Effects of nitrogen addition on nitrogen metabolism and carbon reserves in the temperate seagrass Posidonia oceanica

The effect of repeated N additions on a dense, shallow meadow of Posidonia oceanica (L.) Delile in the NW Mediterranean was studied over a year. N was added biweekly both to the sediment and to the water column as ammonium and nitrate. The most obvious result of these additions was an overall increase in N content (% DW) in all tissues of fertilized plants; this increase was maximum in rhizomes, with values of 5% N reached, which confirmed the storage capacities of these organs.Fertilization affected the different N fractions in distinct ways. The free amino acid (FAA) concentration increased the most, particularly in rhizomes and roots, suggesting the function of these compounds for N storage and, probably, translocation. The non-soluble N fraction also increased greatly. The total soluble protein (TSP) and the inorganic N forms concentrations were less sensitive to fertilization, and only increased moderately in a few cases. N assimilation, assessed through in vivo glutamine synthetase (GS) activity, was maximum in leaves after the peak of growth, which coincided with the lowest N values in both control and fertilized plants. Thus assimilation was probably greatest at the period of highest N deficiency. Growth rates did not respond to N enrichment. Another clear effect of N addition was to decrease carbon reserves. In effect, the concentration of total non-structural carbohydrate (TNC) greatly decreased in rhizomes of fertilized plants, coinciding with the increase in FAA. We conclude that increased nitrogen availability can affect plant survival through the decrease in their carbon reserves, crucial for P. oceanica overwintering. This interaction between N and C metabolism helps to explain changes in benthic vegetation after steadily increasing coastal water eutrophication.     

Does ammonium affect accumulation of starch in rhizomes ofPhragmites australis (Cav.) Trin. ex Steud.?

Reed rhizomes from four habitats differing in soil nitrogen availability were collected and analysed for carbohydrate content during one year. Rhizomes collected from sediments where nitrate was the prevailing form of available nitrogen accumulated predominantly starch. On the other hand, rhizomes buried in sediments rich in ammonium contained predominantly sucrose. These observations together with the fact that ammonium stimulates the activity of enzymes involved in degradation of starch suggest that ammonium affects starch storage by inducing conversion of starch into sucrose. During spring when sprouting takes place, the rates of sucrose or starch mobilization differed remarkably, the sucrose in rhizomes buried in ammonium-rich sediments being depleted much faster than the starch in rhizomes at ammonium-poor habitats. The consequent reduction of total carbohydrate content in rhizomes at ammonium-rich habitats places the plants in a vulnerable situation. Eutrophication and reed decline are postulated to be events causally related via toxicity of ammonium, of which considerable amounts are microbiologically released from decaying organic matter in reed beds under excessive nutrient supply.     

Nitrogen and carbohydrate pools of two rhizome types of Phragmites australis (Cav.) Trin. ex Steudel

Carbohydrate and total nitrogen contents as well as free amino acid (FAA) concentrations were determined in distinct types of rhizomes of two genetically homogeneous stands of reed (Phragmites australis (Cav.) Trin. ex Steudel) differing in morphology, productivity and nutrient supply in order to evaluate the storage capacity of vertical rhizomes and expansion rhizomes. The expansion rhizomes possess significantly higher amounts of FAA and of total nitrogen but similar carbohydrate concentrations in comparison to the vertical rhizomes. However, no significant differences were found for total nitrogen, FAA and total carbohydrates between both investigated stands indicating a comparable storage capacity of rhizomes independent of nutrient availability. Only the composition of the FAA pool varied in the alanine/asparagine ratio probably influenced by the oxygen supply of the rhizome/root system.     

根茎在羊草响应短期NaCl胁迫过程中的作用

根茎是根状茎型克隆植物的特有结构,在养分储存、运输和分蘖茎的形成等方面起关键性作用。然而有关根茎生理学方面的研究却十分匮乏。为了探讨根茎在植物感应环境胁迫中的作用, 本文以羊草(Leymus chinensis)为实验材料,研究和比较了短期NaCl胁迫根、根茎、根和根茎3种处理方式下羊草对盐胁迫的响应。试验结果表明:200 mmol·L^-1 NaCl处理羊草根、根茎、根和根茎24 h,显著(p<0.05)降低羊草叶片净光合速率和蒸腾速率,增加叶片渗透浓度与脯氨酸含量;其中同时处理根和根茎叶片,蒸腾速率和净光合速率的降低程度显著高于分别处理根和根茎。在分别处理根与根茎的情况下,叶片含水量、脯氨酸含量、净光合速率、蒸腾速率均无显著性差异。不论单独胁迫根、根茎还是同时胁迫根和根茎,羊草根、根茎和叶片内Na⁺含量都显著高于对照,而羊草根、根茎和叶片内K⁺含量都显著低于对照。这些结果显示:1)根茎在羊草响应盐胁迫的生理过程中与根系具有类似的功能;2)羊草根茎在盐胁迫条件下能够有效地吸收Na⁺;3)鉴于根茎的生物量和表面积都明显地低于根系,在盐胁迫下羊草根茎吸收Na⁺的效率高于根系。     

Seasonal Changes in Nitrogen Fractions of Coptis japonica, a Temperate Forest Evergreen Chamaephyte, and their Ecological Implications

Seasonal changes in several forms of nitrogen were investigatedin Coptis japonica, an evergreen rosette hemicryptophyte intemperate deciduous forest. The concentration of total nitrogenin rhizomes and roots decreased during the period of new shootgrowth from winter to spring. In the rhizomes, total solubleprotein stored by early summer decreased gradually until winter,coupled with an increase in free amino acids. Nitrogen was largelystored in free amino acids in the roots, especially during summer.The total soluble protein in current-year leaves decreased fromspring to summer and then increased during winter. The seasonalchanges in nitrogen components were coincident with the changein light-saturated photosynthetic rates recorded in a previousstudy. The ratio of total soluble protein to total nitrogendecreased from spring to summer and then increased from latesummer to winter in the current-year leaves. In contrast, chlorophyllcontent and the ratio of chlorophyll to total nitrogen werehigher in summer than in other seasons. The results indicatethat nitrogen was used in a manner that better utilizes thevery weak light in summer and the higher light intensities inother seasons. The major component of the free amino acid poolwas asparagine, in every organ throughout the season, exceptfor the senescent leaves. Since asparagine has a high N:C ratio,we suppose that the asparagine-dominated amino acid pool isadvantageous in the carbon-limited environment of the forestfloor.Copyright 1994, 1999 Academic Press Free amino acid composition, total nitrogen, total soluble protein, photosynthesis, evergreen hemicryptophyte     

Population Dynamics of Pratylenchus penetrans,Paratylenchus sp., and Criconemella xenoplax on Western Oregon Peppermint

Endoparasitic nematode populations are usually measured separately for soil and roots without a determination of the quantitative relation between soil and root population components. In this study, Pratylenchus penetrans populations in peppermint soil, roots, and rhizomes were expressed as the density within a standardized core consisting of 500 g dry soil plus the roots and rhizomes contained therein. Populations of Paratylenchus sp. and Criconemella xenoplax in 500 g dry soil were also determined, thus measuring the total plant-parasitic nematode population associated with the plant. Mean wet root weight per standard core peaked in spring and again in late summer and was lowest early in the growing season and in early fall. Pratylenchus penetrans populations peaked 4 to 6 weeks after root weight peaks. The percentage of the total population in roots reached 70% to 90% in early April, decreased to 20% to 40% in August, and returned to higher percentages during the winter. Rhizomes never contained more than a minor proportion of the population. Mean Paratylenchus sp. populations increased through spring and peaked in late August. Mean C. xenoplax populations fluctuated, peaking in August or September. Populations of all parasitic species were lowest during winter. Evaluation using the standard core method permits assessment of the total P. penetrans population associated with the plant and of changes in root weight as well as the seasonal distribution of P. penetrans.     

Nitrogen Assimilation in Barley (Hordeum vulgare L. cv. Mazurka) in Response to Nitrate and Ammonium Nutrition

Barley plants (Hordeum vulgare L. cv. Mazurka) were grown inaerated solution cultures with 2 mM or 8 mM inorganic nitrogensupplied as nitrate alone, ammonium alone or 1:1 nitrate+ammonium.Activities of the principal inorganic nitrogen assimilatoryenzymes and nitrogen transport were measured. Activities ofnitrate and nitrite reductases, glutamine synthetase and glutamatesynthase were greater in leaves than in roots but glutamatedehydrogenase was most active in roots. Only nitrate and nitritereductases changed notably (4–10 times) in response tothe different nitrogen treatments. Nitrate reductase appearedto be rate-limiting for nitrate assimilation to glutamate inroots and also in leaves, where its total in vitro activitywas closely related to nitrate flux in the xylem sap and wasslightly in excess of that needed to reduce the transportednitrate. Xylem nitrate concentration was 13 times greater thanthat in the nutrient solution. Ammonium nitrogen was assimilatedalmost completely in the roots and the small amount releasedinto the xylem sap was similar for the nitrate and the ammoniumtreatments. The presence of ammonium in the nutrient decreasedboth export of nitrate to the xylem and its accumulation inleaves and roots. Nitrate was stored in stem bases and was releasedto the xylem and thence to the leaves during nitrogen starvation.In these experiments, ammonium was assimilated principally inthe roots and nitrate in the leaves. Any advantage of this divisionof function may depend partly on total conversion of inorganicnitrogen to amino acids when nitrate and ammonium are givenin optimal concentrations. Hordeum vulgare L., barley, nitrate, ammonium, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, nitrogen transport     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.     

增铵营养对小麦光合作用及硝酸还原酶和谷氨酰胺合成酶的影响

采用水培试验研究不同形态氮营养(NH4^+／NO3^-分别为0／100、50／50和100／0)对小麦光合作用及氮代谢关键酶活性的影响．结果表明,增铵营养较单—NO₃^-营养显著提高叶片叶绿素含量、净光合速率及可溶性糖含量,叶、根中可溶性蛋白质含量和叶片硝酸还原酶活性。而对谷铵酰胺合成酶活性影响较小．与单—NO₃^-营养相比。增氨营养下叶片较高的可溶性糖含量与净光合速率的提高相关。而维持较高的叶片和根系可溶性糖／可溶性蛋白质比例有利于氮同化和生长．因此,增铵营养下提高了叶片净光合速率、可溶性糖含量和硝酸还原酶活性。维持较高叶片和根系可溶性糖／蛋白质比例。从而促进小麦生长．     

Sink control of nitrogen uptake and assimilation during regrowth after-cutting of ryegrass (Lolium perenne L.)

Abstract. The ¹⁵N isotope was used to compare the uptake and the assimilation of NH₄⁺ and NO₃ nitrogen in ryegrass ( Lolium perenne L.) during regrowth after cutting. Uptake of nitrate-N, expressed per plant, was at all times greater than ammonium-N uptake and assimilation decreased in roots and stubble while its assimilation was maintained at a high level in leaves. It has been suggested that ammonium assimilation is directly related to the availability of carbohydrates in the sink organ (leaves) resulting from their remobilization from the source organs (roots and stubble). Nitrate reduction decreased in all organs, while the uptake of NO₃ was still high. After this first period of regrowth, nitrogen assimilation both from nitrate and ammonium increased in all the plants. Nitrate reduction capacity (expressed in μg NO₃-N reduced per g D.W. per d) is 7.5 and 22.5 times greater in leaves than in stubble and roots, respectively. Therefore, nitrogen assimilation in stubble and particularly in roots was mainly dependent on ammonium nitrogen.     

The role of organic and inorganic solutes in the osmotic adjustment of drought-stressed Jatropha curcas plants

This study aimed to assess the accumulation of organic and inorganic solutes and their relative contribution to osmotic adjustment in roots and leaves of Jatropha curcas subjected to different water deficit intensity. Plants were grown in vermiculite 50% (control), 40%, 30%, 20% and 10% expressed in gravimetric water content. The water potential, osmotic potential and turgor potential of leaves decreased progressively in parallel to CO₂ photosynthetic assimilation, transpiration and stomatal conductance, as the water deficit increased. However, the relative water content, succulence and water content in the leaves did not show differences between the control and stressed plants, indicating osmotic adjustment associated with an efficient mechanisms to prevent water loss by transpiration through stomatal closure. The K⁺ ions had greater quantitative participation in the osmotic adjustment in both leaves and roots followed by Na⁺ and Cl⁻, while the NO₃⁻ ion only showed minor involvement. Of the organic solutes studied, the total soluble sugars showed the highest relative contribution to the osmotic adjustment in both organs and its concentration positively increased with more severe water deficit. The free amino acids and glycinebetaine also effectively contributed to the osmotic potential reduction of both the root and leaves. The role of proline was quantitatively insignificant in terms of osmotic adjustment, in both the control and stressed roots and leaves. Our data reveal that roots and leaves of J. curcas young plants display osmotic adjustment in response to drought stress linked with mechanisms to prevent water loss by transpiration by means of the participation of inorganic and organic solutes and stomatal closure. Of all the solutes studied, soluble sugars uniquely display a prominent drought-induced synthesis and/or accumulation in both roots and leaves.     

How does glutamine synthetase activity determine plant tolerance to ammonium?

The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants’ capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.     

Nitrogen uptake and metabolism in Populus x canescens as affected by salinity

External salinization can affect different steps of nitrogen (N) metabolism (ion uptake, N assimilation, and amino acid and protein synthesis) depending on the inorganic N source. Here, we assessed the net uptake of N supplied as nitrate or ammonium and N assimilation (combining metabolite analyses with molecular biological approaches) in grey poplar (Populus x canescens) plants grown under saline (75 mM NaCl) and control conditions. The specific (micromol N g(-1) dry weight fine roots h(-1)) and total plant (micromol N per plant h(-1)) N net uptake rates, total plant N content, total plant biomass and total leaf protein concentration were reduced under saline conditions when plants were supplied with ammonium. In both nutritional groups, salt treatment caused pronounced accumulation of soluble N compounds in the leaves. The mRNAs of genes coding for enzymes catalyzing rate-limiting steps of both proline synthesis and degradation (delta-1-pyrroline-5-carboxylate synthase and proline dehydrogenase) as well as for NADH-dependent glutamate synthase were accumulated under saline conditions. Whereas under control conditions the plant N status seemed to be superior when ammonium was supplied, the N balance of ammonium-fed plants was more severely affected by salt stress than that of plants supplied with nitrate. Possible metabolic implications of stress-related accumulation of particular amino acids are discussed.     

Comparison of nitrogen uptake in the roots and rhizomes of <Emphasis Type="Italic">Leymus chinensis</Emphasis>

Leymus chinensis (Trin.) Tzvel is a rhizomatous grass species in the Eastern Eurasian steppe zone that is often limited by low soil nitrogen availability. Although a previous study showed that the rhizomes of L. chinensis have the capacity to take up nitrogen, the importance of such uptake for nitrogen nutrition is unclear. Moreover, little is known regarding the inorganic nitrogen uptake kinetics of roots and rhizomes in response to nitrogen status. Here, we first found that ammonium is preferred over nitrate and glycine for L. chinensis growth. Using the ¹⁵N-labelling method, we found that the rate of ion influx into roots was approximately five-fold higher than into rhizomes under the same nitrogen content, and the ion influxes into roots and rhizomes under 0.05 mM N were greater than in the presence of 3 mM N, especially in the form of NH₄⁺. Using a non-invasive micro-test technique, we characterised the patterns of NH₄⁺ and NO₃^– fluxes in the root mature zone, root tip, rhizome mature zone, and rhizome tip following incubation in the solution with different N compounds and different N concentrations. These results suggest a dynamic balance between the uptake, utilisation, and excretion of nitrogen in L. chinensis.     

Assimilation and translocation of nitrogen and carbon in Curcuma alismatifolia Gagnep

Curcuma or Siam tulip (Curcuma alismatifolia Gagnep.) is an ornamental flowering plant with two underground storage organs, rhizomes and storage roots. Characteristics of N and C assimilation and transport in curcuma were investigated. The plants were treated with ¹⁵NH₄⁺ + ¹⁵NO₃^? and ¹³CO₂ at 10, 13 or 21 weeks after planting. Plants were sampled at several stages up to 32 weeks. The C stored in old storage roots was used rapidly during the first 10 weeks; after which N stored in old rhizomes and old storage roots were used. The daily gain in C depending on photosynthesis was remarkably high between 10 and 21 weeks. However, the daily gain in N was relatively constant throughout the growth period. The ¹⁵N absorbed at 10 weeks was initially accumulated in leaves and roots, but some was transported to flowering organs at 13 weeks. At harvest, 41% of ¹⁵N was recovered in new rhizomes and 17% in new storage roots. After ¹³CO₂ exposure at 10 and 13 weeks, the distribution of ¹³C among organs was relatively constant in subsequent stages. When given ¹³CO₂ at 21 weeks, a large amount of labelled C was recovered in new storage roots and new rhizomes at harvest. Both new rhizomes and new storage roots stored N and C, however, rhizomes played a more important role in supplying N, while storage roots provided C.     

The cytosolic glutamine synthetase GLN1;2 plays a role in the control of plant growth and ammonium homeostasis in Arabidopsis rosettes when nitrate supply is not limiting

Glutamine synthetase (EC 6.3.1.2) is a key enzyme of ammonium assimilation and recycling in plants where it catalyses the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, five GLN1 genes encode GS1 isoforms. GLN1;2 is the most highly expressed in leaves and is over-expressed in roots by ammonium supply and in rosettes by ample nitrate supply compared with limiting nitrate supply. It is shown here that the GLN1;2 promoter is mainly active in the minor veins of leaves and flowers and, to a lower extent, in the parenchyma of mature leaves. Cytoimmunochemistry reveals that the GLN1;2 protein is present in the companion cells. The role of GLN1;2 was determined by examining the physiology of gln1;2 knockout mutants. Mutants displayed lower glutamine synthetase activity, higher ammonium concentration, and reduced rosette biomass compared with the wild type (WT) under ample nitrate supply only. No difference between mutant and WT can be detected under limiting nitrate conditions. Despite total amino acid concentration was increased in the old leaves of mutants at high nitrate, no significant difference in nitrogen remobilization can be detected using (15)N tracing. Growing plants in vitro with ammonium or nitrate as the sole nitrogen source allowed us to confirm that GLN1;2 is induced by ammonium in roots and to observe that gln1;2 mutants displayed, under such conditions, longer root hair and smaller rosette phenotypes in ammonium. Altogether the results suggest that GLN1;2 is essential for nitrogen assimilation under ample nitrate supply and for ammonium detoxification.     

Seasonal Changes of Nitrogen Storage Compounds in a Rhizomatous Grass Calamagrostis epigeios

The seasonal dynamics in content and distribution of N-rich compounds between overwintering organs of Calamagrostis epigeios were examined. Samples were taken both from plants grown in natural conditions and in containers with controlled nutrient supply. There were significant changes in content of nitrate, free amino acids and soluble protein in all investigated plant parts during the course of a year. Amino acids showed both the highest maximum and seasonal fluctuation among the all N compounds observed and, therefore, appear to have a central role in N storage. Their content rises in the autumn, remains stable during winter and declines quickly at the beginning of spring. The most abundant amino acids in the end of winter storage period - asparagine, arginine and glutamine - constituted about 90 % of N in fraction of free amino acids. The portion of N stored in soluble proteins, however, was considerably smaller compare to both amino acids and nitrate. The amount of N stored in rhizomes of C. epigeios was smaller than in roots and stubble base before the onset of spring re-growth. This indicates that roots and stubble base are particularly important for winter N storage in this species.     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Effects of nitrogen forms on dry matter partitioning and nitrogen metabolism in two contrasting genotypes of Catasetum fimbriatum (Orchidaceae)

The effects of either organic (urea and glutamine) or inorganic nitrogen forms (nitrate and ammonium) on dry matter accumulation in shoots and roots and on nitrogen assimilatory enzyme activities were studied in two Catasetum fimbriatum genotypes. Both genotypes, which had inverse patterns of dry matter partitioning between shoots and roots, were aseptically incubated in gelled culture media containing 6 mol m⁻³ of nitrogen and incubated in growth chamber for 30 and 60 days. In vivo nitrate reductase, glutamine synthetase, glutamate dehydrogenase activities as well as free ammonium contents were determined in shoots and roots of plants grown in four different nitrogen sources. Nitrogen assimilatory enzyme activities showed the highest values in the genotype that accumulated dry matter predominantly in the shoots. The nitrogen sources supplied affected dry matter accumulation in shoots and roots of both C. fimbriatum genotypes; however, they were not enough to change the characteristic pattern of dry matter partitioning of each genotype. On the other hand, the differences in the root/shoot ratio found among nitrogen treatments were relatively higher in the genotype that directed dry matter mainly to roots than in the genotype that allocates biomass to shoots. Our results suggest that NADH-dependent glutamate dehydrogenase plays an important role in ammonium assimilation in C. fimbriatum plants, particularly in the root system. Nitrogen metabolism and the dry matter partitioning of the two genotypes are discussed.