High-density linkage maps for <Emphasis Type="Italic">Citrus sunki</Emphasis> and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> using DArTseq markers

The construction of a high-resolution genetic map of citrus would be of great value to breeders and to associate genomic regions with characteristics of agronomic interest. Here, we describe a novel high-resolution map of citrus using a population derived from a controlled cross between Citrus sunki (female parent) and Poncirus trifoliata (male parent). The genetic linkage maps were constructed using DArTseq markers and a pseudo-testcross strategy; only markers showing the expected segregation ratio were considered. To investigate synteny, all markers from both linkage maps were aligned with the genome of Citrus sinensis. The C. sunki map has a total of 2778 molecular markers and a size of 2446.6 cM, distributed across ten linkage groups. The map of P. trifoliata was built with 3084 markers distributed in a total of nine linkage groups, with a total size of 2411.6 cM. These maps are the most saturated linkage maps available for C. sunki and P. trifoliata and have high genomic coverage. We also demonstrated that the maps reported here are closely related to the reference genome of C. sinensis.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

Inheritance and gene mapping of spotted to non-spotted trait gene <Emphasis Type="Italic">CmSp-1</Emphasis> in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L<Emphasis Type="Italic">.</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis> Pangalo)

Melon (Cucumis melo L.) is one of the most popular and highly nutritious vegetable species within Cucurbitaceae. Because appearance is used as an important indicator of quality, the spotted to non-spotted trait associated with this product somewhat influences the buying habits of consumers. We tested a six-generation family to determine the inheritance and genetic basis of this trait. Genetic groups F₁, F₂, BC₁P₁, and BC₁P₂ were from a cross between “IM16559” (non-spotted) and “IM16553” (spotted). Our genetic analysis showed that the spotted to non-spotted trait was controlled by a single dominant gene that we named CmSp-1. Whole-genome resequencing-bulked segregant analysis (WG-BSA) demonstrated that this gene was located on the end of chromosome 2, in the intersections of 22,160,000 to 22,180,000 bp and 22,260,000 to 26,180,000 bp, an interval distance of 3.94 Mb. Insertion-deletion (InDel) markers designed based on WG-BSA data were used to map this gene. Using 13 InDel markers, we produced a genetic map indicating that CmSp-1 was tightly linked to markers I734-2 and I757, with genetic distances of 1.8 and 0.4 cM and an interval distance of 280.872 kb. The closest marker was I757. Testing of 107 different melon genotypes presented an accuracy of 84.11% in predicting the phenotype. By being able to locate CmSp-1 in melon, we can now use the findings to identify potential targets for further marker-assisted breeding and cloning projects.     

The <Emphasis Type="Italic">VKORC1</Emphasis> Asp36Tyr variant and <Emphasis Type="Italic">VKORC1</Emphasis> haplotype diversity in Ashkenazi and Ethiopian populations

The vitamin K epoxide reductase (VKORC1) is a key enzyme in the vitamin K cycle impacting various biological processes. VKORC1 genetic variability has been extensively studied in the context of warfarin pharmacogenetics revealing different distributions of VKORC1 haplotypes in various populations. We previously identified the VKORC1 Asp36Tyr mutation that was associated with warfarin resistance and with distinctive ethnic distribution. In this study, we performed haplotype analysis using Asp36Tyr and seven other VKORC1 markers in Ashkenazi and Ethiopian-Jewish and non-Jewish individuals. The VKORC1 variability was represented by nine haplotypes (V1-V9) that could be grouped into two distinct clusters (V1-V3 and V4-V9) with intra-cluster difference limited to two nucleotide changes. Phylogeny analysis suggested that these haplotypes could have developed from an ancestral variant, the common V8 haplotype (40 % in all population samples), after ten single mutation events. Asp36Tyr was exclusive to the V5 haplotype of the second cluster. Two haplotypes V5 and V4, distinguished only by Asp36Tyr, were prevalent in both Ethiopian population samples. The V2 haplotype, belonging to the first cluster, was the second most prevalent haplotype in the Ashkenazi population sample (15.8 %) but relatively uncommon in the Ethiopian origin (4.5-4.7 %). We discuss the genetic diversity among studied populations and its potential impact on warfarin-dose management in certain populations of African and European origin.     

Insights into the genetic relationships among plants of <Emphasis Type="Italic">Beta</Emphasis> section <Emphasis Type="Italic">Beta</Emphasis> using SNP markers

Key message

Using a much higher number of SNP markers and larger sample sizes than all the previous studies, we characterized the genetic relationships among wild and cultivated plants of section Beta.

Abstract

We analyzed the genetic variation of Beta section Beta, which includes wild taxa (Beta macrocarpa, B. patula, B. vulgaris subsp. adanensis and B. vulgaris subsp. maritima) and cultivars (fodder beet, sugar beet, garden beet, leaf beet, and swiss chards), using 9724 single nucleotide polymorphism markers. The analyses conducted at the individual level without a priori groups confirmed the strong differentiation of B. macrocarpa and B. vulgaris subsp. adanensis from the other taxa. B. vulgaris subsp. maritima showed a complex genetic structure partly following a geographical pattern, which confounded the differences between this taxon and the cultivated varieties. Cultivated varieties were structured into three main groups: garden beets, fodder and sugar beets, and leaf beets and swiss chards. The genetic structure described here will be helpful to correctly estimate linkage disequilibrium and to test for statistical associations between genetic markers and environmental variables.

     

Gene cloning system for sulfonamide-mineralizing <Emphasis Type="Italic">Microbacterium</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strain BR1

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli–Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.     

Assessment of genomic imprinting of <Emphasis Type="Italic">PPP1R9A</Emphasis>, <Emphasis Type="Italic">NAP1L5</Emphasis> and <Emphasis Type="Italic">PEG3</Emphasis> in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kiduey, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it’s expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

Association analysis of the glutelin synthesis genes <Emphasis Type="Italic">GluA</Emphasis> and <Emphasis Type="Italic">GluB1</Emphasis> in a <Emphasis Type="Italic">Japonica</Emphasis> rice collection

Glutelin is the most significant seed storage protein and is regarded as an important nutrient quality trait in rice. Research on the genetic basis of the glutelin content distinction in rice will provide more choices for the diets of people with kidney disease and diabetes. The GluA and GluB1 genes play important roles in the process of glutelin synthesis. In this study, 128 Japonica rice accessions with wide geographic distributions were collected to construct the association panel. Among all the 128 accessions, both sequences of the GluA and GluB1 genes were obtained, and nucleotide polymorphisms were detected. A total of 46 SNPs and eight InDels, six SNPs and four InDels were found in the GluA and GluB1 gene sequences, respectively. Eight haplotypes and two haplotypes were classified based on the SNPs in the coding region of the GluA and GluB1 genes, respectively. Moreover, the association of the polymorphic sites in the two genes with glutelin content in the tested population was estimated. The results revealed that five SNPs in the GluA gene, one SNP and one InDel in the GluB1 gene were associated with glutelin content at a significant level (P < 0.01). Corresponding markers were also designed to check the alleles of GluA and GluB1 genes. These results suggested that polymorphisms in the GluA and GluB1 genes in rice could be utilized in molecular marker-assisted selection to improve the nutrient quality of rice breeding programmes.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Map-based cloning of the dominant genic male sterile <Emphasis Type="Italic">Ms</Emphasis>-<Emphasis Type="Italic">cd1</Emphasis> gene in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

Key message

Using map-based cloning, we delimited the Ms - cd1 gene responsible for the male sterile phenotype in B. oleracea to an approximately 39-kb fragment. Expression analysis suggests that a new predicted gene, a homolog of the Arabidopsis SIED1 gene, is a potential candidate gene.

Abstract

A dominant genic male sterile (DGMS) mutant 79-399-3 in Brassica oleracea (B. oleracea) is controlled by a single gene named Ms-cd1, which was genetically mapped on chromosome C09. The derived DGMS lines of 79-399-3 have been successfully applied in hybrid cabbage breeding and commercial hybrid seed production of several B. oleracea cultivars in China. However, the Ms-cd1 gene responsible for the DGMS has not been identified, and the molecular basis of the DGMS is unclear, which then limits its widespread application in hybrid cabbage seed production. In the present study, a large BC₉ population with 12,269 individuals was developed for map-based cloning of the Ms-cd1 gene, and Ms-cd1 was mapped to a 39.4-kb DNA fragment between two InDel markers, InDel14 and InDel24. Four genes were identified in this region, including two annotated genes based on the available B. oleracea annotation database and two new predicted open reading frames (ORFs). Finally, a newly predicted ORF designated Bol357N3 was identified as the candidate of the Ms-cd1 gene. These results will be useful to reveal the molecular mechanism of the DGMS and develop more practical DGMS lines with stable male sterility for hybrid seed production in cabbage.

     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Genetic Distance in Housekeeping Genes Between <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> and <Emphasis Type="Italic">Plasmodium reichenowi</Emphasis> and Within <Emphasis Type="Italic">P. falciparum</Emphasis>

The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672±0.0088 and 0.0011±0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30±5.40) × 10⁴ and (11.62±7.56) × 10⁴ years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively.This article contains an online supplementary table.Reviewing Editor: Dr. Martin Kreitman     

Genetic mapping of <Emphasis Type="Italic">Stb19</Emphasis>, a new resistance gene to <Emphasis Type="Italic">Zymoseptoria tritici</Emphasis> in wheat

Key message

A new and dominant R gene Stb19 is identified from a soft wheat cultivar ‘Lorikeet’ and was mapped on the distal region of chromosome 1DS. Two tightly linked KASP markers were also discovered and validated for molecular-assisted breeding programs.

Abstract

A new R gene, designated as Stb19, provides resistance to Zymoseptoria tritici in wheat. This new dominant gene resides on the short arm of chromosome 1D, exhibiting complete resistance to three Z. tritici isolates, WAI332, WAI251, and WAI161, at the seedling stage. A genetic linkage map, based on an F_2:3 population of ‘Lorikeet’ and ‘Summit,’ found the Stb19 gene at a 9.3 cM region on 1DS, closely linked with two Kompetitive Allele-Specific PCR markers, snp_4909967 and snp_1218021. Further, the two markers were tested and validated in another F_2:3 population and 266 different wheat accessions, which gave over 95% accuracy of resistance/susceptibility prediction. Combined with the physical location of the identified SNPs and the previous evidence of gene order on chromosome 1DS (centromere–Sr45–Sr33–Lr21–telomere), Stb19 is proposed to be located between Sr33 and Lr21. Thus, the newly discovered Stb19 along with the KASP markers represents an increase in genetic resources available for wheat breeding resistance to Z. tritici.

     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.