Molecular Marker Map Research Articles

Characterization and Mapping of a Rolling Leaf Mutant Allele rlT73 on Chromosome 1BL of Wheat.

Leaf rolling is regarded as an important morphological trait in wheat breeding. Moderate leaf rolling is helpful to keep leaves upright and improve the photosynthesis of plants, leading to increased yield. However, studies on the identification of genomic regions/genes associated with rolling leaf have been reported less frequently in wheat. In this study, a rolling leaf mutant, T73, which has paired spikelets, dwarfism, and delayed heading traits, was obtained from a common wheat landrace through ethyl methanesulfonate mutagenesis. The rlT73 mutation caused an increase in the number of epidermal cells on the abaxial side and the shrinkage of bulliform cells on the adaxial side, leading to an adaxially rolling leaf phenotype. Genetic analysis showed that the rolling leaf phenotype was controlled by a single recessive gene. Further Wheat55K single nucleotide polymorphism array-based bulked segregant analysis and molecular marker mapping delimited rlT73 to a physical interval of 300.29-318.33 Mb on the chromosome arm 1BL in the Chinese Spring genome. We show that a point mutation at the miRNA165/166 binding site of the HD zipper class III transcription factor on 1BL altered its transcriptional level, which may be responsible for the rolling leaf phenotype. Our results suggest the important role of rlT73 in regulating wheat leaf development and the potential of miRNA-based gene regulation for crop trait improvement.

Status, Gaps and Perspectives of Powdery Mildew Resistance Research and Breeding in Cucurbits

Cucurbitaceae, the gourd family of flowering plants, is a very large and diverse family, the order Cucurbitales, contains 95 genera and 950–980 species of food and ornamental plants, and wild and weedy species mostly with high genetic diversity. This review is focused on the most important cucurbit crops (Cucumis sativus, Cucumis melo, Cucurbita spp., Citrullus lanatus, Momordica charantia, Lagenaria siceraria, and Luffa acutangula) and some their wild relatives as hosts of cucurbit powdery mildews (CPM). Powdery mildews (PM) (Ascomycota, Erysiphales) are one of the most frequently encountered and easily visible groups of plant pathogenic fungi with > 900 species. They are obligate biotrophs, they colonize above-ground plant tissues, mostly leaves, though they may also colonize stems, petioles, flowers and fruits, and they are usually debilitators, not killers. These parasitic fungi have been problematic on cucurbits for a long time world-wide, causing serious economic losses in yield and quality. All economically important cucurbit crops host CPM. Seven PM species with different taxonomic positions, host ranges, geographic distributions and ecological requirements are known on cucurbits. CPM species taxonomy and denomination rapidly changed during last few decades through detailed analyses and clarifications. At least three PM species frequently parasitize cucurbits: the endoparasite Leveillula taurica (Lt) with marginal economic importance; and two ectoparasitic species, Golovinomyces orontii (Go), and Podosphaera xanthii (Px), which are economically important world-wide. The two pathogens differ in ecological requirements and distribution, though they may occur together in mixed infections. They are highly variable at the population level for virulence, race identities, and fast adaptation of pathogens. Cucurbit-CPM species interactions are diverse and complicated, and differ between hosts and their respective pathogen genera and species. Here we present a critical overview of obstacles, gaps and recent progress in these matters for six cucurbit genera with respect to resistance resources, genetics of resistance, genetic mapping and development of molecular markers, physiology and mechanisms of resistance, developments in mlo-mediated resistance, patents, and CPM resistance breeding.

Has recombination changed during the recent evolution of the guppy Y chromosome?

Genome sequencing and genetic mapping of molecular markers have demonstrated nearly complete Y-linkage across much of the guppy (Poecilia reticulata) XY chromosome pair. Predominant Y-linkage of factors controlling visible male-specific coloration traits also suggested that these polymorphisms are sexually antagonistic (SA). However, occasional exchanges with the X are detected, and recombination patterns also appear to differ between natural guppy populations, suggesting ongoing evolution of recombination suppression under selection created by partially sex-linked SA polymorphisms. We used molecular markers to directly estimate genetic maps in sires from 4 guppy populations. The maps are very similar, suggesting that their crossover patterns have not recently changed. Our maps are consistent with population genomic results showing that variants within the terminal 5 Mb of the 26.5 Mb sex chromosome, chromosome 12, are most clearly associated with the maleness factor, albeit incompletely. We also confirmed occasional crossovers proximal to the male-determining region, defining a second, rarely recombining, pseudo-autosomal region, PAR2. This fish species may therefore have no completely male-specific region (MSY) more extensive than the male-determining factor. The positions of the few crossover events suggest a location for the male-determining factor within a physically small repetitive region. A sex-reversed XX male had few crossovers in PAR2, suggesting that this region's low crossover rate depends on the phenotypic, not the genetic, sex. Thus, rare individuals whose phenotypic and genetic sexes differ, and/or occasional PAR2 crossovers in males can explain the failure to detect fully Y-linked variants.

Mapping and identification ofmolecular markers forthePc96 gene conferring resistance tocrown rust inoat.

Oat crown rust caused by Puccinia coronata f. sp. avenae P. Syd. & Syd (Pca) is a major constraint to oat (Avena sativa L.) production in many parts of the globe. The objectives of this study were to locate Pc96 on the oat consensus map and to develop SNP markers linked to Pc96 for use in marker-assisted selection. SNP loci linked to the crown rust resistance gene Pc96 were identified by linkage analysis and PACE assays were developed for marker-assisted selection in breeding programs. Pc96 is a race-specific crown rust resistance gene originating from cultivated oat that has been deployed in North American oat breeding programs. Pc96 was mapped in a recombinant inbred line population (n = 122) developed from a cross between the oat crown rust differential known to carry Pc96 and the differential line carrying Pc54. A single resistance locus was identified on chromosome 7D between 48.3 and 91.2 cM. The resistance locus and linked SNPs were validated in two additional biparental populations, Ajay × Pc96 (F2:3, n = 139) and Pc96 × Kasztan (F2:3, n = 168). Based on all populations, the most probable location of the oat crown rust resistance gene Pc96 on the oat consensus map was on chromosome 7D approximately at 87.3 cM. In the Ajay × Pc96 population, a second unlinked resistance gene was contributed by the Pc96 differential line, which mapped to chromosome 6C at 75.5 cM. A haplotype of nine linked SNPs predicted the absence of Pc96 in a diverse group of 144 oat germplasm. SNPs that are closely linked to the Pc96 gene may be beneficial as PCR-based molecular markers in marker-assisted selection.

Candidate genes for height measurements in Campolina horses

Context Genomic tools allow for increased efficiency in animal production and, consequently, greater production gains. Morphometric measurements of height can be used for breed characterisation/registration, and sex distinction, in addition to being associated with balance, posture, and the range of motion of the limbs, influencing the dynamics and quality of the gait. Aims This study was conducted to examine candidate genes related to the morphometric measurement of heights in Campolina horses. Methods The study involved information from 18 876 horses of this breed, containing phenotypic data of the measurements of withers, croup, back, and barrel heights, provided by the Brazilian Association of Campolina Horse Breeders (ABCCCampolina). Of these, 48 horses of the Brazilian Campolina breed were genotyped with the Equine 65 K lllumina® chip. The effects of the single-nucleotide polymorphisms were estimated by the weighted single-step method, using BLUPF90 family programs. Genes in the main chromosomal regions were identified by consultation with the NCBI. Key results A genomic-wide association study for withers height showed that 46% of the variation in this measurement was explained by 20 genomic windows. For croup height, 19 genomic windows were responsible for explaining 44% of the variation. Sixteen genomic windows were responsible for explaining 37.8% of the variation in back height and 18 genomic regions explained 39.7% of the variation in barrel height. Conclusion The ADAM17, SOX11, and KLF11 genes are located in the genomic windows that most explain the variations in the traits under study and have a biological action on the locomotor system. Implications The ADAM17, SOX11, and KLF11 genes are candidates for fine mapping and construction of molecular markers.

Integrated transcriptional and metabolomics signature pattern of pigmented wheat to insight the seed pigmentation and other associated features

Anthocyanin biosynthesis in plants is complex, especially in a polyploid monocot wheat plant. Using whole-genome sequencing, transcriptomics, and LC-MS/MS, we investigated anthocyanin pigmentation patterns in (black, blue, and purple) colored wheat seeds. According to differential gene expression profiling, 2AS-MYC, 7DL-MYB, and WD40 regulatory genes control purple pericarp coloration, 4DL-MYC, 2AS-MYC, 7DL-MYB, WD40 control blue aleurone coloration, and 4DL-MYC, 7DL-MYB, WD40 controls black aleurone color. We hypothesized that at least one MYC and MYB isoform is sufficient to regulate the anthocyanin synthesis in pericarp or aleurone. Transcriptomics and metabolomics revealed that the purple pericarp trait is associated with acylated anthocyanins compared to blue aleurone. Based upon the reduced expressions of the genes belonging to the 4D, SSR molecular marker mapping, variant calling using genome sequencing, and IGV browser gene structure visualization, it was inferred that the advanced black and blue wheat lines were substitution lines (4E{4D}), with very small recombinations. Pericarp anthocyanin pigmentation is controlled by a mutation in chromosome 2AS of purple wheat, and environmental variations influence pigmented pericarp trait. The expression patterns of anthocyanin structural and other genes varied in different colored wheat, corroborating differences in agronomical metrics. Ovate seed shape trait in black and blue wheat dragged with 4E chromosome.

Making data map-worthy—enhancing routine malaria data to support surveillance and mapping of Plasmodium falciparum anti-malarial resistance in a pre-elimination sub-Saharan African setting: a molecular and spatiotemporal epidemiology study

BackgroundIndependent emergence and spread of artemisinin-resistant Plasmodium falciparum malaria have recently been confirmed in Africa, with molecular markers associated with artemisinin resistance increasingly detected. Surveillance to promptly detect and effectively respond to anti-malarial resistance is generally suboptimal in Africa, especially in low transmission settings where therapeutic efficacy studies are often not feasible due to recruitment challenges. However, these communities may be at higher risk of anti-malarial resistance.MethodsFrom March 2018 to February 2020, a sequential mixed-methods study was conducted to evaluate the feasibility of the near-real-time linkage of individual patient anti-malarial resistance profiles with their case notifications and treatment response reports, and map these to fine scales in Nkomazi sub-district, Mpumalanga, a pre-elimination area in South Africa.ResultsPlasmodium falciparum molecular marker resistance profiles were linked to 55.1% (2636/4787) of notified malaria cases, 85% (2240/2636) of which were mapped to healthcare facility, ward and locality levels. Over time, linkage of individual malaria case demographic and molecular data increased to 75.1%. No artemisinin resistant validated/associated Kelch-13 mutations were detected in the 2385 PCR positive samples. Almost all 2812 samples assessed for lumefantrine susceptibility carried the wildtype mdr86ASN and crt76LYS alleles, potentially associated with decreased lumefantrine susceptibility.ConclusionRoutine near-real-time mapping of molecular markers associated with anti-malarial drug resistance on a fine spatial scale provides a rapid and efficient early warning system for emerging resistance. The lessons learnt here could inform scale-up to provincial, national and regional malaria elimination programmes, and may be relevant for other antimicrobial resistance surveillance.

Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat.

Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1-2 nucleotides leading to non-synonymous amino acid substitutions. The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

Identification of Maize Rf4-Restorer Lines and Development of a CAPS Marker for Rf4

Rf4 is one of the dominant restorer genes for maize C-type cytoplasmic male sterility (CMS-C), which has significant value in hybrid maize seed production. However, the highly complex fertility restoration mechanism of CMS-C makes it difficult to screen Rf4-restorer lines, and insufficient Rf4-restorer lines limit its use in current agricultural production. To search for Rf4-restorer lines, in this study, the genotypes of eighteen inbred maize lines at the Rf4 locus were analyzed based on the male fertility investigation of hybrid F1, the genetic analysis of F2 populations, molecular marker mapping, allelic tests, and Rf4 genomic sequence analysis. Our results indicated that of the eighteen maize inbred lines, ten were able to completely rescue CMS-C line CHuangzaosi (CHZS) male sterility. A genetic analysis showed that DAN598, PHT77, 78551S, and LH212Ht only contained one dominant restorer gene each, and the molecular-marker mapping indicated that their restorer genes were located at the short arm of chromosome 8. The allelic testing of the fertility of the restorer (Rf) demonstrated that the restorer gene of twelve inbred lines, including DAN598, PHT77, 78551S, and LH212Ht, was allelic to one restorer gene of A619. Furthermore, the genomic sequence alignment of Rf4 revealed that there were two different amino acids in the coding sequence between the A619 (Rf4Rf4) restorer lines and four CMS-C lines (rf4rf4). For the crucial S1596 site variation (TTT/TAC), DAN598, PHT77, 78551S, and LH212Ht shared the same bases (TTT) with A619 and encoded phenylalanine, while the four CMS-C sterile lines had the TAC and encoded tyrosine. Our results revealed that these tester lines, DAN598, PHT77, 78551S, and LH212Ht, were the Rf4-restorer lines. Additionally, derived from the sequence variants of Rf4, 39 possible Rf4-restorer lines from 129 inbred maize lines were detected. Furthermore, we developed a Cleaved Amplified Polymorphism Sequences (CAPS) marker based on the S1596 variations. The PCR amplification product of S1596 (TAC) was digested by the TatI endonuclease into two bands with sizes of ~260 bp and ~100 bp. In comparison, when S1596 was TTT, the PCR product could not be digested. In conclusion, in this study, we identified various Rf4-restorer lines for maize CMS-C and developed a molecular marker for Rf4. The reported results will contribute to the popularization and application of Rf4 in hybrid maize-seed production.

Genetic and transcriptomic dissection of an artificially induced paired spikelets mutant of wheat (Triticum aestivum L.).

Morphological, genetic and transcriptomic characterizations of an EMS-induced wheat paired spikelets (PS) mutant were performed. A novel qualitative locus WPS1 on chromosome 1D was identified. Grain yield of wheat is significantly associated with inflorescence or spike architecture. However, few genes related to wheat spike development have been identified and their underlying mechanisms are largely unknown. In this study, we characterized an ethyl methanesulfonate (EMS)-induced wheat mutant, wheat paired spikelets 1 (wps1). Unlike a single spikelet that usually develops at each node of rachis, a secondary spikelet appeared below the primary spikelet at most of the rachis nodes of wps1. The microscope observation showed that the secondary spikelet initiated later than the primary spikelet. Genetic analysis suggested that the PS of wps1 is controlled by a single dominant nuclear gene, designated WHEAT PAIRED SPIKELETS 1 (WPS1). Further RNA-seqbased bulked segregant analysis and molecular marker mapping localized WPS1 in an interval of 208.18-220.92Mb on the chromosome arm 1DL, which is different to known genes related to spike development in wheat. By using wheat omics data, TraesCS1D02G155200 encoding a HD-ZIP III transcription factor was considered as a strong candidate gene for WPS1. Transcriptomic analysis indicated that PS formation in wps1 is associated with auxin-related pathways and may be regulated by networks involving TB1, Ppd1, FT1, VRN1, etc. This study laid the solid foundation for further validation of the causal gene of WPS1 and explored its regulatory mechanism in PS formation and inflorescence development, which may benefit to kernel yield improvement of wheat based on optimization or design of spike architecture in the future.

Discerning the Putative U and V Chromosomes of Saccharina japonica (Phaeophyta) by Cytogenetic Mapping of Sex-Linked Molecular Markers

Saccharina japonica, an importantly industrial species in brown seaweeds, has a typical alternate life history of heteromorphic generations and UV sex determination system. But up to now, the sex chromosomes, U and V, in this kelp remain to be evidenced cytologically. In the present study, the female-linked (SJ-f_000170 and MSj68-58-2) and male-linked (SJ-13_001840 and MSj68-16-2) molecular markers developed on the basis of Ectocarpus sex-determining regions (SDRs) were selected after PCR amplification and Southern blotting analysis. Mono-color fluorescence in situ hybridization (FISH) to the kelp metaphase chromosomes with each of these confirmed sex-linked markers as a probe indicated that the hybridization signals were confined to either female or male gametophytes exclusively. The shape and size of hybridized chromosomes and the relative location of these sex-linked markers on them suggested that the marked chromosomes in a sex were the same, which was further confirmed by dual-color FISH observations. SJ-f_000170 and SJ-13_001840, corresponding to each sex, were therefore used to map the sporophyte metaphase chromosomes by dual-color FISH technique, and they were hybridized on different chromosomes as expected. The sporophyte metaphase chromosomes as hybridized by SJ-f_000170 and SJ-13_001840 resembled those in either female or male gametophytes, suggesting that they were the putative U and V, respectively, chromosomes in S. japonica. To further demonstrate the reliability of this inference, genomic information of the screened bacterial artificial chromosome (BAC) clones carrying two female-linked markers, for example, from the constructed BAC libraries of the kelp female gametophytes was provided. The insert sequencing of two selected BAC clones revealed that repetitive elements were rich whereas gene density was poor, which was characterized as non-recombining SDR sequences in brown seaweed Ectocarpus sp. and liverwort Marchantia polymorpha. The present study laid a solid foundation for unveiling the mystery of S. japonica sex chromosomes.

Breeding for Economically and Environmentally Sustainable Wheat Varieties: An Integrated Approach from Genomics to Selection.

Simple SummaryThe BREEDWHEAT project has laid the foundation for future commercial varieties by providing new and original modern molecular tools to breeders who have applied them to: (1) efficiently analyze and structure the genetic diversity; (2) decipher traits of agronomical interest including biotic and abiotic resistance and tolerance; (3) develop methodologies to implement genomic and phenomic selection; (4) store and bring tools to the world wheat community to access all of the results and data. This will provide a helping hand to developing new original and adapted wheat varieties that will be more productive in terms of the quantity and quality in the context of sustainable agriculture using less fertilizers, pesticides, fungicides, and water. This would help in tackling the challenges that we have to face, especially with regard to global change.There is currently a strong societal demand for sustainability, quality, and safety in bread wheat production. To address these challenges, new and innovative knowledge, resources, tools, and methods to facilitate breeding are needed. This starts with the development of high throughput genomic tools including single nucleotide polymorphism (SNP) arrays, high density molecular marker maps, and full genome sequences. Such powerful tools are essential to perform genome-wide association studies (GWAS), to implement genomic and phenomic selection, and to characterize the worldwide diversity. This is also useful to breeders to broaden the genetic basis of elite varieties through the introduction of novel sources of genetic diversity. Improvement in varieties particularly relies on the detection of genomic regions involved in agronomical traits including tolerance to biotic (diseases and pests) and abiotic (drought, nutrient deficiency, high temperature) stresses. When enough resolution is achieved, this can result in the identification of candidate genes that could further be characterized to identify relevant alleles. Breeding must also now be approached through in silico modeling to simulate plant development, investigate genotype × environment interactions, and introduce marker–trait linkage information in the models to better implement genomic selection. Breeders must be aware of new developments and the information must be made available to the world wheat community to develop new high-yielding varieties that can meet the challenge of higher wheat production in a sustainable and fluctuating agricultural context. In this review, we compiled all knowledge and tools produced during the BREEDWHEAT project to show how they may contribute to face this challenge in the coming years.

A consensus and saturated genetic map provides insight into genome anchoring, synteny of Solanaceae and leaf- and fruit-related QTLs in wolfberry (Lycium Linn.)

BackgroundLycium Linn. (Solanaceae) is a genus of economically important plants producing fruits and leaves with high nutritional value and medicinal benefits. However, genetic analysis of this plant and molecular breeding for quality improvement are limited by the lack of sufficient molecular markers.ResultsIn this study, two parental strains, ‘Ningqi No. 1’ (Lycium barbarum L.) and ‘Yunnan Gouqi’ (Lycium yunnanense Kuang et A.M. Lu), and 200 F1 hybrid individuals were resequenced for genetic analysis. In total, 8,507 well-selected SNPs were developed, and a high-density genetic map (NY map) was constructed with a total genetic distance of 2,122.24 cM. A consensus genetic map was established by integrating the NY map and a previously published genetic map (NC map) containing 15,240 SNPs, with a total genetic distance of 3,058.19 cM and an average map distance of 0.21 cM. The 12 pseudochromosomes of the Lycium reference genome were anchored using this consensus genetic map, with an anchoring rate of 64.3%. Moreover, weak collinearities between the consensus map and the pepper, potato, and tomato genomes were observed. Twenty-five stable QTLs were identified for leaf- and fruit-related phenotypes, including fruit weight, fruit longitude, leaf length, the fruit index, and the leaf index; these stable QTLs were mapped to four different linkage groups, with LOD scores ranging from 2.51 to 19.37 and amounts of phenotypic variance explained from 6.2% to 51.9%. Finally, 82 out of 188 predicted genes underlying stable QTLs for fruit-related traits were differentially expressed according to RNA-seq analysis.ConclusionsA chromosome-level assembly can provide a foundation for further functional genomics research for wolfberry. The genomic regions of these stably expressed QTLs could be used as targets for further fine mapping and development of molecular markers for marker-assisted selection (MAS). The present study provided valuable information on saturated SNP markers and reliable QTLs for map-based cloning of functional genes related to yield and morphological traits in Lycium spp.

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods

Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2–2000 μm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 μm) to mesoplankton (180–2000 μm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 μm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.

Identification and candidate gene mining of HvSS1, a novel qualitative locus on chromosome 6H, regulating the uppermost internode elongation in barley (Hordeum vulgare L.).

A novel qualitative locus regulating the uppermost internode elongation of barley was identified and mapped on 6H, and the candidate gene mining was performed by employing various barley genomic resources. The stem of grass crops, such as barley and wheat, is composed of several interconnected internodes. The extent of elongation of these internodes determines stem height, and hence lodging, canopy architecture, and grain yield. The uppermost internode (UI) is the last internode to elongate. Its elongation contributes largely to stem height and facilitates spike exsertion, which is crucial for final grain yield. Despite the molecular mechanism underlying regulation of UI elongation was extensively investigated in rice, little is known in barley. In this study, we characterized a barley spontaneous mutant, Sheathed Spike 1 (SS1), showing significantly shortened UI and sheathed spike (SS). The extension of UI parenchyma cell in SS1 was significantly suppressed. Exogenous hormone treatments and RNA-seq analysis indicated that the suppression of UI elongationis possibly related to insufficient contentof endogenous bioactive gibberellin. Genetic analysis showed that SS1 is possibly controlled by a qualitative dominant nuclear factor. Bulked segregant analysis and further molecular marker mapping identified a novel major locus, HvSS1, in a recombination cold spot expanding 173.44-396.33Mb on chromosome 6H. The candidate gene mining was further conducted by analyzing sequence differences, spatiotemporal expression patterns, and variant distributions of genes in the candidate interval by employing various barley genomic resources of worldwide collections of barley accessions. This study made insight into genetic control of UI elongation in barley and laid a solid foundation for further gene cloning and functional characterization. The results obtained here also provided valuable information for similar research in wheat.

Comparative Cytogenetics in Four Leptodactylus Species (Amphibia, Anura, Leptodactylidae): Evidence of Inner Chromosomal Diversification in Highly Conserved Karyotypes

With 82 species currently described, the genus Leptodactylus is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 Leptodactylus species (L. macrosternum, L. pentadactylus, L. fuscus, and Leptodactylus cf. podicipinus), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for Leptodactylus cf. podicipinus which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in L. pentadactylus), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.

QTL analysis of shoot elongation before winter in relation to vernalization requirement in the doubled haploid population L16\u2009\xd7\u2009Express617 (Brassica napus L.)

In winter oilseed rape shoot elongation before winter is considered as a critical component of the complex trait winter hardiness. Genotypes with an enhanced shoot length before winter are very much prone to frost damage. However, it is not known to which extent shoot elongation before winter is related to vernalization requirement. Shoot length measured 3 months after sowing of winter oilseed rape in spring has been shown to be a good quantitative estimator for vernalization requirement. The main objective of this study was to analyze inheritance of shoot elongation before winter and in spring-sown field experiments using a doubled haploid population derived from the cross between two inbred lines. A SNP based molecular marker map was used to map QTL for shoot length and flowering time. Significant genotypic effects were detected for shoot length before winter and in the spring-sown environment, but there was no correlation between both traits. Six QTL for shoot length before winter contributed 49.2% to the phenotypic variance. For shoot length in the spring-sown environment three QTL were detected that explained 68% of the phenotypic variance. Flowering time genes CONSTANS (BnaC09g41980D), FLC (BnaC09g42060D) and FT (BnaA02g12130D) were identified within C09 and A02 QTL confidence intervals. No collocation of QTL for shoot length before winter and for shoot length in the spring-sown environment was found. Results show that selection for reduced shoot length before winter would be effective and that this trait is inherited largely independent of vernalization requirement.

Molecular patterning of the embryonic cranial mesenchyme revealed by genome-wide transcriptional profiling

In the head of an embryo, a layer of mesenchyme surrounds the brain underneath the surface ectoderm. This cranial mesenchyme gives rise to the meninges, the calvaria (top part of the skull), and the dermis of the scalp. Abnormal development of these structures, especially the meninges and the calvaria, is linked to significant congenital defects in humans. It has been known that different areas of the cranial mesenchyme have different fates. For example, the calvarial bone develops from the cranial mesenchyme on the baso-lateral side of the head just above the eye (supraorbital mesenchyme, SOM), but not from the mesenchyme apical to SOM (early migrating mesenchyme, EMM). However, the molecular basis of this difference is not fully understood. To answer this question, we compared the transcriptomes of EMM and SOM using high-throughput sequencing (RNA-seq). This experiment identified a large number of genes that were differentially expressed in EMM and SOM, and gene ontology analyses found very different terms enriched in each region. We verified the expression of about 40 genes in the head by RNA in situ hybridization, and the expression patterns were annotated to make a map of molecular markers for 6 subdivisions of the cranial mesenchyme. Our data also provided insights into potential novel regulators of cranial mesenchyme development, including several axon guidance pathways, lectin complement pathway, cyclic-adenosine monophosphate (cAMP) signaling pathway, and ZIC family transcription factors. Together, information in this paper will serve as a unique resource to guide future research on cranial mesenchyme development.

Genetic mapping and development of molecular markers for a candidate gene locus controlling rind color in watermelon.

ClCG08G017810 (ClCGMenG) encoding a 2-phytyl-1,4-beta-naphthoquinone methyltransferase protein is associated with formation of dark green versus light green rind color in watermelon. Rind color is an important agronomic trait in watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai], but the underlying molecular mechanism for this trait is not fully known. In the present study, we identified a single locus on chromosome 8 accounting for watermelon rind color (dark green vs. light green). Genetic analysis of F1, F2, and BC1 populations derived from two parental lines (9904 with dark green rind and Handel with light green rind) revealed that the watermelon rind color (dark green vs. light green) is controlled by a single locus, and dark green is dominant to light green rind. Initial mapping revealed a region of interest spanning 2.07Mb on chromosome 8. Genetic mapping with CAPS and SNP markers narrowed down the candidate region to 31.4kb. Gene annotation of the corresponding region in the reference genome revealed the ClCG08G017810 gene sequence encoding the 2-phytyl-1,4-beta-naphthoquinone methyltransferase protein. The sequence alignment of the candidate gene with the two parental lines suggested a nonsynonymous SNP mutation in the coding region of ClCG08G017810, converting an arginine (R) to glycine (G). The SNP might be associated with rind color of 103 watermelon germplasm lines investigated in this study. The qRT-PCR analysis revealed higher expression of ClCG08G017810 in dark green rind than in light green rind. Therefore, ClCG08G017810 is a candidate gene associated with watermelon rind color. The present study facilitates marker-assisted selection useful for the development of cultivars with desirable rind color.

Identification and fine mapping of molecular markers closely linked to fruit spines size ss gene in cucumber (Cucumis sativus L.)

Fruit spine size is one of the importantly external quality traits effected the economic value of cucumber fruit. Morphological–cytological observation of the fruit spine size phenotype indicated that large spine formation arises from an increasing of spiny pedestal cell number caused by cell division, and best periods to accurately score fruit spine size trait was 4th day before flowering to 7th day after flowering according the continuous observation. Genetic analysis showed that a single dominant gene determined the fruit spine size trait in cucumber. BC1 population (189 individuals) of two inbred lines (large spine PI197088 and small spine SA0422) was used for primary mapping of the SS/ss locus with 7 markers covering an interval of 37.1 cM. An F2 segregating population of 1032 individuals constructed from the same two parents (PI197088 and SA0422) was used to fine mapping of the SS/ss locus. Six new markers linked to the gene were successfully screened for construction of a fine linkage map, in which the SS/ss locus was located in the region flanked by marker SE1 (3 recombinants) and SSR43 (2 recombinants) with a 189 kb physical distance. Markers from this study will be valuable for candidate gene cloning and marker-assisted selection for cucumber breeding.