Association mapping of loci linked to copper,phosphorus, and potassium concentrations in the seeds of C. arietinum and C. reticulatum

Due to its high nutritional value, chickpea is one of the most important and cost-effective legumes for human diet. Nutrient elements, such as Cu, P, K have numerous essential functions for the human metabolism. In this study, association mapping of loci linked to the seed Cu, P and K concentrations were performed on a population consisting of 107 Cicer reticulatum and 73 Cicer arietinum individuals in four environments (two locations x two years). A total of 121,840 SNPs were genotyped across 180 individuals by GBS analysis. The association mapping between the SNP markers and the seed Cu, P, K concentrations were identified and eight SNPs were found to be significantly associated with variations in three nutrient elements in more than two environments This research suggests that association mapping is a useful methodology for the identification of loci controlling the Cu, P and K uptake in chickpea seeds for further association mapping, molecular breeding, and marker-assisted selection and plant breeding studies and provides a broader understanding of the relationship between the investigated Cicer species and the effects of environmental conditions.     

The seed proteins of chick pea: Comparative studies ofCicer arietinum,C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatum Ladiz. andC. echinospermum Davis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.     

Starch degradation during germination of Civer arietinum L. seeds.

The variations in starch and soluble sugar content, in phosphorylase and amylase activities in cotyledons of germinating seeds of Cicer arietinum L. are determined. Results from various experiments prove that the alpha-amylases are chiefly responsible for amylase activity. Phosphorylase plays an important r?le during the first two days of germination, but it is relegated to a secondary position as the amylase activity increases. Disc electrophoresis on polyacrylamide gel shows the existence of a phosphorylase throughout germination, and detects two alpha-amylases after 48 and 96 h germination respectively. The increase in alpha-amylase activity during germination is due to de novo synthesis of the two isoenzymes, since both are inhibited by cycloheximide and actinomyces D. This de novo synthesis depends on some embryo produced factor, unreplaceable either by giberellic acid or by kinetin.     

Germination of Cicer arietinum seeds and thiourea-induced phytotoxicity

Thiourea, hydroxyurea, phenylthiourea, methylurea, methylthiourea, thiosemicarbazide and 2,2-dithiodipyridine affected the germination of Cicer arietinum L. cv. Castellana (chick-pea) seeds. Microscopic observations of the subapical zone of the radicle showed that thiourea induced an increase in cell volume and length when compared with control seeds germinated at 25° or 30°C in water. These results emphasize the importance of the processes controlling solute and water uptake during early germination of chick-pea seeds. In contrast to this stimulation of volume increase, the thiourea-treated seedlings were unable to synthesize chlorophyll when exposed to light. This toxic effect was reduced when thiourea was administered only during the first few hours of germination. Thiourea also caused an increase in the uptake of ³H-thymidine and ¹⁴C-leucine but it decreased their incorporation into DNA and protein, respectively. These results suggest a stimulation of plasmalemma exchange activities, but toxic or inhibitory effects on other metabolic processes necessary for normal development of seedlings.     

Changes in the cellular permeability of the embryonic axis inGcer arietinum L. seeds during germination

The effect of short chain fatty acids on the cellular permeability of embryonic axis inGcer arietinum seeds was studied. Octanoic (OCT) and nonanoic (NON) adds, which reduce both germination and growth of the embryonic axis and raise the inhibitor effects of the supraoptimal temperatures (30?C), induce a greater ionic efflux into the medium (conductivity). NON reduces glucose (3-0-MG) and K⁺ (⁸⁶Rb) uptake during the germinative process, this action being counteracted by fusicoccin (FC) at optimal (25?C) and supraoptimal temperatures (30 ?C). Tonoplast and plasmalemma increase their permeability to the K⁺ efflux when NON is present. Addition of NON+FC gives rise to higher values in the efflux rate, the vacuolar compartment being the most affected. Temperatures around zero (2 ?C) notably reduce the isotope efflux from cytosol and vacuole. NON acid does not significantly affect the efflux of³H-ABA, suggesting that it does not cause any important changes in the phytohormone compartmentation.     

Properties of a lectin purified from the seeds of Cicer arietinum

A lectin was isolated from seed extracts of Cicer arietinum by (NH4)2SO4 precipitation and subsequent ion exchange chromatography and gel filtration. Affinity chromatography on desialylated human IgM coupled to AH-Sepharose was also performed, but the amount bound was very low. The lectin has a molecular mass of about 44000 Da, as determined by ultracentrifugation and gel filtration. Dodecyl sulphate polyacrylamide-gel electrophoresis showed one band corresponding to a molecular mass of 26000 Da. N-Terminal amino acid sequence analyses indicate only one type of chain, suggesting that the lectin is probably dimeric. The amino acid composition is given. Papainized human erythrocytes of the different ABO groups were agglutinated equally well by the Cicer lectin, whereas untreated cells reacted weakly and only in the presence of bovine serum albumin. Simple sugars did not inhibit the agglutination, but some glycoproteins did inhibit. The lectin is probably nonmitogenic against human lymphocytes. Antigenic analyses in an enzyme-linked immunosorbent assay (ELISA) showed only a weak cross-reaction between Cicer and the lectins in the Vicieae tribe. Thus, our physicochemical and antigenic studies of the Cicer lectin support the botanical reasons recently given for removing the genus Cicer from the Vicieae tribe.     

Endogenous hormone profiles during early seed development of C. arietinum and C. anatolicum

Cicer anatolicum, a perennial species, has ascochyta blight resistance superior to that found in the cultivated chickpea. However, hybridization barriers during early stages of embryo development curtail access to this trait. Since hormones play an essential role in early embryo development, we have determined the hormone profiles of 4-, 8-, and 12-day old seeds from a Canadian chickpea (Cicer arietinum L.) cv. CDC Xena, from Indian cvs. Swetha and Bharati, and from a perennial accession of C. anatolicum (PI 383626). Indole-3-acetic acid content peaked on day 4 in CDC Xena, on day 8 in both Indian cultivars but only on day 12 in C. anatolicum. The cytokinins, isopentenyladenosine (iPA) and trans zeatin riboside (tZR) were predominant in CDC Xena and Swetha seeds on day 4, whereas cis zeatin riboside was the major component in Bharati. In C. anatolicum, iPA maxed out on day 4 and tZR on day 12. The bioactive gibberellin GA₁ spiked on day 4 in CDC Xena and Bharati, on day 8 in Swetha but only on day 12 in C. anatolicum. Eight-day old seeds had the highest abscisic acid content in the cultivars but spiked on day 12 in the perennial species. The hormone profiles of the perennial species showed delayed spikes in all four hormone groups indicating that there is a mismatch in the hormone requirements of the different embryos. Improving synchronization of early seed hormone profiles of cultivated and perennial chickpea should improve interspecific hybrid production.     

The level of proteins and leakage of solutes in germinating fresh and stored seeds ofCicer arietinum L.

One-year-old seeds of chickpea (Cicer arietinum L. cv. C-235) lost about 23 % germinability and leaked larger quantities of N, P, K, saccharides and proteins into the soaking medium in the first 48 h, as compared with fresh seeds. The protein content in stored seeds decreased more than in fresh seeds, as the soaking progressed.     

Fasting plasma magnesium concentrations and glucose disposal in diabetes.

Fasting plasma concentrations of magnesium were measured by neutron activation analysis in 30 non-diabetics and 87 diabetics (55 non-insulin-treated, 32 insulin treated). Plasma concentrations of magnesium were lowest in the insulin treated group (mean 0.84 (SEM 0.01) mmol/1; 2.0 (0.02) mg/100 ml), intermediate in the non-diabetics (mean 0.89 (SEM 0.01) mmol/1; 2.2 (0.02) mg/100 ml), and highest in the non-insulin-treated diabetics (mean 0.95 (SEM 0.02) mmol/1; 2.3 (0.05) mg/100 ml). In all diabetics plasma magnesium concentrations were inversely related to plasma glucose values (rs = -0.33; p less than 0.01) and in non-insulin-treated patients to plasma insulin concentrations (rs = -0.28; p less than 0.05), the former confirming previous observations. In 67 of the diabetics the KG constant for disposal rate of glucose during a standard intravenous glucose tolerance test was directly related to fasting plasma magnesium concentrations, and this relation persisted after controlling for age, sex, body mass index, type of treatment, and glucose and insulin values. This direct relation of plasma magnesium concentration with glucose disposal was unexplained by its influence on insulin secretion but was related to insulin sensitivity; hence magnesium may be an important determinant of insulin sensitivity in maturity onset diabetes.     

RFLP mapping of manganese efficiency in barley

In many cropping regions of the world, yield is limited by the availability of micronutrients, and micronutrient-efficient cultivars provide a yield advantage. Traditional methods of testing cultivars for micronutrient efficiency are time-consuming and laborious. Molecular markers linked to loci controlling micronutrient efficiency will allow more rapid and efficient selection and introgression of these traits than is currently possible. Using a pot-based bioassay and bulked segregant analysis of an F₂ population, we have identified several RFLPs (grouped distally on chromosome 4HS) linked to a locus for manganese efficiency in barley. This manganese efficiency locus has been designated Mel1. Pot bioassay analysis of intercrosses suggests that three useful sources of manganese efficiency are likely to be allelic at the Mel1 locus. Field evaluation of marker selected F₄ progeny supports the major role of Mel1 in the genetic control of manganese efficiency. Adoption of marker assisted selection for this trait in the Southern Australian barley breeding program has occurred. This has been facilitated by the demonstration that the Mel1 allele of Amagi Nijo can be distinguished from 95 other locally useful varieties and breeder’s lines on the basis of RFLPs identified by just two molecular markers. Received: 20 October 1999 / Accepted: 18 February 2000     

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome

A size-selected genomic library comprising 280,000 colonies and representing ≈18% of the chickpea genome, was screened for (GA)_n, (GAA)_n and (TAA)_n microsatellite-containing clones, of which 389 were sequenced. The majority (～75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%–9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613?cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P?≥?0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.     

MR T1 mapping for quantifying brain manganese deposition in type C hepatic encephalopathy rats

BioMetals - To evaluate magnetic resonance (MR) T1 mapping for quantifying brain manganese (Mn) deposition in type C hepatic encephalopathy (CHE) rats and to investigate the mechanism of magnesium...     

Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase.

The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1). The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+). This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.     

Respiratory activity during germination of seeds of Cicer arietinum L. II. Effect of some metabolic inhibitors and mitochondrial activities

Changes in respiration rate during the early period of germination of Cicer arietinum L. seeds were studied. Three phases were distinguished. During phases I and II the RQ was higher than unity. Sodium fluoride showed no effect on gaseous exchange during phase I while in phases II and III a progressive increase in the inhibition of the respiratory activity was obtained. Iodoacetate inhibited oxygen uptake and carbon dioxide evolution from the beginning of germination. The activities of mitochondrial enzymes increased during the germination period, especially after the first 24 hours. The respiratory control and ADP/O ratio of the isolated mitochondria increased gradually during germination, reaching maximum values at 48 hours, lower, however than the expected theoretical ones.     

Responses to the lowering of magnesium and calcium concentrations in the cerebrospinal fluid of unanesthetized sheep.

A technique for ventriculolumbar perfusion of the cerebrospinal fluid space has been used to study the neuromuscular effects of low concentrations of magnesium and calcium in the cerebrospinal fluid of conscious sheep. Perfusion with synthetic cerebrospinal fluid solutions containing less than 0-6 mg magnesium/100 ml produced episodes of tetany which were abolished by perfusion with a solution of normal magnesium concentration. This suggests that the low cerebrospinal fluid magnesium concentrations reported in cases of hypomagneseamic tetany may result in changes within the central nervous system that could produce the nervous signs. Perfusates with a calcium concentration below 2-0 mg/100 ml caused hyperpnoea and continuous muscle tremors. Magnesium (0-6 mg/100 ml) and calcium (2-0 mg/100 ml) perfused simultaneously acted synergistically to produce signs characteristic of low levels of each of the ions.     

Quantitative trait loci governing carotenoid concentration and weight in seeds of chickpea (Cicer arietinum L.)

Chickpea is a staple protein source in many Asian and Middle Eastern countries. The seeds contain carotenoids such as beta-carotene, cryptoxanthin, lutein and zeaxanthin in amounts above the engineered beta-carotene-containing golden rice level. Thus, breeding for high carotenoid concentration in seeds is of nutritional, socio-economic, and economic importance. To study the genetics governing seed carotenoids in chickpea, we studied the relationship between seed weight and concentrations of beta-carotene and lutein by means of high-performance liquid chromatography in segregating progeny from a cross between an Israeli cultivar and wild Cicer reticulatum Ladiz. Seeds of the cross progeny varied with respect to their carotenoid concentration (heritability estimates ranged from 0.5 to 0.9), and a negative genetic correlation was found between mean seed weight and carotenoid concentration in the F₃. To determine the loci responsible for the genetic variation observed, the population was genotyped using 91 sequence tagged microsatellite site markers and two CytP450 markers to generate a genetic map consisting of nine linkage groups and a total length of 344.6 cM. Using quantitative data collected for beta-carotene and lutein concentration and seed weight of the seeds of the F₂ population, we were able to identify quantitative trait loci (QTLs) by interval mapping. At a LOD score of 2, four QTLs for beta-carotene concentration, a single QTL for lutein concentration and three QTLs for seed weight were detected. The results of this investigation may assist in improving the nutritional quality of chickpea.