Quantitative trait loci for yield and correlated traits under high and low soil nitrogen conditions in tropical maize

The first objective of this study was to map and characterize quantitative trait loci (QTL) for grain yield (GY) and for secondary traits under varying nitrogen (N) supply. To achieve this objective, a segregating F_2:3 population previously developed for QTL mapping under water-limited conditions was used. The population was evaluated in Mexico under low N conditions in the dry winter season and under low and high N conditions in the wet summer season. From eight QTLs identified for GY under low N conditions, two were also detected under high N conditions. Five QTLs were stable across the two low N environments and five co-localized with QTLs identified for the anthesis-silking interval (ASI) or for the number of ears per plant (ENO) under low N conditions. The percentage of the phenotypic variance expressed by all QTLs for ASI and ENO was quite different when evaluated under low N conditions during the dry winter (40% for ASI and 22% for ENO) and the wet summer seasons (22% for ASI and 46% for ENO). The results suggest optimizing different breeding strategies based on selection index depending on the growing season. Good QTL colocalization was observed for ASI (four QTLs) and ENO (three QTLs) when looking at QTL identified under low N and water-limited conditions in the same population. The results suggest that that both secondary traits can be used in breeding programs for simultaneous improvement of maize against low N and drought stresses.     

Two consensus quantitative trait loci clusters controlling anthesis–silking interval, ear setting and grain yield might be related with drought tolerance in maize

Unravelling the molecular basis of drought tolerance will provide novel opportunities for improving crop yield under water-limited conditions. The present study was conducted to identify quantitative trait loci (QTLs) controlling anthesis–silking interval (ASI), ear setting percentage (ESP) and grain yield (GY). The mapping population included 234 F₂ plants derived from the cross X178 (drought tolerant) × B73 (drought susceptible). The corresponding F_2:3 progenies, along with their parents, were evaluated for the above-mentioned traits under both well-watered and water-stressed field conditions in three different trials carried out in central and southern China. Interval mapping and composite interval mapping identified 45 and 65 QTLs for the investigated traits, respectively. Two QTL clusters influencing ASI and ESP on chromosomes 1 (bin 1.03) and 9 (bins 9.03–9.05) were identified in more than two environments, showing sizeable additive effects and contribution to phenotypic variance; these two QTL clusters influenced GY only in one environment. No significant interaction was detected between the two genomic regions. A comparative analysis of these two QTL clusters with the QTLs controlling maize drought tolerance previously described in three mapping populations confirmed and extended their relevance for marker-assisted breeding to improve maize production under water-limited conditions.     

Identification of quantitative trait loci controlling resistance to gray leaf spot disease in maize

Breeding maize for gray leaf spot (GLS) resistance has been hindered by the quantitative nature of the inheritance of GLS resistance and by the limitations of selection under less than optimumal disease pressure. In order to identify the quantitative trait loci (QTLs) controlling GLS resistance, a cross was made between B73 (susceptible) and Va14 (resistant) to generate a large F₂ population. Six GLS disease assessments were made throughout the disease season for over 1000 F₂ plants in 1989, and for 600 F₂-derived F₃ lines replicated in two blocks in 1990. RFLP analysis for78 marker loci representing all ten maize chromosomes was conducted in 239 F₂ individuals including those with the extreme GLS disease phenotypes. The GLS disease scores of the three field evaluations, each averaged over six ratings, were separately used for the interval mapping in order to determine the consistency of the QTL effects. The heavy GLS disease pressure, meticulous disease ratings, and large population size of this study afforded us the sensitivity for detecting QTL effects. QTLs located on three chromosomes (1, 4, and 8) had large effects on GLS resistance, each explaining 35.0–56.0%, 8.8–14.3%, and 7.7–11.0% of the variance, respectively. These three QTL effects were remarkably consistent across three disease evaluations over 2 years and two generations. Smaller QTL effects were also found on chromosomes 2 and 5, but the chromosome-5 effect might be a false positive because it was not repeatable even in the same location. The chromosome-1 QTLs had the largest effect or highest R² reported for any quantitative trait to-date. Except for the chromosome-4 gene, which was from the susceptible parent B73, the resistance alleles at all QTL were derived from Va14. The resistance QTLs on chromosomes 1 and 2 appear to have additive effects, but those on chromosomes 4 and 8 are dominant and recessive, respectively. Significant interaction between the QTLs on chromosomes 1 and 4 was detected in all three evaluations. Cumulatively, the four QTLs identified in this study explained 44, 60, and 68% of the variance in F₂, and in F₃ replications 1 and 2, respectively.     

Transpiration, and nitrogen uptake and flow in two maize (Zea mays L.) inbred lines as affected by nitrogen supply

BACKGROUND AND AIMS: The influence of two nitrogen (N) levels on growth, water relations, and N uptake and flow was investigated in two different inbred lines of maize (N-efficient Zi330 and N-inefficient Chen94-11) to analyse the differences in N uptake and cycling within a plant. METHODS: Xylem sap from different leaves of the inbred lines cultured in quartz sand was collected by application of pressure to the root system. Plant transpiration was measured on a daily basis by weighing five pots of each of the treatments. KEY RESULTS: N-efficient Zi330 had a higher relative growth rate and water-use efficiency at both high (4 mm) and low (0.08 mm) N levels. At a high N level, the amount of N taken up was similar for the two inbred lines; the amount of N transported in the xylem and retranslocated in the phloem was slight greater in Chen94-11 than in Zi330. At a low N level, however, the total amount of N taken up, transported in the xylem and retranslocated in the phloem of Zi330 was 2.2, 2.7 and 2.7 times more, respectively, than that of Chen94-11. Independent of inbred line and N level, the amounts of N transported in the xylem and cycled in the phloem were far more than that taken up by roots at the same time. Low N supply shifted NO(3)(-1) reduction towards the roots. The major nitrogenous compound in the xylem sap was NO(3)(-1), when plants grew at the high N level, while amino acid-N was predominant when plants grew at the low N level. CONCLUSIONS: The N-efficient maize inbred line Zi330 had a higher ability to take up N and cycle N within the plant than N-inefficient Chen94-11 when grown under N-deficiency.     

In an elite cross of maize a major quantitative trait locus controls one-fourth of the genetic variation for grain yield

Quantitative trait loci (QTLs) for grain yield, dry matter content and test weight were identified in an F₂ segregating population derived from a single cross between two elite maize lines (B73 and A7) and testcrossed to two genetically divergent in breds. Most of the QTLs inferred were consistent across locations, indicating that the expression of the genes influencing the traits under investigation was largely independent of the environment. By using two different tester lines we found that QTLs exhibited by one tester may not necessarily be detected with the second one. Only loci with larger effects were consistent across testers, suggesting that interaction with tester alleles may contribute to the identification of QTLs in a specific fashion. Analysis across both testers revealed four significant QTLs for grain yield that explained more than 35% of the phenotypic variation and showed an overall phenotypic effect of more than 2t/ha. The major QTL for grain yield, located in the proximity of the Nucleolus Organiser Region, accounted for 24.5% of the phenotypic variation for grain yield and showed an average effect of allele substitution of approximately 1 t/ha. Marker-assisted introgression of the superior A7 allele at this locus in the B73 genetic background will not differ from qualitative trait introgression and will eventually lead to new lines having superior testcross performance.     

Genetic diversity within and among maize populations: a comparison between isozyme and nuclear RFLP loci

 In order to compare the potential of enzyme and DNA markers to investigate genetic diversity within and among populations, ten maize populations were characterized for (1) 20 isozyme loci and (2) restriction fragment length polymorphism (RFLP) for 35 probe-enzyme combinations. Each population was represented by a sample of at least 30 individuals. The average number of alleles detected per locus was clearly higher for RFLPs (6.3) than for isozymes (2.4). Similarly, total diversity was higher for RFLPs (0.60) than for isozymes (0.23). This difference is consistent with observations on inbred-line collections and can be related to the fact that many variations at the DNA level do not change the amino-acid composition or the global charge of proteins. By contrast, the magnitude of population differentiation, relative to the total diversity, was similar for isozymes (23%) and RFLPs (22%). This suggests that the isozyme and RFLP loci considered in this study are subject to similar evolutionary forces, and that both are mostly neutral. However, RFLPs proved clearly superior to isozymes both to (1) identify the origin of a given individual and (2) reveal a relevant genetic structure among populations. The higher polymorphism observed for RFLP loci and the greater number of these loci contributed to the superior discriminative ability of the RFLP data. Received: 1 June 1997 / Accepted: 3 November 1997     

氮肥运筹对小麦产量、氮素利用效率和光能利用率的影响

连续2年在西南冬麦区的重庆、仁寿、广汉、西昌4个地点,开展3种施氮水平(每公顷纯氮0、120、180 kg,简写为N₀、N₁₂₀、N₁₈₀)和3种氮肥分配模式(N_A:底肥100%;N_B:底肥70%+苗肥30%;N_C:底肥60%+拔节肥40%)的田间试验,监测小麦花后冠层叶片SPAD值、群体光合速率(CAP)、光能利用等生理参数和籽粒产量,计算氮素利用效率、光能利用率等.结果表明: 随施氮水平增加,小麦上三叶SPAD值、CAP、光合有效辐射(PAR)截获率和产量均呈增加趋势,而氮肥农学利用效率、生产效率、吸收效率和利用效率呈降低趋势.氮肥后移的增效作用因施氮水平而异,SPAD于N₁₈₀增效明显,而CAP于N₁₂₀增效明显,不同氮肥管理模式的光能利用率因地点而异.氮肥后移能明显提高小麦氮肥农学效率、生产效率、吸收效率和氮素表观回收率,但氮肥利用效率则略有减少.氮肥后移效果N_C总体优于N_B处理.不同地点比较,广汉的SPAD值、CAP、PAR截获率、氮肥利用参数较高,其产量也相应最高;西昌的产量、SPAD值及氮素利用效率较高,但其光能利用率和CAP较低;重庆和仁寿的SPAD值、光能利用率及氮素利用效率均较低,其产量也最低.小麦生物产量与各地点的籽粒产量、CAP、SPAD值和PAR累积截获量均呈显著或极显著的正相关关系.表明不同生态区域增施氮肥都能促进小麦增产,氮肥后移可进一步优化产量结构、改善氮肥和光能利用效率,但存在年份和地点差异,各地需要制定有针对性的氮肥管理模式.     

Identification of quantitative trait loci controlling sucrose content in soybean (Glycine max)

Sucrose is a primary constituent of soybean (Glycine max) seed; however, little information concerning the inheritance of seed sucrose in soybean is available. The objective of this research was to use molecular markers to identify genomic regions significantly associated with quantitative trait loci (QTL) controlling sucrose content in a segregating F₂ population. DNA samples from 149 F₂ individuals were analyzed with 178 polymorphic genetic markers, including RFLPs, SSRs, and RAPDs. Sucrose content was measured on seed harvested from each of 149 F_2:3 lines from replicated field experiments in 1993 and 1995. Seventeen marker loci, mapping to seven different genomic regions, were significantly associated with sucrose variation at P<0.01. Individually, these markers explained from 6.1% to 12.4% of the total phenotypic variation for sucrose content in this population. In a combined analysis these genomic regions; explained 53% of total variation for sucrose content. No significant evidence of epistasis among QTLs was observed. Comparison of our QTL mapping results for sucrose content and those previously reported for protein and oil content (the other major seed constituents in soybean), suggests that seed quality traits are inherited as clusters of linked loci or that `major' QTLs with pleiotropic effects may control all three traits. Of the seven genomic regions having significant effects on sucrose content, three were associated with significant variation for protein content and three were significantly associated with oil content.     

Response of maize genotypes with different nitrogen use efficiency to low nitrogen stresses

Objectives

This paper aims to compare the property difference of spatial and temporal distribution of different nitrogen use efficiency maize genotypes and discuss the physiological mechanism of nitrogen efficiency of maize.

Quantitative trait loci for plant height in four maize populations and their associations with qualitative genetic loci

Summary We report that plant height quantitative trait loci (QTLs) identified in a given small population are not consistent with QTLs identified in other small populations, and that most QTLs are in close proximity to mapped qualitative genetic loci. These observations provide evidence to support the hypothesis that qualitative genetic loci are the same loci that affect quantitative traits, and affirm that these modest experiments probably identify real QTLs.     

Effects of intercropping on photosynthetic rate and net photosynthetic nitrogen use efficiency of maize under nitrogen addition

研究间作后作物光合碳同化和光合氮利用效率(PNUE)对氮投入的响应, 对阐释间作产量优势的氮调控效应, 指导间作氮肥管理有重要意义。本研究设置玉米(Zea mays)单作、玉米间作两种种植模式的4个氮水平(N0, 0 kg·hm ^-2; N1, 125 kg·hm ^-2; N2, 250 kg·hm ^-2; N3, 375 kg·hm ^-2), 分析间作与施氮量对玉米叶片特征、光合参数、PNUE和产量的影响。结果表明: 与单作相比, 间作显著增加玉米叶片的叶干质量和比叶质量; 各施氮水平(除N3)下, 间作中靠近马铃薯(Solanum tuberosum)侧的玉米叶面积均显著高于单作玉米。单间作对比发现, 间作提高了玉米光饱和点和暗呼吸速率。单作、间作靠玉米侧(I-M)、间作靠马铃薯侧(I-P)的玉米PNUE均随施氮量增加而降低, 降幅以I-P最大; 施氮量低于250 kg·hm ^-2时, 相同施氮量下的玉米PNUE和净光合速率(P_n)均以I-P最高, I-M和单作次之。间作显著提高了玉米产量(土地当量比>1)。该研究中当施氮量≤250 kg·hm ^-2时, 间作I-P的玉米叶片P_n和PNUE显著提高可能是间作玉米产量提高的重要原因。     

间作对氮调控玉米光合速率和光合氮利用效率的影响

研究间作后作物光合碳同化和光合氮利用效率(PNUE)对氮投入的响应, 对阐释间作产量优势的氮调控效应, 指导间作氮肥管理有重要意义。本研究设置玉米(Zea mays)单作、玉米间作两种种植模式的4个氮水平(N0, 0 kg·hm ^-2; N1, 125 kg·hm ^-2; N2, 250 kg·hm ^-2; N3, 375 kg·hm ^-2), 分析间作与施氮量对玉米叶片特征、光合参数、PNUE和产量的影响。结果表明: 与单作相比, 间作显著增加玉米叶片的叶干质量和比叶质量; 各施氮水平(除N3)下, 间作中靠近马铃薯(Solanum tuberosum)侧的玉米叶面积均显著高于单作玉米。单间作对比发现, 间作提高了玉米光饱和点和暗呼吸速率。单作、间作靠玉米侧(I-M)、间作靠马铃薯侧(I-P)的玉米PNUE均随施氮量增加而降低, 降幅以I-P最大; 施氮量低于250 kg·hm ^-2时, 相同施氮量下的玉米PNUE和净光合速率(P_n)均以I-P最高, I-M和单作次之。间作显著提高了玉米产量(土地当量比>1)。该研究中当施氮量≤250 kg·hm ^-2时, 间作I-P的玉米叶片P_n和PNUE显著提高可能是间作玉米产量提高的重要原因。     

Kernel set in maize genotypes differing in nitrogen use efficiency in response to resource availability around flowering

Environmental conditions affect grain yield in maize (Zea mays L.) mainly by altering the kernel number per plant (KNP). This number is determined during a critical period of about 2 weeks around silking. The objectives of this study were to assess how the rate and timing of nitrogen (N) fertilizer applications affect biomass partitioning and KNP in two genotypes with different N use efficiency, and to compare kernel set of these genotypes under varying regimes of carbohydrate and N availability during the critical period for kernel set. In the first field experiment, plant density and the rate of N supply per plant were varied independently. In the second field experiment, N availability was controlled via the application of N fertilizer, and carbohydrate availability was controlled by shading or thinning at silking. In both experiments, low rates of N supply reduced KNP more strongly in the non-efficient genotype when compared to the efficient genotype. The genotypic differences in kernel set were neither associated with N uptake into the above-ground biomass at maturity, nor above-ground biomass at silking. In the non-efficient genotype, application of N fertilizer at silking increased KNP. This increase was not associated with an increase in plant growth but with increased partitioning of biomass towards the reproductive organs during the critical period for kernel set. The genotype which had been selected for its high N use efficiency also showed higher kernel set at high plant density and shading during flowering when compared to the non-efficient genotype. Under conditions of restricted resource availability per plant, plant and ear growth rates during the critical period of about 14 days after onset of flowering declined compared with non-limiting conditions. However, these growth rates were less reduced in the efficient genotype. Pooling treatments of different plant density and different available N, each hybrid showed linear responses of KNP to plant growth rate and to ear growth rate. Furthermore, in the efficient genotype KNP was reduced to a lesser extent in response to decreasing growth rates. We conclude that higher kernel set of the efficient genotype compared to the non-efficient genotype under stressful conditions was associated with low sensitivity of plant growth and dry matter distribution towards reproductive organs to low assimilate availability during the critical period of kernel set, and particularly with low sensitivity of kernel set to decreasing plant and ear growth rates.     

A gene for resistance to the maize streak virus in the African CIMMYT maize inbred line CML202

Resistance to maize streak virus (MSV) is an essential trait of improved maize varieties in sub-Saharan Africa. We mapped quantitative trait loci (QTL) for resistance to MSV in a population of 196 F_2:3 lines derived from a cross between the maize inbred lines CML202 (resistant) from CIMMYT-Zimbabwe and Lo951 (susceptible) from Italy. Field tests were planted at two locations in Zimbabwe, inoculated with viruliferous leaf hoppers (Cicadulina mbila), and scored twice (21 and 83 days after infesting, DAI) on a 1–5 scale. The mean final streak intensity (score 2) of the parent lines was 2.2 (CML202) and 4.8 (Lo951). Genotype × location interaction was large for score 1 but negligible for score 2. Consequently, the heritability was higher for score 2 (0.93) than for score 1 (0.62). By composite interval mapping across locations, using a linkage map with 110 RFLP loci, four significant (LOD 3.0) QTL were identified for score 1 on chromosomes (C) 1, 2, 3, and 4, respectively. All four were contributed by CML202. For score 2, only the QTL on C 1 was significant (LOD =37), explaining 59% of the phenotypic and 64% of the genotypic variance. The QTL's partially dominant gene action was consistent with the nearly intermediate resistance of the F₁ generation (relative heterosis for resistance 12%). The presence of one major QTL is consistent with the bimodal frequency distribution of the mapping population showing a clear 3:1 segregation. This gene seems to be allelic or identical to Msv1, a major resistance gene which was previously identified in the same genomic region in Tzi4, an inbred line from IITA. Inbred CML202 had lower final disease ratings than Tzi4. The greater resistance of CML202 may be due to allelic differences at the msv1 locus or due to the minor QTL on C 2, 3, and 4 which were not detected in Tzi4.z y Trigo (International Maize and Wheat Improvement Center); IITA, International Institute of Tropical Agriculture; IRAT, Institute de Recherches Agronomiques Tropicales et des Cultures Vivrières; KARI, Kenya Agricultural Research Institute; MSV, maize streak virus; QTL, quantitative trait locus/loci     

Increased seminal root number associated with domestication improves nitrogen and phosphorus acquisition in maize seedlings

Background and AimsDomesticated maize (Zea mays ssp. mays) generally forms between two and six seminal roots, while its wild ancestor, Mexican annual teosinte (Zea mays ssp. parviglumis), typically lacks seminal roots. Maize also produces larger seeds than teosinte, and it generally has higher growth rates as a seedling. Maize was originally domesticated in the tropical soils of southern Mexico, but it was later brought to the Mexican highlands before spreading to other parts of the continent, where it experienced different soil resource constraints. The aims of this study were to understand the impacts of increased seminal root number on seedling nitrogen and phosphorus acquisition and to model how differences in maize and teosinte phenotypes might have contributed to increased seminal root number in domesticated maize.MethodsSeedling root system architectural models of a teosinte accession and a maize landrace were constructed by parameterizing the functional–structural plant model OpenSimRoot using plants grown in mesocosms. Seedling growth was simulated in a low-phosphorus environment, multiple low-nitrogen environments, and at variable planting densities. Models were also constructed to combine individual components of the maize and teosinte phenotypes.Key ResultsSeminal roots contributed ~35 % of the nitrogen and phosphorus acquired by maize landrace seedlings in the first 25 d after planting. Increased seminal root number improved plant nitrogen acquisition under low-nitrogen environments with varying precipitation patterns, fertilization rates, soil textures and planting densities. Models suggested that the optimal number of seminal roots for nutrient acquisition in teosinte is constrained by its limited seed carbohydrate reserves.ConclusionsSeminal roots can improve the acquisition of both nitrogen and phosphorus in maize seedlings, and the increase in seed size associated with maize domestication may have facilitated increased seminal root number.     

Modelling postsilking nitrogen fluxes in maize (Zea mays) using 15N-labelling field experiments

In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are two processes that provide amino acids for grain protein synthesis. To study the way in which N is allocated to the grain and to the stover, two different 15N-labelling techniques were developed. 15NO(3-) was provided to the soil either at the beginning of stem elongation or after silking. The distribution of 15N in the stover and in the grain was monitored by calculating relative 15N-specific allocation (RSA). A nearly linear relationship between the RSA of the kernels and the RSA of the stover was found as a result of two simultaneous N fluxes: N remobilization from the stover to the grain, and N allocation to the stover and to the grain originating from N uptake. By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence of protein turnover, a large proportion of the amino acids synthesized from the N taken up after silking were integrated into the proteins of the stover, and these proteins were further hydrolysed to provide N to the grain.     

RFLP mapping of quantitative trait loci controlling abscisic acid concentration in leaves of drought-stressed maize (Zea mays L.)

 Abscisic acid (ABA) concentration in leaves of drought-stressed plants is a quantitatively inherited trait. In order to identify quantitative trait loci (QTLs) controlling leaf ABA concentration (L-ABA) in maize, leaf samples were collected from 80 F_3:4 families of the cross Os420 (high L-ABA)×IABO78 (low L-ABA) tested under drought conditions in field trials conducted over 2 years. In each year, leaf samples were collected at stem elongation and near anthesis. The genetic map obtained with 106 restriction fragment length polymorphism (RFLP) loci covered 1370 cM, which represented approximately 85% of the UMC maize map. Sixteen different QTLs with a LOD>2.0 were revealed in at least one sampling. Across samplings, only four QTLs significantly influenced L-ABA, accounting for 66% of the phenotypic variation and 76% of the genetic variation among families. At these QTLs, the alleles which increased L-ABA were contributed by Os420. The two most important QTLs were mapped on chromosome 2 near csu133 and csu109a. The effects associated with the QTL near csu133 were more pronounced near anthesis. The support intervals of the four primary QTLs for L-ABA did not overlap the presumed map position of mutants impaired in ABA biosynthesis. Received: 27 January 1998 / Accepted: 22 April 1998     

施氮量对不同肥力水平下夏玉米碳氮代谢及氮素利用率的影响

以郑单958为材料,在高产田和中产田两种地力水平下,利用15N标记法研究了施氮量对夏玉米氮素分配率、利用率和碳氮代谢的影响.结果表明:高产田适量施氮可以提高玉米产量,过量施氮没有表现出进一步增产效果,其氮肥利用率较低(29 04%).中产田随施氮量的增加产量提高,但氮素利用率却降低.各个器官15N积累量依次为籽粒>叶片>茎>根>叶鞘>穗轴.在高产田,当施氮量超过300kg·hm-2时,玉米籽粒和叶片中积累15N有所下降,而茎和根中积累15N的量随施N量的增加而增加;在中产田,随着施N量的增加,籽粒和穗轴积累15N量均相应增加.高产田叶片的硝酸还原酶活性、谷氨酰胺合成酶活性和蔗糖磷酸合成酶活性以及籽粒中蔗糖合成酶活性和酸性转化酶活性均是施氮300kg·hm-2时最大,施氮450 kg·hm-2则抑制了其活性的增强,而中产田的各个酶活性则随着施氮量的增加而增加.     

Mapping quantitative trait loci controlling silking date in a diallel cross among four lines of maize

 We describe and apply an interval mapping method for quantitative trait locus (QTL) detection using F₃ and testcross progenies derived from F₂ populations obtained from a diallel cross among four elite lines of maize. Linear model-based procedures were used for the test and estimation of putative QTL effects together with genetic interactions including epistasis. We mapped QTL associated with silking date and explored their genetic effects. Ten QTL were detected, and these explained more than 40% of the phenotypic variance. Most of these QTL had consistent and stable effects among genetic backgrounds and did not show significant epistasis. QTL-by-environment interaction was important for four QTL and was essentially due to changes in magnitude of allelic effects. These results show the efficiency of our method in several genetic situations as well as the power of the diallel design in detecting QTL simultaneously over several populations. Received: 2 September 1996 / Accepted: 20 December 1996