The Amaranth Genome: Genome, Transcriptome, and Physical Map Assembly.

Amaranth ( L.) is an emerging pseudocereal native to the New World that has garnered increased attention in recent years because of its nutritional quality, in particular its seed protein and more specifically its high levels of the essential amino acid lysine. It belongs to the Amaranthaceae family, is an ancient paleopolyploid that shows disomic inheritance (2 = 32), and has an estimated genome size of 466 Mb. Here we present a high-quality draft genome sequence of the grain amaranth. The genome assembly consisted of 377 Mb in 3518 scaffolds with an N of 371 kb. Repetitive element analysis predicted that 48% of the genome is comprised of repeat sequences, of which -like elements were the most commonly classified retrotransposon. A de novo transcriptome consisting of 66,370 contigs was assembled from eight different amaranth tissue and abiotic stress libraries. Annotation of the genome identified 23,059 protein-coding genes. Seven grain amaranths (, , and ) and their putative progenitor () were resequenced. A single nucleotide polymorphism (SNP) phylogeny supported the classification of as the progenitor species of the grain amaranths. Lastly, we generated a de novo physical map for using the BioNano Genomics' Genome Mapping platform. The physical map spanned 340 Mb and a hybrid assembly using the BioNano physical maps nearly doubled the N of the assembly to 697 kb. Moreover, we analyzed synteny between amaranth and sugar beet ( L.) and estimated, using analysis, the age of the most recent polyploidization event in amaranth.

Construction of black (Rubus occidentalis) and red (R. idaeus) raspberry linkage maps and their comparison to the genomes of strawberry, apple, and peach.

The genus Rubus belongs to the Rosaceae and is comprised of 600-800 species distributed world-wide. To date, genetic maps of the genus consist largely of non-transferable markers such as amplified fragment length polymorphisms. An F(1) population developed from a cross between an advanced breeding selection of Rubus occidentalis (96395S1) and R. idaeus 'Latham' was used to construct a new genetic map consisting of DNA sequence-based markers. The genetic linkage maps presented here are constructed of 131 markers on at least one of the two parental maps. The majority of the markers are orthologous, including 14 Rosaceae conserved orthologous set markers, and 60 new gene-based markers developed for raspberry. Thirty-four published raspberry simple sequence repeat markers were used to align the new maps to published raspberry maps. The 96395S1 genetic map consists of six linkage groups (LG) and covers 309 cM with an average of 10 cM between markers; the 'Latham' genetic map consists of seven LG and covers 561 cM with an average of 5 cM between markers. We used BLAST analysis to align the orthologous sequences used to design primer pairs for Rubus genetic mapping with the genome sequences of Fragaria vesca 'Hawaii 4', Malus × domestica 'Golden Delicious', and Prunus 'Lovell'. The alignment of the orthologous markers designed here suggests that the genomes of Rubus and Fragaria have a high degree of synteny and that synteny decreases with phylogenetic distance. Our results give unprecedented insights into the genome evolution of raspberry from the putative ancestral genome of the single ancestor common to Rosaceae.

Characterization of Salt Overly Sensitive 1 (SOS1) gene homoeologs in quinoa (Chenopodium quinoa Willd.).

Salt tolerance is an agronomically important trait that affects plant species around the globe. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth of plants in saline environments. Quinoa (Chenopodium quinoa Willd.) is a halophytic, allotetraploid grain crop of the family Amaranthaceae with impressive nutritional content and an increasing worldwide market. Many quinoa varieties have considerable salt tolerance, and research suggests quinoa may utilize novel mechanisms to confer salt tolerance. Here we report the cloning and characterization of two homoeologous SOS1 loci (cqSOS1A and cqSOS1B) from C. quinoa, including full-length cDNA sequences, genomic sequences, relative expression levels, fluorescent in situ hybridization (FISH) analysis, and a phylogenetic analysis of SOS1 genes from 13 plant taxa. The cqSOS1A and cqSOS1B genes each span 23 exons spread over 3477 bp and 3486 bp of coding sequence, respectively. These sequences share a high level of similarity with SOS1 homologs of other species and contain two conserved domains, a Nhap cation-antiporter domain and a cyclic-nucleotide binding domain. Genomic sequence analysis of two BAC clones (98 357 bp and 132 770 bp) containing the homoeologous SOS1 genes suggests possible conservation of synteny across the C. quinoa sub-genomes. This report represents the first molecular characterization of salt-tolerance genes in a halophytic species in the Amaranthaceae as well as the first comparative analysis of coding and non-coding DNA sequences of the two homoeologous genomes of C. quinoa.

Analyses of a multi-parent population derived from two diverse alfalfa germplasms: testcross evaluations and phenotype-DNA associations.

In a previous study, we showed that the genetic variation present in the Medicago sativa subsp. sativa Peruvian and M. sativa subsp. falcata WISFAL germplasms could be used to improve forage yields when favorable alleles were recombined and used in hybrid combination with cultivated alfalfa. In this paper, we present testcross forage yield and fall growth data for two seasons of a C0 population generated after intermating the Peruvian x WISFAL population for several generations. In addition, we conducted marker-trait association analysis as an attempt to identify Peruvian and WISFAL genomics regions affecting the targeted traits. Five and seven genomic regions were found significantly associated with forage yield and fall growth, respectively. In the case of fall growth, alleles from both accessions were positively associated with plant height. However, more alleles from WISFAL were positively associated with forage yield than from Peruvian. WISFAL is known for its winter hardiness and genomic regions with large effects on winter survival may have masked the effect of forage yield from Peruvian. The fact that most of the genomic regions discovered in this study have been previously associated with traits involved in winter hardiness validates our findings and suggests that associations between DNA fragments and agronomic traits can be detected without the necessity of developing bi-parental mapping populations.

Recent advances using streptokinase for acute coronary thrombosis.

Most important in comparison to earlier European trials, streptokinase (STK) is administered now at the earliest time possible after acute coronary thrombosis. In this series, STK was started 2.5 (+/- 1.5) h after onset of chest pain, with reperfusion achieved approximately 1 h later in 6 (55%) of 11 patients treated. Posttreatment angiograms will not be required to identify thrombolysis if noninvasive indicators will provide this information correctly. Early creatine kinase enzyme peaking 8 to 15 h after chest pain appears to be the most accurate marker available. Among untreated and unsuccessfully treated patients, creatine kinase peaking usually occurs 18-36 h after chest pain. A large intravenous STK loading dose of 1,500,000 IU produces a plasma concentration of approximately 500 IU/ml, equal to that concentration employed originally by intracoronary infusions. Such large doses have been employed in 60 patients thus far, without an unusual incidence or severity of hemorrhages. High dose, ultrashort-term treatment for only 1 h is being investigated now. Systemic STK penetrates most "blind coronary pouches" and gains access to acute thrombi, as identified by radiocontrast material washout during angiography in patients with severe coronary occlusions. Streptokinase exerts a significant anticoagulant effect, not previous considered, which may be beneficial in the prevention of new clot formation and the rapid dissolution of acute coronary thrombi.

Noninvasive markers of intravenous streptokinase coronary thrombolysis.

Early creatine kinase (CK) enzyme peaking, rapid electrocardiographic (EKG) changes toward normal, reperfusion arrhythmias, pain disappearance, and 201thallium myocardial scintigraphy appear useful to identify the success or failure of intravenous (i.v.) thrombolytic therapy in patients with acute myocardial infarction (AMI). Most patients with AMI are treated currently in community hospitals which do not possess coronary angiographic capabilities. Recent evidence indicates that early intravenous streptokinase results in coronary thrombolysis in the majority of patients treated. A composite of noninvasive markers of coronary reperfusion was assessed in two similar patients with transmural AMI. One received intravenous streptokinase (STK) 750,000 U 90 min after AMI onset; the other received intracoronary (i.c.) STK 4000 U/min 140 min after onset. Within one hour each showed a sudden change in elevated EKG ST segments toward normal, followed by frequent premature ventricular beats and pain disappearance. Posttreatment angiograms documented recanalization of each infarct-related artery. Early CK peaking occurred at 10 hours after the onset of chest pain in the first patient and at 12 hours in the second. This contrasts with delayed CK peaking at 26.4 hours among 384 patients reviewed with untreated AMI. Early CK peaking appears the most accurate indirect marker of successful coronary thrombolysis.

A new atrial transseptal pacing electrode.

The atrial septum provides a convenient structure to which a transvenous electrode can be attached securely. The Brokenbrough cardiac catheterization technique has been used to gain access to the left atrium. A new electrode has been fabricated which can be inserted into the left atrium through the lumen of a Brockenbrough catheter. The electrode is composed of a stainless steel coil attached inside the proximal end of a platinum cylinder at the cardiac terminal. Three Elgiloy wires, having a wing configuration, are attached inside the distal end of the platinum cylinder. The coil and cylinder are insulated by silicone rubber and the metallic surface area of the distal electrode tip equals 5 mm2. This electrode has been implanted without difficulty or complications in five patients with tachycardia-bradycardia syndrome.

Predictive implications of ventricular premature contractions associated with treadmill stress testing.

Follow-up data on future coronary events was collected on 6,500 patients who had undergone stress testing; 1,327 of these exhibited ventricular premature contractions (VPCs) associated with one or more phases of treadmill stress testing (TSTs). Eighty-three percent of all patients tested had known or suspected cardiovascular disease. The annual incidence of new coronary events (myocardial infarction, angina, cardiac death) during a five year follow-up was 1.7% among 1,067 patients without VPCs or ischemic ST changes, 6.4% in 758 patients with VPCs alone, 9.5% among 609 patients with ischemic ST changes alone and 11.4% in 569 patients with VPCs plus ischemic ST changes. The significance of VPCs associated with the TSTs rests largely upon the clinical status of the persons tested. VPCs observed among patients referred for TSTs for the evaluation of known or suspected cardiovascular disease, with or without ischemic ST abnormalities, represent a definite risk factor for future coronary events.

Intrasubject variation of warfarin binding to protein in serum of patients with cardiovascular disease.

A study of 31 patients with cardiovascular disease who were taking warfarin regularly had shown pronounced intersubject differences in serum protein binding of warfarin and a highly significant correlation between the body clearance of warfarin and the free fraction of the drug in serum. Similar observations have been made in experimental animals and are consistent with predictions based on theoretical considerations. The purpose of this investigation was to determine the intrasubject variation in the free fraction of warfarin in serum. Samples of serum were obtained from 23 of the 31 patients previously studied. The time interval between the two studies was 3.4 to 5.7 mo. The daily dose of warfarin had been changed by 10.6% on the average. With two exceptions, there was no change in concurrent medications. The ratio of free fraction values of warfarin in serum, second/first study, was 0.948 +/- 0.297 (mean +/- S.D.), and there was a highly significant correlation (P less than 0.001) between the individual free fraction values in the first and second studies.

Patient selection for anticoagulant therapy in coronary heart disease.

Short-term anticoagulant therapy given after an acute myocardial infarction is directed toward preventing thromboembolism and is fairly safe. Long-term anticoagulant therapy prevents coronary thrombosis in selected patients with coronary heart disease (CHD), but carries an appreciable risk of hemorrhage. A decision for or against short-term therapy should be based on an assessment of the immediate risk of thromboembolism. Similarly, the risk of coronary thrombosis should be the major determinant in a decision for or against long-term anticoagulation. The most important information emerging from the clinical trials of long-term anticoagulant therapy in CHD concerns the significant benefit observed among patients with advanced disease.

Serum protein binding as a determinant of warfarin body clearance and anticoagulant effect.

The serum protein binding and elimination kinetics of warfarin were determined in 31 patients with cardiovascular disease who were taking warfarin regularly. The free fraction of warfarin in the serum ranged from 0.00436 to 0.0189, indicating 98.11% to 99.56% protein binding. There was no apparent relationship between the extent of protein binding of warfarin and the concentration of albumin or total protein in the serum. The estimated total body clearance of warfarin in the patients ranged from 1.16 to 4.35ml/hr/kg of body weight and correlated significantly with the free fraction of warfarin in serum. This correlation has been predicted on theoretical grounds and shows that serum protein binding is a major determinant of the elimination kinetics of warfarin in man and an important cause of interindividual variations in its body clearance. The interindividual variation of free warfarin concentrations in the serum of patients with similar prothrombin times was somewhat smaller than the variations in total serum-warfarin concentrations and in the daily dose of warfarin. There was no correlation between prothrombin time and the concentration of free warfarin in serum, indicating that variables other than protein binding also affect the anticoagulant response of patients.

Clinical implications of warfarin interactions with five sedatives.

The intensity, uniformity and time course of anticoagulant interference by phenobarbital, secobarbital, glutethimide, chloral hydrate and methaqualone were systematically investigated in 16 patients receiving coumarin therapy. Each subject received an individualized fixed daily dose of warfarin and served as his own pre- and postsedative treatment control. Prothrombin times were measured four times weekly during five long-term experiments. Anticoagulant inhibition was observed during the administration of phenobarbital, secobarbital and glutethimide; there was no significant change in prothrombin test results during the trials of chloral hydrate and methaqualone. Barbiturates and glutethimide should not be administered to patients receiving coumarin drugs. The concurrent use of drugs from these groups is decreasing according to a survey of 200 hospital medical records. Chloral hydrate and methaqualone interact pharmacologically with orally administered anticoagulant agents, but the effect is not clinically significant. It is concluded that chloral hydrate and methaqualone may be administered safely without additional caution in prothrombin test monitoring during oral anticoagulant therapy.