Molecular marker analysis of genetic variation characterizing a grasspea (Lathyrus sativus) collection from central Italy

Levels of genetic similarity characterizing 20 grasspea (Lathyrus sativus L.) populations collected in central Italy (17 populations in the Marche region and three populations in the Abruzzo region) were analysed with amplified fragment length polymorphism (AFLP) molecular markers. Two main clusters were found: one included large‐seeded populations from farms that were not market‐oriented (named Household populations) and the second, small‐seeded populations, cultivated in market‐oriented farms (named Commercial populations). Relationships among populations collected in different regions were found, although one population of the Abruzzo region was placed between the two main clusters, suggesting a possible further genetic differentiation within this grasspea germplasm collection. Principal component analysis based on AFLP marker frequency was effective in identifying polymorphic markers showing high discriminating ability between clusters H and C. In particular, seven markers showing high positive and three markers with low negative PC1 scores showed an almost cluster‐specific distribution. These results will be useful for enhancing Italian grasspea germplasm use in plant‐breeding programmes and for extending grasspea cultivation within the sustainable agricultural systems of central Italy.     

Variation and association of morphological and biochemical characters in grass pea (Lathyrus sativus L.)

Fifty grass pea land race populations of Ethiopia that were selected based on administrative regions and different altitude classes were used in this study to determine variability in morphological characters, Oxalyl Diamino Propanoic acid(ODAP) – a causative agent of lathyrism, and protein contents and their associations. Highly significant differences were observed among the populations for most of the morphological characters. Populations collected from Gondar region and the higher altitude group (>2550 m) showed the highest mean and coefficient of variation indicating the presence of high genetic diversity in this region and altitude group. ODAP analysis from single plants showed significant variation both within a population and between populations. Four individual plants with low ODAP contents ranging from 0.149% to 0.182% (range within safe level) were identified in populations collected from different regions, indicating the diversity and variation of this trait with in one population and between regions. These low ODAP lines are considered important for further breeding. The association of ODAP with grain yield, biomass, plant height and seed size was negative, suggesting that the selection of tall and late maturing varieties with large seed size and high grain yield potential will enable the development of varieties with low ODAP content. This revised version was published online in August 2006 with corrections to the Cover Date.     

Current status and future strategy in breeding grasspea (Lathyrus sativus)

Efforts in grasspea (Lathyrus sativus) improvement have increased since the development of lines that are very low in the neurotoxin Beta-N-oxalyl-L-alpha-beta-diamino propionic acid (ODAP); also referred to as Beta-N oxalyl-amino-L-alanine (BOAA). Many programs now address several related aspects of improvement simultaneously. These include reduced ODAP concentrations, insect and disease resistance, nitrogen fixation, agronomic practices, fodder and forage production, and components for increased yielding ability. The coordinated, multidisciplinary approach now being applied to the genetic improvement of grasspea should allow the potential of this largely neglected grain legume to be fully realized.     

The Gene Pools of the Grasspea (Lathyrus sativus L.)

Interspecific hybridization between the grasspea, Lathyrus sativus, and 15 wild species in Section Lathyrus is reported. Only two species, L. amphicarpos and L. cicera produced viable F¹ hybrids with low fertility when crossed with L. sativus as male parent. Crosses with six other species produced pods following pollination when L. sativus was the male, but seedlings were inviable, seeds did not germinate or pods were empty or had totally shrivelled seeds. When L. sativus was the female parent, only one cross with L. gorgoni produced an F¹, but the seedling was inviable. The germplasm resources of the grasspea are identified, with L. amphicarpos and L. cicera placed in the secondary gene pool and the other species in the tertiary gene pool. The definition of these germplasm resources is discussed in terms of grasspea improvement through plant breeding.     

Vitamin C,B5, and B6 contents of segregating potato populations detected by GC‐MS: a method facilitating breeding potatoes with improved vitamin content

Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography‐mass spectrometry (GC‐MS). Two diploid potato populations derived from two different crosses were analyzed. Plants were grown at two locations under different environmental conditions. Measurements of vitamin content of tubers showed high variation in both populations. A weak correlation was found between the vitamin concentrations of tubers harvested at the two locations. Correlations between the mean values of vitamins in field‐grown tubers and in different tissues derived from in vitro or greenhouse‐grown potato plants were also determined. A very high correlation between the vitamin content of field‐ and greenhouse‐grown tubers, and between field‐grown tubers and sink leaves of greenhouse‐grown plants was detected. This finding can facilitate breeding by preselection of individuals with improved vitamin content under greenhouse conditions at their early developmental stage.     

Inheritance of neurotoxin (ODAP) content, flower and seed coat colour in grass pea (Lathyrus sativus L.)

Summary A strong epidemiological association is known to exist between the consumption of grass pea and lathyrism. A neurotoxin, -N-Oxalyl-L-, -diaminopropanoic acid (ODAP) has been identified as the causative principle. This study was undertaken to investigate the mode of inheritance of the neurotoxin ODAP, flower and seed coat colour in grass pea. Five grass pea lines with low to high ODAP concentration were inter-crossed in all possible combinations to study the inheritance of the neurotoxin. Parents, F₁ and F₂ progenies were evaluated under field condition and ODAP analyzed by an ortho-phthalaldehyde spectrophotometric method. Many of the progenies of low x low ODAP crosses were found to be low in ODAP concentration indicating the low ODAP lines shared some genes in common for seed ODAP content. The F₁ progenies of the low ODAP x high ODAP crosses were intermediate in ODAP concentration and the F₂ progenies segregated covering the entire parental range. This continuous variation, together with very close to normal distribution of the F₂ population both of low x low and low x high ODAP crosses indicated that ODAP content was quantitatively inherited. Reciprocal crosses, in some cases, produced different results indicating a maternal effect on ODAP concentration. Blue and white flower coloured lines of grass pea were inter-crossed to study the inheritance of flower colour. Blue flower colour was dominant over the white. The F₂ progenies segregated in a 13:3 ratio indicating involvement of two genes with inhibiting gene interactions. The gene symbol LB for blue flower colour and LW for white flower colour is proposed.     

Rapid screening and selection of low-caffeine-containing tea (Camellia sinensis) trees by a colorimetric method

A simple and rapid colorimetric method for the determination of caffeine has been developed. Tea tree lines from the Hadong region in South Korea differ widely in their caffeine contents and this colorimetric method facilitated an efficient screening strategy for the selection of low‐caffeine‐containing lines. A high correlation was observed between the values obtained with colorimetric and high‐performance liquid chromatographic analysis. Among the selected tree lines, the caffeine content for the lowest caffeine‐containing tea tree line (H‐19) was 178.35 μg/g dry weight basis, which was 61‐fold less productive than the highest caffeine‐producing tea tree line (H‐82). Caffeine contents of the selected tea tree lines remained similar during the corresponding collection periods. The colorimetric method is of great practical value in screening low‐caffeine‐containing tea trees.     

Genotype-environment interaction for seed yield and ODAP concentration of Lathyrus sativus L. and L. cicera L. in Mediterranean-type environments

In 1994, 407 L. sativus and 96 L. cicera lines were evaluated for phenology, seed yield and seed ODAP (a neurotoxin) concentration in the Mediterranean-type climate of south-western Australia. A selected number of lines from this study were grown at three sites in south-western Australia in 1995 and 1996, to examine genotype-environment interactions on seed yield and ODAP concentration in the seed of the two Lathyrus spp. Principal components analysis showed that L. sativus lines grown in 1995 and 1996 could be divided into two geographical origins: Indian sub-continent and Mediterranean/European. Those lines of Mediterranean/European origin were consistently higher yielding (both in 1994 and 1995/96), with much larger seeds and later phenology. In L. cicera seed yield was closely associated with greater mean seed weight and to a lesser extent with early phenology. The genotype-environment study indicated that higher yielding lines of both species generally responded to favourable environments; in the case of L. sativus the Mediterranean/European lines and for L. cicera the larger seeded, earlier phenology lines. For both species, genotype was the most important determinant of ODAP concentration and environment had less influence. Genotype-environment interactions had no effect on seed ODAP concentrations. In terms of seed yield, both species showed substantial potential in the environments tested. Further improvements in adaptation, seed yield and reduction in ODAP are possible in both species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Variation in cold tolerance and spring growth among Italian white clover populations

Seven populations collected at different altitudes in northern Italy, two Ladino breeding populations and two control cultivars (AberHerald and Grasslands Huia) of white clover (Trifolium repens L.) were included in a series of experiments analysing: (i) levels of cold tolerance using artificial and field-based methods; (ii) relationships between these measures of cold tolerance; (iii) components of spring yield, various physiological traits, and their relationships with cold tolerance. Rates of seedling and growing point mortality in the populations over winter, assessed in separate field experiments, were closely related (r = 0.85). Grasslands Huia showed the highest death rates, and material originating from high altitudes the lowest. The LT₅₀ value, i.e., the temperature at which 50% of the growing points would die, estimated by an artificial freezing test, was significantly correlated with field-based measures of seedling (r = 0.64) and growing point (r = 0.84) mortality. The existence of these correlations is of potential interest for the development of indirect selection criteria for complex and expensive-to-evaluate traits such as winter survival in field plots. Besides being reliable, in this study the artificial assessment was also sensitive, providing a greater degree of separation of the populations means than field-based measures. Of the several physiological traits (water content,concentrations of water soluble and total non-structural carbohydrate, and water soluble protein content of stolons) measured at a mid-winter sampling date under field conditions, the only character showing significant variation between populations was soluble protein content. There was a slight trend for material with a higher protein content to exhibit greater field-based values of cold tolerance. High altitude populations tended to have low spring yields. The highest spring yield was found in one of the Ladino populations. The study identified two populations which combined, to differing extents, cold tolerance and spring yield characteristics that would be of potential use in breeding for specified agronomic/climatic zones. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phenotypic polymorphism of six enzymes in the grasspea (Lathyrus sativus L.)

Summary The phenotype variation in six enzymes, 6-phosphogluconate dehydrogenase (6-PGD), malate dehydrogenase (MDH), peroxidase (PRX), isocitrate dehydrogenase (IDH), galactose dehydrogenase (GD) and glutamate oxaloacetate transaminase (GOT), was investigated using horizontal starch gel electrophoresis in 52 accessions of the grasspea, Lathyrus sativus. Phenotypic polymorphism was observed for all six enzymes. High phenotypic polymorphism (Pj) was observed for PRX and 6-PGD, while there was little polymorphism for GOT, with only two accessions showing variation. There was no correlation between phenotypic polymorphism and region of origin, or groupings of accessions made on the basis of flower colour. Tentative genetic interpretations of banding patterns are given for five of the enzyme systems. The level of apparent heterozygosity was higher than expected in this predominantly autogamous species. The level of variation in the grasspea is discussed in terms of its potential for exploitation through plant breeding.     

Contents of total protein and β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP) in a collection of Lathyrus cicera of the Bank of Plant Germplasm of Cuenca (Spain)

One hundred and thirty-four accessions of Lathyrus cicera from the Bank of Plant Germplasm of Cuenca (BGV-Cuenca, Spain) have been analysed for total protein and β- N -oxalyl- l -α,β-diaminopropionic acid (ODAP) contents in order to identify those with a suitable nutritional quality for animal feeding. There was a large variability in both traits, the content of total protein ranged from 22.95% to 30.98% with a mean value of 27.62%, and the ODAP from 0.09% to 0.30% with a mean value of 0.17%. The accessions from Castilla y León and Castilla-La Mancha showed higher percentages of protein and ODAP as compared with those from Andalucía, Greece and Extremadura. Nevertheless, total protein and ODAP contents showed a low positive correlation ( r  = 0.396, P = 0.01). Several accessions showed a content of ODAP similar to that of the variety 'Chalus' and two accessions seemed very suitable for animal feeding because of their high content of protein and low ODAP.     

Inheritance of sesquiterpene carboxylic acid synthesis in crosses of Lycopersicon hirsutum with insect-susceptible tomatoes

Sesquiterpene carboxylic acids (SCA) from Lycopersicon hirsutum f. typicum Humb, & Bonpl, accession LA 1777 confer host‐plant resistance to Helicoverpa zea (Boddie) and Spodoptera exigua (Hübner), two Lepidopteran (Noctuidae) insect pests of the cultivated tomato. L. esculcntum Mill. Hybrids were made between LA 1777 and two accessions of insect‐susceptible tomatoes that do not produce SCA, L. hirsutum LA 1033 and the L. L. esculentum cultivar Chico III, F₂ and backcross progeny of both crosses were evaluated for variation in amounts of total SCA in replicated field and greenhouse plantings. Analysis of variance of total SCA in a set of F₂ plants of the cross LA 1033 × LA 1777 demonstrated significant variation in SCA attributable to genotype, environment and their interaction. Segregation for high levels of SCA in the populations suggests polygenic inheritance. Broad sense heritability was 0.84, which indicates that phenotypic selection for plants with high SCA is feasible in a backcross‐breeding programme for the introgression of SCA‐mediated insect resistance into tomato cultivars.     

Yield and Osmotic Adjustment Capacity of Barley Under Terminal Water-Stress Conditions

This paper reports the influence of the osmotic adjustment capacity and turgor potential (TP) on tolerance to drought in 12 cultivated genotypes, six breeding lines and six cultivars of barley (Hordeum vulgare L.) under terminal water‐stress conditions. When the crops reached the flag leaf stage, half of the experimental plots in which they were grown were submitted to water stress treatment and the remainder maintained under optimal irrigation conditions. Differences were seen in the osmotic adjustment, relative water content (RWC) and water potential (WP) of the different genotypes. Two of the breeding lines showed the greatest osmotic adjustment capacities, maintaining their TPs better than the other genotypes studied. A positive, significant correlation was found between yield and osmotic adjustment capacity, RWC and WP under water‐stress conditions. Osmotic adjustment capacity and TP were linearly related, indicating that as water stress increases osmotic adjustment favours the maintenance of higher TPs. Significant correlations were found between osmotic adjustment capacity and grain filling rate and grain yield. Under conditions of terminal water stress, yield was negatively correlated with the number of days to ear emergence; no correlation was found between osmotic adjustment capacity and earliness. Early ear emergence, a good osmotic adjustment capacity and high RWC values all contributed to yield increases under terminal water‐stress conditions.     

Genetic analysis and fine mapping of tms9, a novel thermosensitive genic male‐sterile gene in rice (Oryza sativa L.)

Two‐line hybrid rice as a novel hybrid breeding method has huge potential for yield increasing and utilization of intersubspecific heterosis, and it is of major significance for the food security of rice‐consuming populations. Zhu1S is a thermosensitive genic male‐sterile line of rice with low critical temperature and excellent combining ability, which has been widely exploited as a female parent in Chinese two‐line hybrid rice breeding. Here, genetic mapping in F₂ populations was used to show that its male sterility is inherited as a single recessive gene and that responsible gene (termed tms9) lies on the short arm of chromosome 2. A high‐resolution linkage analysis which was based on the Zhu1S/R173 F₂ population found that the thermosensitive genic male‐sterile gene tms9 of Zhu1S was fine mapped between insertion–deletion (Indel) markers Indel 37 and Indel 57, and the genetic distance from the tms9 to the two markers was 0.12 and 0.31 cM, respectively. The physical distance between the two markers was about 107.2 kb. Sequence annotation databases showed that the two Indel markers (Indel 37 and Indel 57) were located on two BAC clones (B1307A11 and P0027A02). There are sixteen open reading frames (ORF) present in this region. The results of this study are of great significance for further cloning tms9 and molecular marker–assisted selection.     

Marker analysis for quantitative traits in sugar beet

Many economically important traits are inherited quantitatively and are analysed by breeders in replicated field trials. If dense maps are available, chromosomal regions containing quantitative trait loci (QTL) can be identified and this opens up the possibility of preselecting for quantitative traits in the laboratory. In this study, QTL analysis for yield and yield components in sugar beet is used in two different populations tested in several environments in both populations, QTL were detected for all traits investigated, and their predictive value in breeding schemes was analysed by correlating predicted with observed values. Tolerance to Rhizomania, caused by a gene on chromosome 3, was the main source of genotype‐environment interaction in one population, allowing selection on a QTL basis within macro‐environments with or without Rhizomania infestation, respectively. No clear results were found for the second population tested in environments with and with‐out Cercospora infestation. Consequences for breeding strategies are discussed.     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

Intra- and inter-population variations in grain protein percentage in wild tetraploid wheat,Triticum turgidum var. dicoccoides

Summary Grain protein percentage (GPP) was studied in 910 accessions of the wild tetraploid wheat, Triticum turgidum var. dicoccoides, collected from 22 populations representing different ecogeographical conditions in Israel. High values of GPP were found, ranging from 19.7% to 28.0% for population means, and from 14.1% to 35.1% for single accessions. Marginal populations had usually lower GPP and smaller intra-population variation than central ones. Repeated sampling of some central populations for four consecutive years revealed relatively large intra-population fluctuations. A high and significant genetic component of variation was found within and between populations by a nested analysis of variance in two nurseries. However, the regression coefficients of parents vs. offsprings were relatively low, indicating a smaller genetic component of variation which may be accounted for by a significant genotype × environment interaction. No correlation was found between GPP and ecological factors, except for soil type: accessions growing on terra-rossa had higher GPP than those growing on basaltic soil. Accessions with black glumes, or with glabrous auricles, or with large grains exhibited high GPP values. A strategy for collecting accessions with high GPP is presented, and the potential use of high GPP genotypes in breeding programs is discussed.     

Carbon Isotope Discrimination, Leaf Ash Content and Grain Yield in Bread and Durum Wheat Grown under Full-Irrigated Conditions

Integrative physiological criteria, such as carbon isotope discrimination (Δ) and (mineral) ash content (m_a) have been found to be very useful, under drought conditions, to elucidate the association between yield gains and variation of photosynthesis‐related traits and orientate future breeding efforts. Information on this association is scarce under irrigated conditions. The relationships between Δ, m_a and yield were studied in bread wheat (Triticum aestivum L.) and durum wheat (Triticum turgidum L. var. durum) under optimal (drip) irrigation in the arid conditions of north‐west Mexico. Carbon isotope discrimination was analysed on leaves at booting stage and anthesis and on grain at maturity, whereas ash content was measured on the flag leaf at anthesis and maturity. At anthesis, there were differences between bread and durum wheat during grain filling for Δ, but not for m_a. No relationship was found between grain yield and Δ. Leaf ash content at anthesis and maturity showed a broad variability within each species and were associated with grain yield. These results suggest that ash content in leaves could be also used as predictive criteria for yield not only under drought, but also under irrigated conditions, particularly when evaporative demand is high.     

Identification of genetic variation in Brassica napus seeds for tocopherol content and composition using near‐infrared spectroscopy technique

Tocopherol is an essential fat‐soluble nutrient for humans. Increasing the tocopherol content in Brassica napus seeds can add value to rapeseed vegetable oil; this has become an important breeding target. However, there is no efficient and non‐destructive method for selecting rapeseed accessions with high tocopherol contents. Here, we report the first near‐infrared reflectance spectroscopy (NIRS)‐based technique for predicting rapeseed tocopherol content. Individual seed tocopherol compositions were estimated from 373 rapeseed genotypes of different origins. This method and chemical methods produced comparable predicted values of the tocopherol constituents in the seeds. Three equations were generated for the prediction of tocopherol content by using a modified partial least squares (MPLS) model. The total tocopherol content for the determination coefficient of cross‐validation (R²_cv) (0.74), determination coefficient (RSQ) (0.76) and one minus the ratio of unexplained variance to total variance (1‐VR) (0.65) values indicates a strong correlation between the calibration and validation sets. Overall, our model confirmed the NIRS method as feasible for predicting tocopherol content in rapeseed and as an efficient screening tool for future breeding programs.     

Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F₂ and F₃ populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.