Developing an integrated genomic selection approach beyond biomass for varietal protection and nutritive traits in perennial ryegrass (Lolium perenne L.).

KEY MESSAGE: Breeding target traits can be broadened to include nutritive value and plant breeder's rights traits in perennial ryegrass by using in-field regression-based spectroscopy phenotyping and genomic selection. Perennial ryegrass breeding has focused on biomass yield, but expansion into a broader set of traits is needed to benefit livestock industries whilst also providing support for intellectual property protection of cultivars. Numerous breeding objectives can be targeted simultaneously with the development of sensor-based phenomics and genomic selection (GS). Of particular interest are nutritive value (NV), which has been difficult and expensive to measure using traditional phenotyping methods, resulting in limited genetic improvement to date, and traits required to obtain varietal protection, known as plant breeder's rights (PBR) traits. In order to assess phenotyping requirements for NV improvement and potential for genetic improvement, in-field reflectance-based spectroscopy was assessed and GS evaluated in a single population for three key NV traits, captured across four timepoints. Using three prediction approaches, the possibility of targeting PBR traits using GS was evaluated for five traits recorded across three years of a breeding program. Prediction accuracy was generally low to moderate for NV traits and moderate to high for PBR traits, with heritability highly correlated with GS accuracy. NV did not show significant or consistent correlation between timepoints highlighting the need to incorporate seasonal NV into selection indexes and the value of being able to regularly monitor NV across seasons. This study has demonstrated the ability to implement GS for both NV and PBR traits in perennial ryegrass, facilitating the expansion of ryegrass breeding targets to agronomically relevant traits while ensuring necessary varietal protection is achieved.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species.

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

Developmental outcome of the use of etamsylate for prevention of periventricular haemorrhage in a randomised controlled trial.

OBJECTIVE: To compare neurodevelopmental outcome of survivors of the multicentre trial of etamsylate (the iRNN for ethamsylate) for prevention of periventricular haemorrhage in very low birthweight infants. DESIGN: Double blind, single observer, prospective follow up of placebo controlled study. SETTING: Six neonatal intensive care units in the United Kingdom. Neurodevelopmental outcome was assessed in health premises or children's homes. SUBJECTS: 268 of 276 survivors of the original study were seen between 3.5 and 4.2 years of age. All were inborn and weighed 1500 g or less at birth. INTERVENTION: Etamsylate 12.5 mg/kg or placebo six hourly from within one hour of delivery for four days. MAIN OUTCOME MEASURES: McCarthy scales of children's abilities, standardised neurological examination, full physical examination, functional assessment, seven letter Stycar vision test, and audiometry. RESULTS: There was no difference between the groups in neuromotor outcome (cerebral palsy) or in the general cognitive index (GCI) of the McCarthy scales (mean GCI was 93.3 for the etamsylate group (n = 133) and 89.7 for the placebo group (n = 131); p = 0.10). There were more children with GCI < 70 (9 v 19; p = 0.047) or

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.).

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

Confidential enquiry into deaths due to prematurity.

The aim of this study was to audit the management of neonatal respiratory distress syndrome (RDS) in a geographically defined population using a retrospective peer review of case notes. The subjects were 49 infants of 24-36 wk gestation with a birthweight >499 g, and dying as a consequence of prematurity at <1 y of age in Wales during 1996. Forty-four infants (90%) were delivered in a unit with staff experienced in the management of preterm birth. Of the 30 infants <30 weeks' gestation, 29 (97%) received neonatal intensive care on a (sub)regional unit. Predelivery corticosteroids were indicated in 34 cases and administered in 31 (91%). Resuscitation at birth was indicated in 47 infants and conducted satisfactorily in 42 (89%). Temperature on admission to the neonatal unit was not recorded in 7 infants; in the other 42 it was >35.5 degrees C in 21 (50%). Early surfactant therapy was administered to 31/34 (91%) infants still intubated 120 min after birth, but was given within 30 min to only 8 (24%). Mechanical ventilation was assessed in 41 infants and considered to be good in 23 (56%). Cardiovascular therapy was evaluated in 40 infants requiring active support and considered to be good in 31 (78%). We concluded that neonatal RDS was generally well managed, thermal care during resuscitation was poor, surfactant should be administered more promptly, and deficiencies in the management of ventilation were common and related mainly to poor anticipation and a slow response to problems.

Early attachment experience and recovery from psychosis.

OBJECTIVES: Two studies were carried out to investigate the relationship between coping styles and co-morbid depression in people adjusting to the onset of psychosis. Evaluative thinking and early attachment experiences were also examined. METHODS AND DESIGN: In Study 1, a 39-item Recovery Style Questionnaire (RSQ) was developed to measure recovery style in people with psychosis, based on McGlashan, Levy & Carpenter's (1975) interview measure of recovery style. Fifty-six participants completed both the RSQ and McGlashan's interview-based measure. Study 2 explores the relationship between these styles of recovery, depression and early childhood attachment experiences. Thirty-six people participated. RESULTS: The RSQ was both reliable and correlated highly with McGlashan's interview-based measure. We found that the RSQ, in keeping with the interview-based measure, was bimodally distributed, thus supporting McGlashan's contention that they define two distinct recovery styles termed 'integration' and 'sealing over'. As predicted, 88 per cent of the 'sealers' were moderately to severely depressed compared to 52 per cent of the 'integrators' who were mildly depressed with no members of the 'integration' group experiencing moderate to severe depression (p < .0003). Patients who employed the sealing over recovery style also made significantly more negative self-evaluations than did patients in the integration group and also perceived their parents to be significantly less caring than those in the integration group. CONCLUSIONS: These findings are explained in terms of a multi-axial model incorporating personality structure and development as well as mental disorder. It is suggested that those individuals with a poorly developed sense of self defend against the threat of psychosis using denial. Clinical implications are discussed and more research is suggested to further investigate the links between evaluative and inferential thinking in co-morbid depression, and how such thinking relates to early childhood experience.

QIAamp spin columns as a method of DNA isolation for forensic casework.

The Detroit Police Crime Lab has historically used Chelex as a method to isolate DNA for amplification and typing of bloodstains at the HLADQA1, PM and D1S80 loci. However, preliminary validation of several STR systems for casework has demonstrated that the Chelex procedure is not the best method of DNA isolation for STR amplifications for our purposes. Long term storage at -20 degrees C in the presence of unbuffered Chelex beads (approximately 1 year), combined with multiple freeze thaws, resulted in signal loss at a locus for many database samples. Therefore, we have employed the QIAamp spin column as an alternative method of DNA isolation for amplification and typing of STR loci currently being validated for use in the laboratory. Moreover, we determined that QIAamp isolated DNA is also suitable for HLADQA1, PM and D1S80 typing. A matrix study was performed to determine if the QIAamp DNA procedure would give better results on bloodstains deposited on "problem surfaces" such as leather, dirt and various dyed fabrics. Again, QIAamp isolated DNA was more readily typeable than Chelex isolated DNA. We successfully replaced the phenol/chloroform extraction steps utilized in our laboratory for differential extractions, a commonly used method for separating sperm and non-sperm fractions of sexual assault evidence, with the QIAamp spin columns. The QIAamp extracted DNA performed as well in all PCR amplification and typing procedures tested (PM, HLADQA1, D1S80, and STR (PowerPlex)) as the phenol/chloroform Centricon isolated or EtOH precipitated DNAs. Thus we concluded that QIAamp spin columns are a superior method for isolating DNA to be typed for a variety of loci.

Screening for hearing loss in an at-risk neonatal population using evoked otoacoustic emissions.

The present study examines the performance of a transient evoked otoacoustic emissions hearing screening programme for neonates with risk factors for hearing loss. The programme has run for 5 years, and 772 babies with various risk factors have been screened using the Programmable Otoacoustic Emissions Measurement System (POEMS) designed by the Medical Research Council Institute of Hearing Research. Fourteen cases of significant hearing loss have been detected, with no false negative results. However, a very high false positive rate is reported, especially in babies with a birthweight of less than 1500 g.

Effect of different chloride infusion rates on plasma base excess during neonatal parenteral nutrition.

Routine biochemical monitoring of parenterally fed newborn infants revealed plasma chloride levels which were higher than widely quoted reference ranges. The parenteral nutrition solutions were reformulated to reduce the chloride infusion rate. In a historically controlled study, 28 infants receiving the original formulation were compared with 31 infants receiving the new formulation. The mean plasma chloride level was 4.8 mmol/l lower in the new formulation group than in the original formulation group (95% confidence interval 2.5-7.2 mmol/l). The mean base excess level was 3.1 mmol/l higher in the new formulation group than in the original formulation group (95% confidence interval 1.9-4.8 mmol/l). A reduction in the chloride load by using acetate salts can be safely achieved and may decrease the plasma chloride levels and decrease acidosis during the first seven days of life.

Ductus arteriosus blood flow during first 48 hours of life.

A volumetric Doppler technique was used to measure net ductus arteriosus shunt during the first 48 hours of life in 30 infants of less than 33 weeks' gestation, and in 10 full term infants. In the full term infants a left to right shunt of 62 ml/kg/minute (95% confidence limits 43-82) shortly after birth decreased rapidly over the first 12 hours and was not measurable by 48 hours. The preterm infants had smaller left to right shunts shortly after birth--49 ml/kg/minute (95% confidence limits 38-59). There was no obvious subsequent change in the mean shunt, although by 48 hours there was greater variation in the size of the shunt. The respiratory distress syndrome did not affect the size of the ductal shunt, but the shorter the gestation period the larger the shunt by 48 hours. A ductal flow of greater than 70 ml/kg/minute at 48 hours of age predicted the subsequent development of a ductal murmur with 75% sensitivity and 100% specificity.

Vasoactivity of the major intracranial arteries in newborn infants.

Blood flow to the head and arms was measured by a new volumetric Doppler technique in 30 preterm infants and 10 full term infants. At least 80% of this blood flow is considered to perfuse the brain. At the same time mean blood velocity in the middle and anterior cerebral arteries was measured by Duplex Doppler scanning. While blood flow to the head and arms remained substantially constant in both groups over the first 48 hours of life (60 ml/100 g brain weight/min), blood velocity doubled in both cerebral arteries in the preterm group. The term infants had higher blood velocities in both arteries at all times, but the velocities also increased over 48 hours, although to a lesser extent than in the preterm group. These findings imply that the major intracranial arteries are themselves vasoactive, being dilated at birth and subsequently constricting, possibly as an autoregulatory response to rising arterial blood pressure. While vasodilated, the cerebral arteries will be less efficient at damping pressure transients, placing preterm infants at particular risk of periventricular haemorrhage during the early hours of life. The implications for possible approaches to the prevention of cerebral haemorrhage are discussed.

Multicentre trial of ethamsylate for prevention of periventricular haemorrhage in very low birthweight infants.

The effectiveness of ethamsylate in the prevention of periventricular haemorrhage (PVH) in very low birthweight infants was evaluated by means of a multicentre, placebo-controlled, double-blind trial. In 330 infants without evidence of PVH on initial cranial ultrasound examination there was little difference between ethamsylate and placebo groups with respect to subependymal haemorrhage, but intraventricular and parenchymal haemorrhages developed in 30/162 infants (18.5%) in the treated group, compared with 50/168 (29.8%) in the control group (p less than 0.02). The incidence of intraventricular and parenchymal haemorrhage in survivors was 20/137 (14.6%) in the ethamsylate group and 37/146 (25.3%) in the controls (p less than 0.05). In 30 infants with evidence of PVH on the initial scan, ethamsylate treatment seemed to limit parenchymal extension. Analysis of the total cohort of 360 infants showed that the proportion of infants in whom an increase of two or more grades of severity of PVH was recorded during the trial was lower in the treated than in the placebo group (p less than 0.01). No adverse effects were attributed to ethamsylate therapy. The reported incidence of patent ductus arterious was lower in the treated than in the placebo group (p less than 0.02). Mortality was similar in the two groups.

Doppler ultrasound in the neonate.

The use of Doppler ultrasound to assess both the changing haemodynamics of the neonatal circulation and the perfusion of the brain is reviewed. The brain is particularly susceptible to both ischaemic and haemorrhagic injury in preterm and asphyxiated infants. However, the unique characteristics of the transitional neonatal circulation, and of the cerebral vasculature, pose considerable problems in the interpretation of Doppler signals from intracranial arteries. A volumetric Doppler method which eliminates some of those problems is discussed. The same method allows full assessment of the cardiovascular status of the newborn infant, including estimation of ductal shunting and left ventricular output. Doppler ultrasound, if used with a full understanding of the inherant assumptions and limitations of the particular methodology, is likely to prove invaluable in investigating pathological cerebral and cardiac vascular events in the newborn.

Slow, natural reduction in blood lead level after chelation therapy for lead poisoning in childhood.

Lead poisoning is treated with chelating agents. We report the natural decline of blood lead (Pb-B) concentration after treatment(s) (1967 to 1972) in 74 patients whose maximal Pb-B level ranged from 100 to 471 micrograms/dL (4.83 to 22.73 mumol/L). These longitudinal data (range, nine to 17 years) disclose a predictable decrease in Pb-B levels after treatment that is independent of the maximal Pb-B level before therapy. The correlation between age in months and the logarithm of the Pb-B level was significant, and the equation defined by the regression line allows one to predict Pb-B levels at specific ages after chelation therapy. It is important to recognize the slow, natural decline of Pb-B levels after chelation therapy once the level is stable and below 70 micrograms/dL (3.38 mumol/L). Multiple repeated courses of calcium disodium edetate are unlikely to influence the natural decline of the Pb-B level in asymptomatic children.

Renal function 9 to 17 years after childhood lead poisoning.

Renal function was studied in 74 subjects who, between 1966 and 1972 (ages 1 to 6 years) had had blood lead levels (PbB) greater than or equal to 100 micrograms/dl (range 100 to 471 micrograms/dl, median 142 micrograms/dl) and in 21 sibling controls. PbB measured in 1983 in study subjects remained significantly higher than in sibling controls (mean +/- 1 SD 14.5 +/- 4.5 vs 11.6 +/- 2.6 micrograms/dl, P less than 0.01). The two groups did not differ in development of hematuria or leukocyturia. The frequency of elevated serum creatinine concentration, depressed creatinine clearance, elevated protein excretion, low urinary osmolality, elevated serum beta 2-microglobulin (beta 2-M), and elevated fractional excretion beta 2-M % X 100 was similar in the two groups. Mean values for these tests were similar in study subjects compared with sibling controls. Mean systolic blood pressure was significantly higher in study subjects compared with that in sibling controls (117 +/- 12 vs 109 +/- 10 mm Hg), but the control group contained a preponderance of females and the study group had more overweight females; mean diastolic blood pressure was similar in the two groups. We conclude that in our adolescent subjects who had had lead poisoning 9 to 17 years earlier, there is little if any evidence of chronic nephropathy.