Maleita, C. M., Simoes, M. J., Egas, C, Curtis, R. H. C, and Abrantes, I. M. de O. 2012. Biometrical, biochemical, and molecular diagnosis of Portuguese Meloidogyne hispánica isolates. Plant Dis. 96:865-874.Meloidogyne hispánica infects many economically important crops worldwide. The accurate identification of this pathogen is essential for the establishment of efficient and sustainable integrated pest management programs. Portuguese M. hispánica isolates were studied by biometrical, biochemical, and molecular characteristics. Biometrical characteristics of M. hispánica females, males, and second-stage juveniles were similar to the original description. Biochemical studies revealed a unique enzyme pattern (Hi4) for M. hispánica esterases that allowed for species differentiation. Molecular analysis of the mtDNA region from COII and 16S rRNA genes resulted in amplification products (1,800 bp) similar to M. hispánica, M. ethiopica, and M. javanica, and the described Hinñ was unable to discriminate M. hispánica from the other two species. Analysis of the mtDNA sequences revealed altered nucleotides among the isolates that created new restriction sites for Alul and Dralll. The resulting restriction patterns successfully discriminated between the three species, providing a new tool for Meloidogyne identification. Finally, the phylogenetic relationship between M. hispánica and several Meloidogyne spp. sequences was analyzed using mtDNA, confirming the divergence between meiotic and mitotic species and revealing the proximity of M. hispánica to closely related species. Based on the studies conducted, the application of isozyme or polymerase chain reaction restriction fragment length polymorphism analysis would be a useful and efficient methodology for M. hispánica identification.The "Seville root-knot nematode", isolated from peach rootstock {Prunus pérsica (L.) Batsch) in Spain, was studied for the first time by Dalmasso and Berge (17) and described later as Meloidogyne hispánica Hirschmann, 1986 (27). This species has a woridwide distribution, and has been reported infecting economically important crops in Africa (22,34,48), Asia (21), Australia (21), Europe (4,21,31,49), and North, Central, and South America (10,15,21).M. hispánica is cytologically similai-to the diploid race of M. arenaria, and morphologically very close to M. arenaria, M. floridensis, and M. incognita (13,25,27). When tested using the North Carolina differential host test, M. hispánica isolates have host responses similar to M. arenaria race 2 or M. javanica (4,13), M. arenaria race 1 or M. incognita race 2 (4,27), and M. incognita race 3 (4), showing an intraspecific variability among the isolates of this species. The identification of M. hispánica only on the basis of morphological characteristics, especially on perineal patterns, or on the pattern of disease reacfions induced in the North Carolina differential hosts, is very difficult. One isolate of M. hispánica from South Africa was erroneously associated with M. arenaria thamesi on the basis ...

The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification.

Purpose: To explore phenotype-genotype correlations that may contribute to a better understanding of diabetic retinopathy (DR). Procedures: An exploratory association study was performed to identify genetic variants associated with non-proliferative DR (NPDR) in 307 type 2 diabetic patients who were previously stratified into 3 different phenotypes of NPDR progression. The 307 patients were genotyped for 174 single nucleotide polymorphisms of 11 candidate genes (ACE, AGER, AKR1B1, ICAM1, MTHFR, NOS1, NOS3, PPARGC1A, TGFB1, TNF and VEGFA). Results: Significant associations were observed for PPARGC1A rs16874120 with phenotype A (odds ratio, OR = 0.60, 95% confidence interval, CI 0.36-0.99), ICAM1 rs1801714 with phenotype B (OR = 3.32, 95% CI 1.05-10.50) and both PPARGC1A rs10213440 (OR = 2.00, 95% CI 1.07-3.73) and MTHFR rs1801133 (OR = 1.84, 95% CI 1.08-3.11) with phenotype C. Conclusions: Results indicate that specific gene variants in ICAM1, PPARGC1A and MTHFR are associated with different NPDR phenotypes, being likely candidates to explain different disease mechanisms underlying the different phenotypes. This is the first study to show correlations between specific gene variants and NPDR phenotypes, opening new perspectives on DR.

PURPOSE. Affecting children by age 3, primary congenital glaucoma (PCG) can cause debilitating vision loss by the developmental impairment of aqueous drainage resulting in high intraocular pressure (IOP), globe enlargement, and optic neuropathy. TEK haploinsufficiency accounts for 5% of PCG in diverse populations, with low penetrance explained by variable dysgenesis of Schlemm's canal (SC) in mice. We report eight families with TEK-related PCG, and provide evidence for SVEP1 as a disease modifier in family 8 with a higher penetrance and severity. METHODS. Exome sequencing identified coding/splice site variants with an allele frequency less than 0.0001 (gnomAD). TEK variant effects were assayed in constructtransfected HEK293 cells via detection of autophosphorylated (active) TEK protein. An enucleated eye from an affected member of family 8 was examined via histology. SVEP1 expression in developing outflow tissues was detected by immunofluorescent staining of 7-day mouse anterior segments. SVEP1 stimulation of TEK expression in human umbilical vascular endothelial cells (HUVECs) was measured by TaqMan quantitative PCR. RESULTS. Heterozygous TEK loss-of-function alleles were identified in eight PCG families, with parent-child disease transmission observed in two pedigrees. Family 8 exhibited greater disease penetrance and severity, histology revealed absence of SC in one eye, and SVEP1:p.R997C was identified in four of the five affected individuals. During SC development, SVEP1 is secreted by surrounding tissues. SVEP1:p.R997C abrogates stimulation of TEK expression by HUVECs. CONCLUSIONS. We provide further evidence for PCG caused by TEK haploinsufficiency, affirm autosomal dominant inheritance in two pedigrees, and propose SVEP1 as a modifier of TEK expression during SC development, affecting disease penetrance and severity.

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