L.‘haifa’)为材料,依次设置10℃、5℃、0℃、-5℃、-10℃、-15℃等6个处理,研究低温下叶绿素、可溶性糖和丙二醛等的变化,对材料的抗寒性进行评价。结果表明,变温对白三叶的叶绿素、可溶性糖和丙二醛等的影响较大。随着温度的降低,白三叶的叶绿素含量总体呈现降低的趋势,在较低(≤0℃)温度处理下,08-2的叶绿素含量降低速度小于海法的;08-2的可溶性糖含量在0℃时达到最高,海法的在5℃达到最高;丙二醛含量和电导率逐渐升高,但08-2变化幅度低于海法。综合分析表明,新品系08-2耐寒性优于海法白三叶。     

Characterization of novel inbred lines of white clover (Trifolium repens L.). II. Variation in N2 fixation, NO3- uptake and their interactions

N₂ fixation, nitrate uptake, and tissue N levels werecompared in four novel self-fertile inbred lines (referred to as A, B, C andD) of white clover (Trifolium repens L.) believed to differ markedly intheir nitrogen relations. Plants were inoculated with a mixture of Rhizobiumstrains and grown in flowing nutrient solutions without mineral N for 18 dprior to supplying half the plants with 20 μM over a 36 d treatment period. Net uptakeof was measured automatically on a dailybasis. The remaining plants constituted ‘controls’ solely dependent uponN₂ fixation for acquisition of N. Total uptake of over 36 days was 27.4 (A), 23.8 (B), 13.8(C) and 30.1 (D) mmol N/plant, compared with N₂ fixation of7.2 (A), 16.9 (B), 26.2 (C) and 6.2 (D) mmol N/plant by the -fed lines. Total N₂ fixationby corresponding ‘control’ plants was 15.6 (A), 35.6 (B), 35.3 (C) and 2.2(D) mmol N/plant. Line D plants exhibited a 3:1 segregation with respect toeither ‘nil’ or ‘partially impaired’ N₂ fixation; thecorresponding ratio for line A was 1:3. Line C plants had a ‘low uptake’ phenotype characterised by specific uptake rates approximately half those ofthe other lines, higher N₂ fixation in the presence of , and low leaf + petiole concentrations of , although concentrations of in stolon and root fractions were similarin all lines. These inbred lines provide a tool for (1) the analysis of thegenetic and physiological basis of variation in the efficacy with which uptake down-regulates N₂fixation, and (2) testing ecological hypotheses for temporal and spatialvariation in clover/grass coexistence in pastures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen Fixation by Hybrids of White Clover (Trifolium repens L.) and Trifolium nigrescens

Development of hybrids between white clover ( Trifolium repens L.) and Trifolium nigrescens provides a novel route for genetically improving the reproductive capacity of white clover, provided the hybrids are agronomically viable, particularly with respect to N₂ fixation. A comparative study of growth and rates of N₂ fixation over 21 days was conducted with the parental species, F ₁ hybrids and backcross hybrids, in flowing solution culture, without a supply of mineral N to the plants. T. nigrescens was unable to fix N₂ in association with the strains of Rhizobium leguminosarum biovar. trifolii selected for inoculation. Rates of N₂ fixation per plant increased in the order T. nigrescens < F ₁ hybrid < T. repens < backcross 1. Specific rates of N₂ fixation (days 0–21) increased in the order T. nigrescens < F ₁ hybrid < backcross 1 <  T. repens . Dry matter production and nodule biomass per plant increased at a higher rate in backcross 1 hybrids than in T. repens. The results suggest that the potential for N₂ fixation by backcross 1 hybrids is at least as great as that by T. repens .     

Bulked segregant AFLP analysis to identify markers for the introduction of the rhizomatous habit from Trifolium ambiguum into T. repens (white clover)

Bulked segregant analysis (BSA) using the technique of amplified fragment length polymorphism (AFLP^TM) was carried out on backcross hybrids between the important pasture species white clover and its relative Trifolium ambiguum. The introgression of the rhizomatous trait fromTrifolium ambiguum, previously shown to be associated with enhanced drought tolerance but which takes eighteen months to manifest itself, was analysed in a series of third generation backcross families with white clover as the recurrent parent. Eleven families differed in the proportions of plants classified according to rhizome content. Three of these families were selected for BSA-AFLP based on the creation of separate DNA pools from plants with large amounts of rhizomes or no rhizomes. Following an initial screen with119 primer combinations, 53 combinations were selected on the basis of the number of clear, reproducible polymorphisms they produced. Polymorphic bands were found that distinguished between rhizomatous and non-rhizomatous bulks in the three families. However, only one polymorphic band was found that unambiguously reflected the presence of the rhizomatous trait in individual plants in these families. This polymorphic band was also shown to be present in the BC2 parent, the BC1 and the original T. ambiguum parent. Given the aneuploid nature of the BC2 plants this band would appear to be a chromosome-specific marker tightly linked to the locus controlling rhizome development. The identification of a consistent marker for the rhizomatous trait opens up the possibility of applying marker assisted introgression and thereby facilitating the rapid incorporation of this trait into diverse germplasm. This revised version was published online in July 2006 with corrections to the Cover Date.     

白三叶种质资源主要数量性状的变异与相关性研究

为了探讨白三叶(Trifolium repens L.)种质资源农艺性状变异和相关性,揭示各种质材料的特征特性和种质资源群间的遗传关系,本研究对78份白三叶种质的12个主要数量性状的变异和相关性进行了分析。结果表明：这些种质材料存在着丰富的遗传多样性,各个数量性状间均呈正相关,植株高度与小叶长度、小叶宽度、小叶大小、叶柄长度呈显著正相关;小叶长度与小叶宽度、小叶大小呈极显著正相关,与叶柄长度、叶柄直径呈显著正相关;小叶宽度与小叶大小呈极显著正相关,与叶柄长度、花柄直径呈显著正相关;小叶大小与叶柄长度、叶柄直径、花柄直径呈显著正相关。该批种质材料能为各类白三叶育种和遗传研究提供较为丰富的亲本材料。