Genetic Intervals Research Articles

Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.

Molecular identification and validation of four stable QTL for adult-plant resistance to powdery mildew in Chinese wheat cultivar Bainong 64.

Four stable QTL for adult-plant resistance (APR) to powdery mildew were identified on chromosome arms 1DL, 2BS, 2DL, and 6BL in the widely grown Chinese wheat cultivar Bainong 64. These QTL had no effect on response to stripe rust or leaf rust. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a devastating fungal disease. Seedlings of Chinese wheat Bainong 64 are susceptible to Bgt, but adult plants have maintained resistance since it was released in 1996. A population of 171 recombinant inbred lines (RILs) developed from cross Jingshuang 16/Bainong 64 (JS16/BN64) was used to dissect genetic components of powdery mildew resistance. A genetic map comprising 5383 polymorphic markers was constructed using the 15K SNP chip and kompetitive allele-specific PCR (KASP) markers. Composite interval mapping identified four stable QTL with favorable alleles all from BN64 on chromosome arms 1DL, 2BS, 2DL, and 6BL in at least four environments. They accounted for 8.3%, 13.8%, 14.4%, and 9.0% of the total phenotypic variation explained (PVE) in maximum, respectively. QPmjbr.caas-1DL, situated about 22Mb from centromere, is probably a new QTL. QPmjbr.caas-2DL located near the end of arm 2DL and explained the largest PVE. Using genetic maps populated with KASP markers, QPmjbr.caas-2BS and QPmjbr.caas-6BL were fine mapped to a 1.8cM genetic intervals spanning 13.6Mb (76.0-89.6Mb) and 1.7cM and 4.9Mb (659.9-664.8Mb), respectively. The four QTL independent of stripe rust and leaf rust resistance were validated for powdery mildew resistance in another RIL population related to BN64 and a cultivar panel using representative KASP markers. Since BN64 has been a leading cultivar and an important breeding parent in China, the QTL and markers reported in this study will be useful for marker-assisted selection of APR.

Meta-QTL and haplo-pheno analysis reveal superior haplotype combinations associated with low grain chalkiness under high temperature in rice.

Chalk, an undesirable grain quality trait in rice, is primarily formed due to high temperatures during the grain-filling process. Owing to the disordered starch granule structure, air spaces and low amylose content, chalky grains are easily breakable during milling thereby lowering head rice recovery and its market price. Availability of multiple QTLs associated with grain chalkiness and associated attributes, provided us an opportunity to perform a meta-analysis and identify candidate genes and their alleles contributing to enhanced grain quality. From the 403 previously reported QTLs, 64 Meta-QTLs encompassing 5262 non-redundant genes were identified. MQTL analysis reduced the genetic and physical intervals and nearly 73% meta-QTLs were narrower than 5cM and 2Mb, revealing the hotspot genomic regions. By investigating expression profiles of 5262 genes in previously published datasets, 49 candidate genes were shortlisted on the basis of their differential regulation in at least two of the datasets. We identified non-synonymous allelic variations and haplotypes in 39 candidate genes across the 3K rice genome panel. Further, we phenotyped a subset panel of 60 rice accessions by exposing them to high temperature stress under natural field conditions over two Rabi cropping seasons. Haplo-pheno analysis uncovered haplotype combinations of two starch synthesis genes, GBSSI and SSIIa, significantly contributing towards the formation of grain chalk in rice. We, therefore, report not only markers and pre-breeding material, but also propose superior haplotype combinations which can be introduced using either marker-assisted breeding or CRISPR-Cas based prime editing to generate elite rice varieties with low grain chalkiness and high HRY traits.

Inheritance and Linkage of Virulence Genes of Puccinia striiformis f. sp. hordei.

Puccinia striiformis f. sp. hordei (Psh) causing barley stripe rust has only recently been known to be heteroecious, for which reason the inheritance of its virulence has not been analyzed. Herein, we selfed a Psh isolate, XZ-19-972, on Berberis aggregata and obtained 53 progenies. The virulence phenotypes (VPs) for these progenies were identified on 11 barley differentials, and their genotypes were assessed with 22 Kompetitive allele specific PCR-single nucleotide polymorphism (KASP-SNP) markers. In total, 18 VPs were detected among progenies, 17 (VP2-VP18) of which, corresponding to 43 isolates, were different from the parental isolate showing VP1. Of the 53 progenies, 8 exhibited increased virulence and 34 decreased virulence. One progeny, belonging to VP18, showed a different virulence formula but without a virulence increase or decrease. The parental isolate and all progenies were avirulent to yrc6 but virulent to yrc7. The parental isolate was heterozygous in terms of avirulence/virulence to nine barley resistance gene loci. KASP-SNP marker analysis identified 36 multilocus genotypes, based on which a linkage map was constructed, with total genetic distance intervals of 516.07 cM, spanning 16 avirulence or virulence loci. Taken together, our results provide important insights into the inheritance and virulence diversity of Psh.

Identification of QTLs linked with watermelon fruit and seed traits using GBS-based high-resolution genetic mapping

Watermelon is an economic horticultural crop of the Cucurbitaceae family. An in-depth genetic understanding of fruit and seed associated traits is important for the effective breeding of watermelon. In the present study, we conducted genotyping by sequencing (GBS)-based high-resolution genetic mapping and performed QTL mapping of twelve horticultural traits using segregated mapping populations (F2 and F2:3) derived from the breeding of two contrasting inbred parent lines. The developed genetic linkage map comprised of 6,164 SNP loci and 1,004 bins, which spanned a total genetic length of 1,261.13 cM with an average interval of 1.26 cM (329.31 kb) between adjacent whole-genome markers. Overall, a total of 34 QTLs were identified, and 16 QTLs exhibited moderate and major-effect QTLs; however, 25 were novel QTLs that were not identified in earlier studies. The mapped genetic loci of RC, RSP, and FFC traits were identified as closely overlapping with previously reported loci and exhibited delimited genetic intervals located on chromosomes 8, 6, and 4, respectively. The identified novel QTL (qfd2.1) of FD and QTL (qfl2.1) of FL were tightly localized with QTL (qfsi2.1) of FSI over chromosome 2, but a minor QTL (qfw3.2) positioned on chromosome 3 was consistent with previously published QTLs (qfd3.1, qfl3.1, and qfsi3.1) of fruit shape. A total of five minor-effect QTLs (qbcc2.1, qbce2.1, qbce2.2, qbcc5.1, and qbce5.1) identified in the novel genetic region which controlled the average Brix content of edible fruit. The seed width QTL (qsw10.1) was found to be localized with 20-seed weight QTL (q20swt10.1) on chromosome 10. The collected phenotypic dataset of two environmental experiments across the two years also revealed highly significant correlations among fruit and seed traits. Hence, we concluded that our GBS-based identified novel QTLs positions would expand powerful molecular breeding prospects for the genetic improvement of quality-related traits of watermelon.

Consensus genomic regions associated with multiple abiotic stress tolerance in wheat and implications for wheat breeding

In wheat, a meta-analysis was performed using previously identified QTLs associated with drought stress (DS), heat stress (HS), salinity stress (SS), water-logging stress (WS), pre-harvest sprouting (PHS), and aluminium stress (AS) which predicted a total of 134 meta-QTLs (MQTLs) that involved at least 28 consistent and stable MQTLs conferring tolerance to five or all six abiotic stresses under study. Seventy-six MQTLs out of the 132 physically anchored MQTLs were also verified with genome-wide association studies. Around 43% of MQTLs had genetic and physical confidence intervals of less than 1 cM and 5 Mb, respectively. Consequently, 539 genes were identified in some selected MQTLs providing tolerance to 5 or all 6 abiotic stresses. Comparative analysis of genes underlying MQTLs with four RNA-seq based transcriptomic datasets unravelled a total of 189 differentially expressed genes which also included at least 11 most promising candidate genes common among different datasets. The promoter analysis showed that the promoters of these genes include many stress responsiveness cis-regulatory elements, such as ARE, MBS, TC-rich repeats, As-1 element, STRE, LTR, WRE3, and WUN-motif among others. Further, some MQTLs also overlapped with as many as 34 known abiotic stress tolerance genes. In addition, numerous ortho-MQTLs among the wheat, maize, and rice genomes were discovered. These findings could help with fine mapping and gene cloning, as well as marker-assisted breeding for multiple abiotic stress tolerances in wheat.

Analyses and identifications of quantitative trait loci and candidate genes controlling mesocotyl elongation in rice

Rice direct seeding has the significant potential to save labor and water, conserve environmental resources, and reduce greenhouse gas emissions tremendously. Therefore, rice direct seeding is becoming the major cultivation technology applied to rice production in many countries. Identifying and utilizing genes controlling mesocotyl elongation is an effective approach to accelerate breeding procedures and meet the requirements for direct-seeded rice (DSR) production. This study used a permanent mapping population with 144 recombinant inbred lines (RILs) and 2 828 bin-markers to detect quantitative trait loci (QTLs) associated with mesocotyl length in 2019 and 2020. The mesocotyl lengths of the rice RILs and their parents, Lijiangxintuanheigu (LTH) and Shennong 265 (SN265), were measured in a growth chamber at 30°C in a dark environment. A total of 16 QTLs for mesocotyl length were identified on chromosomes 1(2), 2(4), 3(2), 4, 5, 6, 7, 9, 11(2), and 12. Seven of these QTLs, including qML1a, qML1b, qML2d, qML3a, qML3b, qML5, and qML11b, were reproducibly detected in both years via the interval mapping method. The major QTL, qML3a, was reidentified in two years via the composite interval mapping method. A total of 10 to 413 annotated genes for each QTL were identified in their smallest genetic intervals of 37.69 kb to 2.78 Mb, respectively. Thirteen predicted genes within a relatively small genetic interval (88.18 kb) of the major mesocotyl elongation QTL, qML3a, were more thoroughly analyzed. Finally, the coding DNA sequence variations among SN265, LTH, and Nipponbare indicated that the LOC_Os03g50550 gene was the strongest candidate gene for the qML3a QTL controlling the mesocotyl elongation. This LOC_Os03g50550 gene encodes a mitogen-activated protein kinase. Relative gene expression analysis using qRT-RCR further revealed that the expression levels of the LOC_Os03g50550 gene in the mesocotyl of LTH were significantly lower than in the mesocotyl of SN265. In conclusion, these results further strengthen our knowledge about rice's genetic mechanisms of mesocotyl elongation. This investigation's discoveries will help to accelerate breeding programs for new DSR variety development.

Morphometric and Genetic Description of Trophic Adaptations in Cichlid Fishes.

Simple SummarySkull and jaw shape are critical to how an animal eats. The goal of this work was to examine how facial variation evolves and the genetic basis of these changes. We used two species of Lake Malawi cichlids with different facial shapes, one which has evolved to eat prey by suction feeding, a second that bites algae from rocks, as well as hybrid individuals generated by artificial mating of the two species. We found a series of changes in craniofacial structure including the shape of the lower jaw and throat region that impact how animals perform at suction feeding and biting. We then identified genetic regions that regulate these facial shapes. These genetic regions suggested that evolution of the senses, among other traits, may play an important role in facial evolution. Also, evolution of different parts of the face are controlled by distinct genetic regions. Despite this, cichlids that eat similar ways have similar facial shapes, suggesting that the function of jaw movement places certain limits on facial evolution in cichlid fishes. Overall, this work provides insights into how the face evolves, how these changes relate to feeding, and the genes and molecules that regulate craniofacial variation.Since Darwin, biologists have sought to understand the evolution and origins of phenotypic adaptations. The skull is particularly diverse due to intense natural selection on feeding biomechanics. We investigated the genetic and molecular origins of trophic adaptation using Lake Malawi cichlids, which have undergone an exemplary evolutionary radiation. We analyzed morphological differences in the lateral and ventral head shape among an insectivore that eats by suction feeding, an obligate biting herbivore, and their F2 hybrids. We identified variation in a series of morphological traits—including mandible width, mandible length, and buccal length—that directly affect feeding kinematics and function. Using quantitative trait loci (QTL) mapping, we found that many genes of small effects influence these craniofacial adaptations. Intervals for some traits were enriched in genes related to potassium transport and sensory systems, the latter suggesting co-evolution of feeding structures and sensory adaptations for foraging. Despite these indications of co-evolution of structures, morphological traits did not show covariation. Furthermore, phenotypes largely mapped to distinct genetic intervals, suggesting that a common genetic basis does not generate coordinated changes in shape. Together, these suggest that craniofacial traits are mostly inherited as separate modules, which confers a high potential for the evolution of morphological diversity. Though these traits are not restricted by genetic pleiotropy, functional demands of feeding and sensory structures likely introduce constraints on variation. In all, we provide insights into the quantitative genetic basis of trophic adaptation, identify mechanisms that influence the direction of morphological evolution, and provide molecular inroads to craniofacial variation.

ABCB1 and OPRM1 single-nucleotide polymorphisms collectively modulate chronic shoulder pain and dysfunction in South African breast cancer survivors.

Background: Chronic shoulder pain/disability is a well-recognized side effect of treatment for breast cancer, with ∼40% of patients experiencing this, despite receiving pain management. To manageacute and chronic pain, several opioids are commonly prescribed. Pharmacogenomics have implicated genes within the opioid signaling pathway, including ABCB1 and OPRM1, to contribute to an individual's variable response to opioids. Aim: To evaluate ABCB1 (rs1045642G>A, rs1128503 G>A) and OPRM1 (rs1799971 A>G, rs540825 T>A) single-nucleotide polymorphisms (SNPs) in chronic shoulder pain/disability in BCS. Materials & methods: TaqManTMassays were used to genotype ABCB1 and OPRM1 SNPswithin the BCS (N=252) cohort. The Shoulder Pain and Disability Index was used to evaluate pain and disability features associated with shoulder pathologies. Participants end scores for each feature (pain, disabilityand combined [pain and disability]) were categorized into no-low (>30%) and moderate-high (≥30%) scores. Statistical analysis was applied, and significance was accepted at p < 0.05. Results: Of participants, 27.0, 19.0 and 22.0% reported moderate-high pain, disability and combined (pain and disability) scores, respectively. ABCB1:rs1045642-(A/A) genotype was significantly associated with disability (p = 0.028: no-low [14.9%] vs mod-high [4.3%]) and combined (pain and disability) (p = 0.011: no-low [15.9%] vs mod-high [5.7%]). The ABCB1:rs1045642-(A) allele was significantly associated with disability (p = 0.015: no-low [37.9%] vs mod-high [23.9%]) and combined (pain and disability) (p = 0.003: no-low [38.5%] vs mod-high [23.6%]). The inferred ABCB1 (rs1045642G>A - rs1128503G>A): A-G (p=0.029; odds ratio [OR]: 0.0; 95% CI: 0.0-0.0) and the OPRM1 (rs1799971A>G- rs540825T>A): G-T (p=0.019; OR: 0.33; 95% CI: 0.14-0.75) haplotypes were associated with disability and pain, respectively. Gene-gene interactions showed the ABCB1 (rs1045642 G>A) -OPRM1 (rs540825 T>A) combinations, (A-T) (p=0.019; OR: 0.62; 95% CI: 0.33-1.16) and (G-A) (p=0.021; OR: 1.57; 95% CI: 0.30-3.10) were associated with disability. Conclusion: The study implicated ABCB1 with shoulder pain and disability; and haplotype analyses identified specific genetic intervals within ABCB1 and OPRM1 to associate with chronic shoulder pain and disability. Evidence suggests that potentially gene-gene interactions between ABCB1 and OPRM1 contribute to chronic shoulder pain and disability experienced in this SA cohort.

Charakterystyka serologiczna populacji wsi Wielkie Drogi w pobliżu Krakowa

Serological characteristic of the population of Wielkie Drogi village near Cracow was made on the material of 83 persons of both sexes. The ABO, Rh(D) and Kell systems were examined. The frequencies of genes and phenotypes were evalusted and then the genetic intervals were computed for the investigated group in relation to other populations from Poland, Ukraine and Moravia.

A Meta-Analysis of Quantitative Trait Loci Associated with Stay-Green in Sorghum

A strong association between stay-green and post-flowering drought tolerance in sorghum has been established previously. In this study, a meta-analysis was conducted to find relevant genetic intervals that regulate stay-green in sorghum and thereby affect drought resilience. Meta-QTL analysis was performed to combine 115 QTLs mapped in previous studies and locate regions where recurrent QTLs occurred across experiments. This approach overcomes the constraints of a single QTL association experiment by increasing heterogeneity in populations and environments. The analysis included the construction of consensus maps, QTL projection, and QTL clustering. We report 32 Meta-QTL for stay-green and their position relative to genetic markers on the sorghum genome. The results from this study will facilitate future attempts aiming to improve and understand drought tolerance in Sorghum by the use of fine mapping and marker-assisted selection.

Mapping pre-harvest sprouting resistance loci in AAC Innova \xd7 AAC Tenacious spring wheat population

BackgroundPre-harvest sprouting (PHS) is a major problem for wheat production due to its direct detrimental effects on wheat yield, end-use quality and seed viability. Annually, PHS is estimated to cause > 1.0 billion USD in losses worldwide. Therefore, identifying PHS resistance quantitative trait loci (QTLs) is crucial to aid molecular breeding efforts to minimize losses. Thus, a doubled haploid mapping population derived from a cross between white-grained PHS susceptible cv AAC Innova and red-grained resistant cv AAC Tenacious was screened for PHS resistance in four environments and utilized for QTL mapping.ResultsTwenty-one PHS resistance QTLs, including seven major loci (on chromosomes 1A, 2B, 3A, 3B, 3D, and 7D), each explaining ≥10% phenotypic variation for PHS resistance, were identified. In every environment, at least one major QTL was identified. PHS resistance at most of these loci was contributed by AAC Tenacious except at two loci on chromosomes 3D and 7D where it was contributed by AAC Innova. Thirteen of the total twenty-one identified loci were located to chromosome positions where at least one QTL have been previously identified in other wheat genotype(s). The remaining eight QTLs are new which have been identified for the first time in this study. Pedigree analysis traced several known donors of PHS resistance in AAC Tenacious genealogy. Comparative analyses of the genetic intervals of identified QTLs with that of already identified and cloned PHS resistance gene intervals using IWGSC RefSeq v2.0 identified MFT-A1b (in QTL interval QPhs.lrdc-3A.1) and AGO802A (in QTL interval QPhs.lrdc-3A.2) on chromosome 3A, MFT-3B-1 (in QTL interval QPhs.lrdc-3B.1) on chromosome 3B, and AGO802D, HUB1, TaVp1-D1 (in QTL interval QPhs.lrdc-3D.1) and TaMyb10-D1 (in QTL interval QPhs.lrdc-3D.2) on chromosome 3D. These candidate genes are involved in embryo- and seed coat-imposed dormancy as well as in epigenetic control of dormancy.ConclusionsOur results revealed the complex PHS resistance genetics of AAC Tenacious and AAC Innova. AAC Tenacious possesses a great reservoir of important PHS resistance QTLs/genes supposed to be derived from different resources. The tracing of pedigrees of AAC Tenacious and other sources complements the validation of QTL analysis results. Finally, comparing our results with previous PHS studies in wheat, we have confirmed the position of several major PHS resistance QTLs and candidate genes.

Time-sequential detection of quantitative trait loci and candidate genes underlying the dynamic growth of Salix suchowensis.

Elucidating the genetic factors underlying long-term biological processes remains challenging since the relevant genes and their effects may vary across different developmental stages. In this study, we carried out a large-scale field trial of the progeny of an F1 full-sib pedigree of Salix suchowensis and measured plant height and ground diameter periodically over a time course of 240days. With the obtained data, we characterized plant growth rhythms and performed time-sequential analyses of quantitative trait loci underlying the dynamic growth of the plants. The dynamic mapping of quantitative trait loci revealed that stem height and ground diameter were under the control of four quantitative trait loci, and the effects of these quantitative trait loci varied greatly throughout the growth process, in which two quantitative trait loci were found to exert a pleiotropic effect determining the correlation between stem height and ground diameter. The analysis of candidate genes in the target genetic intervals showed that the pleiotropic effect of the two quantitative trait loci arises from the colocalization of genes with independent effects on stem height and ground diameter. Further examination of the expression patterns of the candidate genes indicated that height and circumference growth involve different activities of leaf and cambium tissues. This study provides unprecedented information to help us understand the dynamic growth of plants and presents an applicable strategy for elucidating the genetic mechanism underlying a long-term biological process by using plant growth as an example.

Congenital Thrombocytopenia and Myelofibrosis Due to Germline Mutations in G6b-B— a Megakaryocyte-Specific Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Receptor

Unlike primary myelofibrosis (PMF) in adults, which is one of the three classical BCR-ABL-negative myeloproliferative neoplasms (MPN), myelofibrosis in children is rare, occurring most commonly in patients with acute leukemia, particularly acute megakaryoblastic leukemia. Congenital or inherited forms of myelofibrosis (cMF) are exceedingly rare, and may occur in the context of patients with inherited platelet disorders. Here we describe 3 families with inherited thrombocytopenia and cMF due to germline null mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM) receptor G6b-B.We studied 3 families with a total of 6 affected children, each of which presented with macrothrombocytopenia, mild anemia, mild leukocytosis and a distinctive pattern of bone marrow reticulin fibrosis centered around clusters of atypical megakaryocytes. The disease appeared to segregate as an autosomal recessive trait. All affected children had mild to moderate bleeding symptoms and required frequent platelet and occasional red cell transfusions. None showed evidence of extramedullary hematopoiesis, such as splenomegaly, and all were negative for MPN associated mutations in JAK2, MPL, and CALR.To identify the disease gene, we performed Affymetrix SNP 6.0 genotyping on peripheral blood DNA from three clinically affected children and 7 first and second-degree relatives from the index family, a complex consanguineous pedigree from Kuwait. Shared regions of homozygosity by descent were assessed using a custom designed platform; multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (MHC) on chromosome 6p (LOD=2.01), were identified as potential candidate genetic intervals. We focused on this region because affected individuals in the index family and one of the two other families, which were not known to be related, shared a common homozygous human leukocyte antigen (HLA) type and had congenital adrenal hyperplasia (CAH) due to the identical 21-hydroxylase (CYP21A2) mutation; the CYP21A2 and HLA loci are located at 6p21.33 and 6p21.32-6p22.1. Whole exome sequencing was performed on all three nuclear families and revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor G6b (MPIG6B, previously G6B-B, C6orf25), encoded within the candidate linkage region. We identified 5 individuals (4 affected, one previously unaffected) with a MPIG6B frameshift mutation (c.61_61+1dup; p.20fs) in two families from Kuwait. In addition we confirmed a second MPIG6B frameshift mutation at c.147insT (p.49fs) in two individuals (one proband, one mildly affected sibling) in a family from Saudi Arabia. No other homozygous mutations that segregated with the phenotype in all three families were identified. MPIG6B is a member of the immunoglobulin superfamily and is located in the MHC class III region on chromosome 6. To validate MPIG6B as a potential disease-causing gene, we evaluated G6b-B expression in bone marrow (BM) biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody specific to G6b-B. G6b-B was strongly and selectively expressed in megakaryocytes of control BM samples, but completely absent in megakaryocytes of the clinically affected individuals.Interestingly, a murine knockout that lacks G6b-B has a strikingly similar phenotype to these patients, the animals having macrothrombocytopenia, myelofibrosis and aberrant platelet production and function. Thus, in addition to uncovering the molecular basis of this rare disease, the clinical phenotype highlights the potential importance of MPIG6B and aberrant megakaryocytic activation in mediating other forms of BM fibrosis. DisclosuresNo relevant conflicts of interest to declare.

Autoimmune experimental orchitis and chronic glomerulonephritis with end stage renal disease are controlled by Cgnz1 for susceptibility to end organ damage

Cgnz1 on chromosome 1 mapped into a 1.34 Mb region of chromosome 1 in NZM2328 confers the progression of immune complex (IC)-mediated glomerulonephritis (GN) from acute GN (aGN) to chronic GN (cGN) with severe proteinuria and end stage renal disease in female mice. This genetic locus mediates podocyte susceptibility to IC-mediated damage. Taking advantage of the published observation that Cgnz1 is derived from NZW and that NZW is susceptible to orchitis, epididymitis and vasitis while C57L/J is resistant to these diseases, the possibility that this genetic region also confers germ cells susceptible to damage with aspermatogenesis and sterility in an active experimental autoimmune orchitis (EAO) model was investigated. Male mice from multiple intrachromosome (chromosome 1) recombinant strains were subjected to immunization with a sperm homogenate in CFA with concomitant administration of Bordetella pertussis toxin. There was concordance of the progression from aGN to cGN, severe proteinuria and end stage renal disease with susceptibility of EAO in NZM2328 and its congenic strains with various chromosome 1 genetic intervals introgressed from C57L/J to NZM2328. Both resistant and susceptible strains made comparable anti-testis and anti-sperm Abs. Thus the genetic interval that determines susceptibility to EAO is identical to that of Cgnz1 and mapped to the 1.34 Mb region in chromosone 1. This region likely confers germ cells in the male gonad susceptible to damage by immunologically mediated inflammation. This region has been tentatively renamed Cgnz1/Eaoz1. These observations further emphasize the importance of end organ susceptibility to damage in the pathogenesis of both systemic and organ specific autoimmune diseases.

QTL mapping for soybean (Glycine max L.) leaf chlorophyll-content traits in a genotyped RIL population by using RAD-seq based high-density linkage map

BackgroundDifferent soybean (Glycine max L.) leaf chlorophyll-content traits are considered to be significantly linked to soybean yield. To map the quantitative trait loci (QTLs) of soybean leaf chlorophyll-content traits, an advanced recombinant inbred line (RIL, ZH, Zhonghuang 24 × Huaxia 3) population was adopted to phenotypic data acquisitions for the target traits across six distinct environments (seasons and soybean growth stages). Moreover, the restriction site-associated DNA sequencing (RAD-seq) based high-density genetic linkage map of the RIL population was utilized for QTL mapping by carrying out the composite interval mapping (CIM) approach.ResultsCorrelation analyses showed that most traits were correlated with each other under specific chlorophyll assessing method and were regulated both by hereditary and environmental factors. In this study, 78 QTLs for soybean leaf chlorophyll-content traits were identified. Furthermore, 13 major QTLs and five important QTL hotspots were classified and highlighted from the detected QTLs. Finally, Glyma01g15506, Glyma02g08910, Glyma02g11110, Glyma07g15960, Glyma15g19670 and Glyma15g19810 were predicted from the genetic intervals of the major QTLs and important QTL hotspots.ConclusionsThe detected QTLs and candidate genes may facilitate to gain a better understanding of the hereditary basis of soybean leaf chlorophyll-content traits and may be valuable to pave the way for the marker-assisted selection (MAS) breeding of the target traits.

Reproductive tissues-specific meta-QTLs and candidate genes for development of heat-tolerant rice cultivars.

By integrating genetics and genomics data, reproductive tissues-specific and heat stress responsive 35 meta-QTLs and 45 candidate genes were identified, which could be exploited through marker-assisted breeding for fast-track development of heat-tolerant rice cultivars. Rice holds the key to future food security. In rice-growing areas, temperature has already reached an optimum level for growth, hence, any further increase due to global climate change could significantly reduce rice yield. Several mapping studies have identified a plethora of reproductive tissue-specific and heat stress associated inconsistent quantitative trait loci (QTL), which could be exploited for improvement of heat tolerance. In this study, we performed a meta-analysis on previously reported QTLs and identified 35 most consistent meta-QTLs (MQTLs) across diverse genetic backgrounds and environments. Genetic and physical intervals of nearly 66% MQTLs were narrower than 5cM and 2Mb respectively, indicating hotspot genomic regions for heat tolerance. Comparative analyses of MQTLs underlying genes with microarray and RNA-seq based transcriptomic data sets revealed a core set of 45 heat-responsive genes, among which 24 were reproductive tissue-specific and have not been studied in detail before. Remarkably, all these genes corresponded to various stress associated functions, ranging from abiotic stress sensing to regulating plant stress responses, and included heat-shock genes (OsBiP2, OsMed37_1), transcription factors (OsNAS3, OsTEF1, OsWRKY10, OsWRKY21), transmembrane transporters (OsAAP7A, OsAMT2;1), sugar metabolizing (OsSUS4, α-Gal III) and abiotic stress (OsRCI2-7, SRWD1) genes. Functional data evidences from Arabidopsis heat-shock genes also suggest that OsBIP2 may be associated with thermotolerance of pollen tubes under heat stress conditions. Furthermore, promoters of identified genes were enriched with heat, dehydration, pollen and sugar responsive cis-acting regulatory elements, proposing a common regulatory mechanism might exist in rice for mitigating reproductive stage heat stress. These findings strongly support our results and provide new candidate genes for fast-track development of heat-tolerant rice cultivars.

Identification of nodulation-related genes in Medicago truncatula using genome-wide association studies and co-expression networks.

Genome‐wide association studies (GWAS) have proven to be a valuable approach for identifying genetic intervals associated with phenotypic variation in Medicago truncatula. These intervals can vary in size, depending on the historical local recombination. Typically, significant intervals span numerous gene models, limiting the ability to resolve high‐confidence candidate genes underlying the trait of interest. Additional genomic data, including gene co‐expression networks, can be combined with the genetic mapping information to successfully identify candidate genes. Co‐expression network analysis provides information about the functional relationships of each gene through its similarity of expression patterns to other well‐defined clusters of genes. In this study, we integrated data from GWAS and co‐expression networks to pinpoint candidate genes that may be associated with nodule‐related phenotypes in M. truncatula. We further investigated a subset of these genes and confirmed that several had existing evidence linking them nodulation, including MEDTR2G101090 (PEN3‐like), a previously validated gene associated with nodule number.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Development and application of indica\u2013japonica SNP assays using the Fluidigm platform for rice genetic analysis and molecular breeding

Molecular markers are efficient and essential genotyping tools for molecular breeding and genetic analysis of rice. We developed two 96-plex indica–japonica single nucleotide polymorphism (SNP) genotyping sets for genetic analysis and molecular breeding in rice using the Fluidigm platform. Informative SNPs between indica and japonica were selected from SNP data of the Rice Diversity database, HapRice world SNP data of the Q-TARO database, and our 40 rice cultivar resequencing dataset. SNPs in set 1 were evenly distributed across all 12 rice chromosomes at a spacing of 4–5 Mb between adjacent SNPs. SNPs in set 2 mapped to the long genetic intervals in set 1 and included 14 functional or linked SNPs in genes previously cloned and associated with agronomic traits. Additionally, we used the SNP sets developed in this study to perform genetic diversity analysis of various cultivated and wild rice accessions, construction and validation of a subspecies diagnostic subset, linkage map construction and quantitative trait locus (QTL) analysis of a japonica × indica F2 population, and background profiling during marker-assisted backcrossing. Furthermore, we identified subspecies-specific SNPs and discuss their distribution and association with agronomic traits and subspecies differentiation. Our results indicate that these subspecies-specific SNPs were present in wild rice prior to domestication. This genotyping system will serve as an efficient and quick tool for genetic analysis and molecular breeding in rice.