Characterization of a flint maize (Zea mays var. indurata) Italian landrace, II. Genetic diversity and relatedness assessed by SSR and Inter-SSR molecular markers

A comparative characterization of 10 field populations of the maize (Zea mays var. indurata) landrace Nostrano di Storo was carried out using different types of PCR-based markers. The inbred line B73 and three synthetics (VA143, VA154 and VA157) selected from as many landraces were also used. Genetic diversity and relatedness were evaluated over 84 SSR and 53 I-SSR marker alleles using a total of 253 individual DNAs. Up to 23 alleles per SSR locus were scored while the average effective number of alleles per population was 6.99. Nei's total genetic diversity as assessed with SSR markers was H_T = 0.851 while the average diversity within populations was H_S = 0.795. The overall Wright's fixation index F_ST was as low as 0.066. Thus, more than 93% of the total variation was within population. Unique alleles over all SSR loci were found for six populations. An average of 17.7 marker alleles per I-SSR primer were scored with an effective number of marker alleles per locus of 1.34. The Shannon's diversity information index over all populations and I-SSR loci was 0.332, varying from 0.286 to 0.391. The extent of differentiation between populations was as low as G_ST = 0.091. Dice's genetic similarity matrices were estimated for both SSR and I-SSR markers. The mean genetic similarity coefficients within and between populations were respectively 0.269 and 0.217, for SSR markers, and 0.591 and 0.564, for I-SSR markers. UPGMA dendrograms displayed all field populations but one clustered into a distinct group, in which the synthetic VA154, selected from the Marano Vicentino landrace, was also included. One field population and the other two synthetics were clustered separately as well B73. The matrix correlation assayed by the Mantel's correspondence test was as high as 0.908. Findings suggest that, although a high variability can be found among plants, most plant genotypes belong to the same landrace called Nostrano di Storo. Although gene flow from commercial hybrids might have occurred, the large number of polymorphisms and the presence of both unique alleles and alleles unshared with B73 and synthetics are the main factors underlying the value of this flint maize landrace as a source of genetic variation and peculiar germplasm traits. Because of its exclusive utilization for human consumption, such a molecular marker characterization will be a key step for obtaining the IGP mark and so promote the in situ conservation and protection of the landrace Nostrano di Storo.     

Molecular characterization of emmer (Triticum dicoccon Schrank) Italian landraces

Emmer (Triticum dicoccon Schrank,2n = 4x = 28) is a hulled wheatspecies [more] widely spread in the Mediterranean basin. In Italy it survives as acrop in a few marginal areas and peculiar ecological niches in different regionsof central and southern Italy. A renewed interest has occurred during the lastdecade toward local varieties belonging to this species. As a matter of fact,local varieties have the highest genetic variation and adaptation to the naturaland anthropological environment from where they originated. Results on thegenetic diversity within and relationships among 11 Italian local varieties ofemmer as assessed with 17 RAPD marker loci are here reported. The proportion ofthe among-local variety genetic diversity was as high as 48% (G_ST =0.479). Thus, about 52% of the total variation was within population. Localvarieties of emmer proved to be formed by a variable number of lines geneticallydistinguishable from each other, and the vast majority of individuals overpopulations proved to be different multilocus genotypes. Landraces of emmer fromcentral and southern Italy showed distinctive molecular traits. In particular,local varieties classified as «Central Italy» types were characterized by a common set of RAPD marker alleles and proved to bedistinguishable from both the «Southern Italy» and the«Garfagnana» accessions. The overall results confirm the highvariability that can be found within landrace populations, underlining thevalues of landraces as an irreplaceable bank of genetically diversified andhighly co-adapted genotypes. Information for an appropriate insitu conservation and management of this valuable source of emmergermplasm is discussed.     

RAPD and AFLP assessment of genetic variation in a landrace of pepper (Capsicum annuum L.), grown in North-West Italy

In several regions of Italy as well as other parts of southern Europe, the heterogeneity of the land, the climate and the soil favour the survival in cultivation of a large number of landraces specifically adapted to local conditions. Knowledge on the level and distribution of their genetic variation can help to develop appropriate strategies, in order to suistainably manage in situ these germplasm resources at risk of genetic erosion. C. annuum is an herbaceous diploid species and is considered to be self-pollinating, although different rates of out-crossing have been recorded. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess genetic diversity within and between five populations of a landrace of Capsicum annuum L., grown in a limited area in north-west Italy and locally known as Cuneo pepper. Partitioning the genetic variation with Shannon's diversity index revealed that 41.6% occurred between and 58.4% within populations. Analogous results were obtained when the analysis was based only on RAPD or AFLP markers. However, AFLP was more reliable, since a lower range of variation was observed among primer combinations in detecting the two components of genetic variation. Notwithstanding the rather high level of within genetic variation detected, the five populations were clearly differentiated and differed in the frequency of alleles exclusive and/or present at very low frequencies. Our results show the need for accurate estimation of allele frequencies, in order to identify populations to which priority should be given for dynamic conservation of landraces.     

Ammonium-excreting Azospirillum sp. become intracellularly established in maize (Zea mays) para-nodules

Maize seedlings develop nodule-like tumour knots (para-nodules) along primary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Inoculated NH ₄ ⁺ -excreting Azospirillum brasilense cells were shown to colonize these tumours, mostly intracellularly, promoting a high level of N₂ fixation when microaerophilic conditions were imposed. The nitrogenase activity inside the para-nodules was less sensitive to free O₂ than in non-para-nodulating roots. Both light and electron microscopy showed a dense bacterial population inside intact tumour cells, with the major part of the cell infection along a central tumour tissue. The bacteria colonized the cytoplasm with a close attachment to inner cell membranes. In an auxin-free growth medium, young 2,4-D-induced para-nodules grew further to become mature differentiated root organs in which introduced bacteria survived with a stable population. These results provide evidence that gramineous plants are potentially able to create a symbiosis with diazotrophic bacteria in which the NH ₄ ⁺ -excreting symbiont will colonize para-nodule tissue intracellularly, thus becoming well protected.     

Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L.) growing on different French soils

Summary We studied the dominant diazotrophs associated with maize roots and rhizosphere soil originating from three different locations in France. An aseptically grown maize plantlet, the spermosphere model, was used to isolate N₂-fixing (acetylene-reducing) bacteria. Bacillus circulans was the dominant N₂-fixing bacterium in the rhizosphere of maize-growing soils from Ramonville and Trogny, but was not found in maize-growing sandy soil from Pissos. In the latter soil, Enterobacter cloacae, Klebsiella terrigena, and Pseudomonas sp. were the most abundant diazotrophs. Azospirillum sp., which has been frequently reported as an important diazotroph accociated with the maize rhizosphere, was not isolated from any of these soils. The strains were compared for their acetylene-reducing activity in the spermosphere model. The Bacillus circulans strains, which were more frequently isolated, also exhibited significantly greater acetylene-reducing activity (3100 nmol ethylene day^-1 plant^-1) than the Enterobacteriaceae strains (180 nmol ethylene day^-1 plant^-1). This work indicates for the first time that Bacillus circulans is an important maizerhizosphere-associated bacterium and a potential plant growth-promoting rhizobacterium.     

Effect of farmers' seed selection on genetic variation of a landrace population of pepper (Capsicum annuum L.), grown in North-West Italy

Recognition by the European Community of élite landraces encourages farmers to grow these and earn more than is possible by growing modern varieties. However, farmers often exercise strong selection by collecting seed from a limited number of plants which best embody a few characters of prime interest. This, in the long run, may be responsible for considerable genetic erosion. In Southern Piedmont, North-West Italy, a local landrace of pepper (Capsicum annuum L.) known as Cuneo is grown; it deserves particular attention because of its hardiness, late production and fruit quality. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess the effect, on genetic composition of a population, of seed sampling carried out using the selection criteria adopted by the farmer. After two reproductive cycles using selection, it was already possible to detect loss in genetic variation and a change in allele frequencies, while no significant effect was found after two cycles of random sampling. Over this period, farmer selection pressure led to disappearance of eight low-frequency alleles, while only three alleles were lost in randomly sampled populations. Our results stress the need to adopt appropriate techniques for seed sampling in order to avoid genetic erosion of local landrace gene pools.     

Response of maize (Zea mays) to Azotobacter inoculation under fertilized and unfertilized conditions

Summary Inoculated seeds of maize (Zea mays) with 11 Azotobacter strains, sown in the fields receiving no fertilizer and fertilizers (N and P at the rate of 125 and 40 kg ha^–1 respectively) increased the grain yield by 19.63% and 15.89% respectively over the corresponding control. The effect was greater in unfertilized than in fertilized soil. The increase in yield due to fertilizers was 21.2% without inoculation and 37.09% with inoculation. The correlations between total yield, and N, P and K uptake were highly significant and comparable among themselves. This indicated that increase in yield due to inoculation was not due to N₂ fixation but that some other mechanisms like production of growth hormones by this bacterium may be responsible.     

Collecting maize (<Emphasis Type="Italic">Zea mays</Emphasis> L. convar. <Emphasis Type="Italic">mays</Emphasis>) with potential technological ability for bread making in Portugal

The Portuguese maize bread (“broa”) manufactured from traditional maize landraces still plays an important economic and social role on Central and Northern rural communities of the country. However the traditional maize landraces agricultural systems are changing. Local maize landraces are in risk of disappearing because of the progressive adoption of hybrid varieties not suitable for bread production. These changes are contributing to a major loss of genetic diversity. An expedition took place in the Central region of Portugal (Beira Interior and Beira Litoral) with the purpose of collecting enduring maize landraces with technological ability for bread production and to access the possibility of establishing a participatory plant breeding and conservation program. A total of 51 different maize landraces and 175 other varieties of associated crops were collected. Maize landraces showed to maintain high diversity and potential for improvement. The production relayed on small farms with polycrop, quality oriented, sustainable systems. A participatory plant breeding and conservation program would be possible on this region with the proviso that local authorities would be involved. This program would allow a higher valuation of these maize populations, contributing to halt the current loss of these unique Portuguese maize landraces.     

Case studies on breeding systems and its consequences for germplasm conservation: 3. Electrophoretic mobility of phaseolins in wild populations of Lima beans (Phaseolus lunatus L.) in the Central Valley of Costa Rica

We studied the variation in the electrophoretic mobility of phaseolins of 202 individual seeds from thirty-eight wild populations of Lima beans (Phaseolus lunatus) in the Central Valley of Costa Rica. We found four main polypeptides or banding regions with molecular weights which ranged from 21.5 to 31 kDaltons. Two of these peptides were polymorphic, and their combination resulted in four distinct genotypes. The frequency of the different genotypes varies among populations, but two of them, namely B¹¹C¹¹, B²²C²², were found in most of the populations studied (37 out of 38 populations examined). Overall, there was a lack of heterozygotes among the individuals examined, which strongly suggests that there is gametic disequilibrium for the encoding regions involved in the synthesis of these polypeptides. When we arranged the populations into eight groups according to their proximity and phenology, we found that the distribution of the peptides was also variable among them. The possible association between the distribution of the patterns of electrophoretic mobility observed and climatic factors, such as mean annual rainfall, mean annual temperature and mean annual relative humidity is discussed.     

Growth and nitrogen nutrition of maize (Zea mays L.) in soil treated with the nitrification-inhibiting insecticide Baythroid

A pot experiment was conducted to compare the uptake and dry matter production potential of NH _inf4 ^sup+ and NO _inf3 ^sup- and to study the effect of Baythroid, a contact poison for several insect pests of agricultural crops, on growth and N uptake of maize (Zea mays L.). Nitrogen was applied as (¹⁵NH₄)₂SO₄, K¹⁵NO₃, or ¹⁵NH₄NO₃ and in one treatment Baythroid was combined with ¹⁵NH₄NO₃. Source of N had, in general, a nonsignificant effect on dry matter and N yield, but uptake of NO _inf3 ^sup- was significantly higher than that of NH _inf4 ^sup+ when both N sources were applied together. Substantial loss of N occurred from both the sources, with NH _inf4 ^sup+ showing greater losses. Baythroid was found to have a significant positive effect on dry matter yield of both root and shoot; N yield also increased significantly. Uptake of N from both the applied and native sources increased significantly in the presence of Baythroid and a substantial added nitrogen interaction (ANI) was determined. The positive effect of Baythroid was attributed to: (1) a prolonged availability of NH _inf4 ^sup+ due to inhibition of nitrification, (2) an increased availability of native soil N through enhanced mineralization, and (3) an enhanced root proliferation.     

Conservation in a changing world: <Emphasis Type="Italic">in situ</Emphasis> conservation of the giant maize of Jala

Using survey data from Jala, Mexico, this case-study evaluates in situ maize conservation of the variety ‘Jala’ (Zea mays L.). Though historically ‘Jala’ was the dominant variety grown in the valley of Jala, today less than 20% of farmers grow it on only 5% of the maize area. Younger growers of the ‘Jala’ variety specialize in it, growing relatively large amounts for niche markets. Older, diversified farmers grow small areas for household use and to compete in a local contest. Conservation of the ‘Jala’ variety has been heavily influenced by shifting ideal concepts of maize, as determined by market and consumption demands and by a contest designed to promote in situ conservation. The current move away from nationalized purchasing may favor ‘Jala’s’ continued conservation.     

Kinetics of zinc uptake by mycorrhizal (VAM) and non-mycorrhizal corn (Zea mays L.) roots

Summary The kinetics of Zn absorption were studied in mycorrhizal (Glomus macrocarpum) and non-mycorrhizal roots of corn (Zea mays L.) at pH 6.0 at Zn concentrations of 75 mol to 1.07 mol m^-3. Five concentration-dependent phases of Zn absorption were recognized; phase 0 (1.5–4.0 mmol m^-3) was linear but the other four phases (4.0 mmol to 1.07 mol m^-3) obeyed Michaelis-Menten kinetics. At low concentrations (less than 4 mmol m^-3), sigmoidal kinetics of Zn absorption were observed. The absorption of Zn by mycorrhizal maize was greater at low concentrations but decreased at higher levels. This appeared to be a result of a higher maximal uptake rate in phase 1 and lower K _m values in the subsequent phases. Kinetic models yielding continuous isotherms could not account for the observed multiphasic pattern.Research paper no. 6820 through the Director, Experiment Station, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, UP, India     

Influence of the inoculation density of the rhizopseudomonad strain 7NSK2 on the growth and the composition of the root microbial community of maize (Zea mays) and barley (Hordeum vulgare)

Summary At least 10⁵–10⁶ viable cells of the rhizopseudomonad strain 7NSK2^* had to be applied per seed of maize cultivar Beaupré and barley cultivar Iban in order to obtain a beneficial effect on plant growth under greenhouse conditions. In pot experiments where an increase in plant growth, varying between 15% and 25%, was observed, the introduced strain 7NSK2^* constituted at least 20% of the bacterial root colonizers. This colonization provoked a shift in the fungal rhizospheric community. Due to the inoculation with 7NSK2, Penicillium spp. became the dominant isolates, while Trichoderma spp. were the dominant isolates in pot experiments with low and inefficient inoculum levels of 7NSK2^*.     

Cassava Genetic Resources: Extinct Everywhere in Brazil

Wild cassava, Manihot spp. are sources of many useful characters. Their occurrence in natural habitats is diminishing day after day and a number of them almost have come to extinction. It is suggested that in situ conservation should be followed. It is appealed to concerned authorities to make an effort for their salvation.     

Cadmium bioavailability to Nicotiana tabacum L., Nicotiana rustica L., and Zea mays L. grown in soil amended or not amended with cadmium nitrate

Summary Mature (flowering) tobacco (Nicotiana tabacum cv. PBD6, Nicotiana rustica cv. Brasilia) and maize (Zea mays cv. INRA 260) plants were grown in an acid sandy-clay soil, enriched to 5.4 mg Cd kg^–1 dry weight soil with cadmium nitrate. The plants were grown in containers in the open air. No visible symptoms of Cd toxicity developed on plant shoots over the 2-month growing period. Dry-matter yields showed that while the Nicotiana spp. were unaffected by the Cd application the yield of Z. mays decreased by 21%. Cd accumulation and distribution in leaves, stems and roots were examined. In the control treatment (0.44 mg Cd kg^–1 dry weight soil), plant Cd levels ranged from 0.4 to 6.8 mg kg^–1 dry weight depending on plant species and plant parts. Soil Cd enrichment invariably increased the Cd concentrations in plant parts, which varied from 10.1 to 164 mg kg^–1 dry weight. The maximum Cd concentrations occurred in the leaves of N. tabacum. In N. rustica 75% of the total Cd taken up by the plant was transported to the leaves, and 81% for N. tabacum irrespective of the Cd level in the soil. In contrast, the Cd concentrations in maize roots were almost five times higher than those in the leaves. More than 50% of the total Cd taken up by maize was retained in the roots at both soil Cd levels. The Cd level in N. tabacum leaves was 1.5 and 2 times higher at the low and high Cd soil level, respectively, than that in N. rustica leaves, but no significant difference was found in root Cd concentrations between the two Nicotiana spp.Cd bioavailability was calculated as the ratio of the Cd level in the control plants to that in the soil or as the ratio of the additional Cd taken up from cadmium nitrate to the amount of Cd applied. The results showed that the plant species used can be ranked in a decreasing order as follows: N. tabacum > N. rustica > Z. mays.     

Morphological and farmers cognitive diversity of barley (Hordeum vulgare L. [Poaceae]) at Bale and North Shewa of Ethiopia

Twenty two accessions of barley landrace/farmers' varieties collected from Bale and North Shewa in situ conservation zones were characterised using 18 qualitative and quantitative morphological traits. Phenotypic frequencies for individual qualitative characters across in situ conservation zones, districts, and strategic sites (localities) have shown mixed and some peculiar patterns. Varieties from Bale conservation zone are predominantly white-yellow seeded whereas varieties from North Shewa are purple-black seeded. White-yellow seeded varieties are more frequent in the lower altitude examined, and the purple-black seeded varieties are more frequent at the highest altitude ( 2650 ). While six row types occur at higher frequencies in almost all the sites in North Shewa, the irregular types are dominant at Bale. The highest frequency of six row types is found at an altitude 2650 , whereas the two rowed and irregular types are found below 2650 . Two row types occur at low frequency at both sites. At Bale, glume awn shorter than glume is the most frequent character whereas glume awn longer than glume is more frequent at North Shewa. Glume awn longer than glume appears to increase with increasing altitude. Caryopsis cover is relatively monomorphic with respect to distribution across zones. The covered types are most frequent in almost all altitudinal ranges. Estimates of diversity index (H) for individual qualitative characters suggest that polymorphism is common in varying degrees for most characters, which indicates the existence of wide range of variation. On the basis of cross validation using discriminant function among the quantitative characters, the landrace varieties from North Shewa seem to be more diverse than the ones from Bale. From the results of multiple regression analysis, the zonal variation is significantly associated with all the quantitative morpho-agronomic characters except plant height. The great majority of the varieties from Bale were perfectly identified and named by farmers. The reliability analysis confirmed that there was a remarkable positive degree of consistency between farmers naming of landrace varieties.     

A preliminary study of the genetic diversity of Bolivian oca (<Emphasis Type="Italic">Oxalis tuberosa</Emphasis> Mol.) varieties maintained<Emphasis Type="Italic"> in situ</Emphasis> and<Emphasis Type="Italic"> ex situ</Emphasis> through the utilization of ISSR molecular markers

ISSR molecular markers have been used to investigate genetic diversity of oca (Oxalis tuberosa Mol.), an Andean neglected tuber crop species. Sampling procedure allowed a preliminary study of the genetic diversity at the intra- and intervarietal levels. Twenty tuber lots conserved in situ in the microcentre of Candelaria and ex situ in the Toralapa Centre (Bolivia) were identified. Four ISSR primers amplified a total of 25 fragments of which 17 (68%) were polymorphic. These experiments show that the structure of oca varieties is mainly based upon vernacular names with a greater differentiation among tuber lots than within them, supporting agromorphological data. ISSR technique enlightened the existence of heterogeneous varieties in oca and divergence between in situ and ex situ conservation strategies. These observations are potentially linked to the different ways of management of tubers in these two conservation systems.     

Ammonia volatilization from nitrogen fertilizers surface-applied to corn (Zea mays) and grass pasture (Dactylis glomerata)

Summary The major agronomic concern with NH₃ loss from urea-containing fertilizers is the effect of these losses on crop yields and N fertilizer efficiency. In this 2-year study, NH₃ volatilization from surface-applied N fertilizers was measured in the field, and the effects of the NH₃ losses detected on corn (Zea mays L.) and orchardgrass (Dactylis glomerata L.) yield and N uptake were determined. For corn, NH₄NO₃ (AN), a urea-AN solution (UAN), or urea, were surface-broadcast at rates of 0, 56 and 112 kg N ha^–1 on a Plano silt loam (Typic Argiudoll) and on a Fayette silt loam (Typic Hapludalf). Urea and AN (0 and 67 kg N ha^–1) were surface-applied to grass pasture on the Fayette silt loam. Significant NH₃ losses from urea-containing N sources were detected in one of four corn experiments (12%–16% of applied N) and in both experiments with grass pasture (9%–19% of applied N). When these losses occurred, corn grain yields with UAN and urea were 1.0 and 1.5 Mg ha^–1, respectively, lower than yields with AN, and orchardgrass dry matter yields with urea were 0.27 to 0.74 Mg ha^–1 lower than with AN. Significant differences in crop N uptake between N sources were detected, but apparent NH₃ loss based on N uptake differences was not equal to field measurements of NH₃ loss. Rainfall following N application markedly influenced NH₃ volatilization. In corn experiments, NH₃ loss was low and yields with all N sources were similar when at least 2.5 mm of rainfall occurred within 4 days after N application. Rainfall within 3 days after N application did not prevent significant yield reductions due to NH₃ loss from urea in grass pasture experiments.     

Evaluation of genetic diversity of bread wheat landraces from Pakistan by AFLP and implications for a future collection strategy

We used amplified-fragment-length polymorphism (AFLP) markers to evaluate genetic variation in a set of bread wheat (Triticum aestivum L.) landraces and improved materials. Landraces collected from different geographic and agro-ecological zones in Pakistan in 1987, 1989 and 1991 were separated into two groups based on their geographic origins: northern (Himalaya) and south-western (Balochistan) Pakistan. Six AFLP primer combinations detected 453 AFLP markers in the 43 landrace accessions and four high-yield varieties (HYVs). Of these, 225 (49.67%) were rare (shared with < 5% of all accessions). Among these rare alleles, 23 (10.22%) were common in the Himalaya (shared with > 10% of accessions collected there) but were not found in Balochistan. We conclude that there is a higher probability of collecting rare alleles at overall, but which are in contrast locally common ones in the Himalayan region. Gene diversity was 0.17 in the Himalayan group and 0.15 in the Balochistan group. Considerable genetic variability was found in both groups. Accessions from different agro-ecological zones were indistinguishable by cluster analysis, indicating intensive seed trading within the country. Cluster analysis indicated that the landraces and the HYVs are genetically distinct; suggesting that genetic erosion of wheat landraces has been unlikely taken in place. This study provides an example of how analysis of existing materials and data, can serve as a basis for future collection planning and conservation policies.