An ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5 gene mutation confers light green peel in cucumber

The peel color of fruit is an important commercial trait in cucumber,but the underlying molecular basis is largely unknown.A mutant showing light green exocarp was discovered from ethyl methane sulfonate(EMS) mutagenized cucumber line 406 with dark green exocarp.Genetic analysis showed the mutant phenotype is conferred by a single recessive gene,here designated as lgp(light green peel).By re-sequencing of bulked segregants,we identified the candidate gene Csa7Go51430 encoding ACCUMULATION AND REPLICATION OF CHLOROPLASTS 5(ARC5) that plays a vital role in chloroplast division in Arabidopsis.A single nucleotide polymorphism(SNP) causing amino acid alteration in the conserved GTPase domain of Csa7G051430 showed co-segregation with the altered phenotype.Furthermore,the transient RNA interference of this gene resulted in reduced number and enlarged size of chloroplasts,which were also observed in the Igp mutant.This evidence supports that the non-synonymous SNP in Csa7G051430 is the causative mutation for the light green peel.This study provides a new allele for cucumber breeding for light green fruits and additional resource for the study of chloroplast development.     

Development and utilization of a new chemically‐induced soybean library with a high mutation density

Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate (EMS)‐induced soybean (Glycine max) population, consisting of 21,600 independent M₂ lines, was developed. Over 1,000 M_{4 (5)} families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll (Chl) content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to MinnGold, where a different single nucleotide polymorphism variation in the Mg‐chelatase subunit gene (ChlI1a) results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker‐assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.     

exonsampler: a computer program for genome‐wide and candidate gene exon sampling for targeted next‐generation sequencing

The computer program exonsampler automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next‐generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User‐adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of exonsampler to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon‐capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16 000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection.     

SNP discovery and genotyping using restriction‐site‐associated DNA sequencing in chickens

Single nucleotide polymorphisms (SNPs) are essential to the understanding of population genetic variation and diversity. Here, we performed restriction‐site‐associated DNA sequencing (RAD‐seq) on 72 individuals from 13 Chinese indigenous and three introduced chicken breeds. A total of 620 million reads were obtained using an Illumina Hiseq2000 sequencer. An average of 75 587 SNPs were identified from each individual. Further filtering strictly validated 28 895 SNPs candidates for all populations. When compared with the NCBI dbSNP (chicken_9031), 15 404 SNPs were new discoveries. In this study, RAD‐seq was performed for the first time on chickens, implicating the remarkable effectiveness and potential applications on genetic analysis and breeding technique for whole‐genome selection in chicken and other agricultural animals.     

Evolution and function of MADS‐box genes involved in orchid floral development

Orchids are known for their beauty and complexity of flower and ecological strategies. The evolution in orchid floral morphology, structure, and physiological properties has held the fascination of botanists for centuries, from Darwin through to the present. In floral studies, MADS‐box genes contributing to the now famous ABCDE model of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization offers an opportunity to discover new variant genes and different levels of complexity to the ABCDE model. Recently, several remarkable research reports on orchid MADS‐box genes, especially B‐class MADS‐box genes, have revealed the evolutionary track and important functions on orchid floral development. Diversification and fixation of both paleoAP3 gene sequences and expression profiles might be explained by subfunctionalization and even neofunctionalization. Knowledge about MADS‐box genes encoding ABCDE functions in orchids will give insights into the highly evolved floral morphogenetic networks of orchids.     

A 63-bp insertion in exon 2 of the porcine KIF21A gene is associated with arthrogryposis multiplex congenita

A recessive form of arthrogryposis multiplex congenita (AMC) was detected 20 years ago in the Swiss Large White (SLW) pig population. A diagnostic marker test enabled the identification of carrier animals, but the underlying causal mutation remains unknown. To identify the mutation underlying AMC, we collected SNP chip genotyping data for 11 affected piglets and 23 healthy pigs. Association testing using 47 829 SNPs confirmed that AMC maps to SSC5 (P = 9.4 × 10⁻¹³). Subsequent autozygosity mapping revealed a common 6.06 Mb region (from 66 757 970 to 72 815 151 bp) of extended homozygosity in 11 piglets affected by AMC. Using WGS data, we detected a 63-bp insertion compatible with the recessive inheritance of AMC in the second exon of KIF21A gene encoding Kinesin Family Member 21A. The 63-bp insertion is predicted to introduce a premature stop codon in KIF21A gene (p.Val41_Phe42insTer) that truncates 1614 amino acids (~97%) from the protein. We found that this deleterious allele still segregates at a frequency of 0.1% in the SLW pig population. Carrier animals can now be detected unambiguously and excluded from breeding.     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.     

Trans‐species variation in Dmrt1 is associated with sex determination in four European tree‐frog species

Empirical studies on the relative roles of occasional XY recombination versus sex‐chromosome turnover in preventing sex‐chromosome differentiation may shed light on the evolutionary forces acting on sex‐determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex‐determination systems that keep sex‐chromosome identity): both models predict X and Y alleles at sex‐linked genes to cluster by species. However, the XY‐recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex‐determining region (SDR) with trans‐species polymorphism associated to sex. Here we combine a candidate‐gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N‐terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex‐chromosome homomorphy in this clade results only from XY recombination, and take an important step toward the identification of the sex‐determining locus. Moreover, the sex‐diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.     

High‐resolution array‐CGH in patients with oculocutaneous albinism identifies new deletions of the TYR,OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene

Oculocutaneous albinism (OCA) is caused by mutations in six different genes, and their molecular diagnosis encompasses the search for point mutations and intragenic rearrangements. Here, we used high‐resolution array‐comparative genome hybridization (CGH) to search for rearrangements across exons, introns and regulatory sequences of four OCA genes: TYR, OCA2, TYRP1, and SLC45A2. We identified a total of ten new deletions in TYR, OCA2, and SLC45A2. A complex rearrangement of OCA2 was found in two unrelated patients. Whole‐genome sequencing showed deletion of a 184‐kb fragment (identical to a deletion previously found in Polish patients), whereby a large portion of the deleted sequence was re‐inserted after severe reshuffling into intron 1 of OCA2. The high‐resolution array‐CGH presented here is a powerful tool to detect gene rearrangements. Finally, we review all known deletions of the OCA1–4 genes reported so far in the literature and show that deletions or duplications account for 5.6% of all mutations identified in the OCA1–4 genes.     

Next‐generation sequencing for identification of candidate genes for Fusarium wilt and sterility mosaic disease in pigeonpea (Cajanus cajan)

To map resistance genes for Fusarium wilt (FW) and sterility mosaic disease (SMD) in pigeonpea, sequencing‐based bulked segregant analysis (Seq‐BSA) was used. Resistant (R) and susceptible (S) bulks from the extreme recombinant inbred lines of ICPL 20096 × ICPL 332 were sequenced. Subsequently, SNP index was calculated between R‐ and S‐bulks with the help of draft genome sequence and reference‐guided assembly of ICPL 20096 (resistant parent). Seq‐BSA has provided seven candidate SNPs for FW and SMD resistance in pigeonpea. In parallel, four additional genotypes were re‐sequenced and their combined analysis with R‐ and S‐bulks has provided a total of 8362 nonsynonymous (ns) SNPs. Of 8362 nsSNPs, 60 were found within the 2‐Mb flanking regions of seven candidate SNPs identified through Seq‐BSA. Haplotype analysis narrowed down to eight nsSNPs in seven genes. These eight nsSNPs were further validated by re‐sequencing 11 genotypes that are resistant and susceptible to FW and SMD. This analysis revealed association of four candidate nsSNPs in four genes with FW resistance and four candidate nsSNPs in three genes with SMD resistance. Further, In silico protein analysis and expression profiling identified two most promising candidate genes namely C.cajan_01839 for SMD resistance and C.cajan_03203 for FW resistance. Identified candidate genomic regions/SNPs will be useful for genomics‐assisted breeding in pigeonpea.     

CsSPL functions as an adaptor between HD‐ZIP III and CsWUS transcription factors regulating anther and ovule development in Cucumis sativus (cucumber)

Anther and ovule genesis preconditions crop fertilization and fruit production; however, coordinative regulation of anther and ovule development and underlying molecular pathways remain largely elusive. Here, we found that SPOROCYTELESS (SPL)/NOZZLE (NZZ) expression was nearly abolished in a Cucumis sativus (cucumber) mutant with severely defective anther and ovule development. CsSPL was expressed specifically in the developing anthers and ovules. Knock‐down of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development. Importantly, CsSPL directly interacted with CsWUS (WUSCHEL) in the nucellus and YABBY family genes in integuments, and positively regulated CsWUS expression, meanwhile the HD‐ZIP III gene CsPHB (PHABULOSA), expressed specifically in the nucellus, promoted CsSPL expression by binding to the CsSPL promoter. Thus, CsSPL acts as an adaptor to link CsPHB and CsWUS functioning, underpinning a previously unidentified regulatory pathway orchestrating sex organ development in planta. In addition, auxin accumulation was reduced in the reproductive organs of CsSPL knock‐down plants. Biochemical analyses further showed that CsSPL stimulated the expression of AUXIN RESPONSE FACTOR 3 (CsARF3), and was positively regulated by CsARF13 during reproductive organ development, indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.     

Effect of parthenocarpy on ovule development in cucumber (Cucumis sativus L.) after pollination with normal and irradiated pollen

In nonparthenocarpic cucumber, analysis of the interaction between season and female receptivity demonstrated two different modes of fruit development and seed set. Spring climatic conditions induced significantly longer ovaries at anthesis with an autonomous fruit set as in natural parthenocarpy. Autumn climatic conditions induced smaller ovaries with fruit set influenced by the pollination stage of the female flower. These differences involve, during fruit development, a regulation of the empty seed number especially at both ends of female receptivity period. Indeed, seasons have no effect on ovule receptivity and fertility measured by full seed number. It is supposed that priority is given to seeds with an embryo in the competition for limited maternal resources in spring as well as in autumn. In spring, the use of irradiated pollen during the whole female receptivity period did not modify the seed set profile. Moreover, a cytological study of 6-day-old ovules pollinated by irradiated pollen has revealed a similar precocious embryogenesis compared to the control. In cucumber, pollination with irradiated pollen involves stenospermocarpy rather than induced parthenocarpy. However, we have not been able to determine whether the early abortive embryogenesis induced by irradiated pollen is gynogenetic or abnormal zygotic.     

Sex‐specific floral morphology,biomass, and phytohormones associated with altitude in dioecious Populus cathayana populations

Relationships between sex‐specific floral traits and endogenous phytohormones associated with altitude are unknown particularly in dioecious trees. We thus examined the relationships between floral morphology or biomass and phytohormones in male and female flowers of dioecious Populus cathayana populations along an altitudinal gradient (1,500, 1,600, and 1,700 m above sea level) in the Xiaowutai Nature Reserve in northern China. The female and male flowers had the most stigma and pollen at 1,700 m, the largest ovaries and least pollen at 1,500 m, and the smallest ovaries and greater numbers of anthers at 1,600 m altitude. The single‐flower biomass was significantly greater in males than in females at 1,600 or 1,700 m, but the opposite was true at 1,500 m altitude. The biomass percentages were significantly higher in anthers than in stigmas at each altitude, while significantly greater gibberellin A3 (GA₃), zeatin riboside (ZR), indoleacetic acid (IAA), and abscisic acid (ABA) concentrations were found in female than in male flowers. Moreover, most flower morphological traits positively correlated with IAA in females but not in males. The biomass of a single flower was significantly positively correlated with ABA or IAA in males but negatively with ZR in females and was not correlated with GA₃ in both females and males. Our results demonstrate a distinct sexual adaptation between male and female flowers and that phytohormones are closely related to the size, shape, and biomass allocation in the pollination or fertilization organs of dioecious plants, although with variations in altitude.     

Compound heterozygosity for TNXB genetic variants in a mixed‐breed dog with Ehlers‐Danlos syndrome

The Ehlers‐Danlos syndromes (EDSs) are a heterogeneous group of inherited connective tissue disorders characterized by skin hyperextensibility, joint hypermobility and tissue fragility. Inherited disorders similar to human EDS have been reported in different mammalian species. In the present study, we investigated a female mixed‐breed dog with clinical signs of EDS. Whole‐genome sequencing of the affected dog revealed two missense variants in the TNXB gene, encoding the extracellular matrix protein tenascin XB. In humans, TNXB genetic variants cause classical‐like EDS or the milder hypermobile EDS. The affected dog was heterozygous at both identified variants. Each variant allele was transmitted from one of the case's parents, consistent with compound heterozygosity. Although one of the variant alleles, XM_003431680.3:c.2012G>A, p.(Ser671Asn), was private to the family of the affected dog and absent from whole‐genome sequencing data of 599 control dogs, the second variant allele, XM_003431680.3:c.2900G>A, p.(Gly967Asp), is present at a low frequency in the Chihuahua and Poodle population. Given that TNXB is a functional candidate gene for EDS, we suggest that compound heterozygosity for the identified TNXB variants may have caused the EDS‐like phenotype in the affected dog. Chihuahuas and Poodles should be monitored for EDS cases, which might confirm the hypothesized pathogenic effect of the segregating TNXB variant.     

Indel‐seq: a fast‐forward genetics approach for identification of trait‐associated putative candidate genomic regions and its application in pigeonpea (Cajanus cajan)

Identification of candidate genomic regions associated with target traits using conventional mapping methods is challenging and time‐consuming. In recent years, a number of single nucleotide polymorphism (SNP)‐based mapping approaches have been developed and used for identification of candidate/putative genomic regions. However, in the majority of these studies, insertion–deletion (Indel) were largely ignored. For efficient use of Indels in mapping target traits, we propose Indel‐seq approach, which is a combination of whole‐genome resequencing (WGRS) and bulked segregant analysis (BSA) and relies on the Indel frequencies in extreme bulks. Deployment of Indel‐seq approach for identification of candidate genomic regions associated with fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea has identified 16 Indels affecting 26 putative candidate genes. Of these 26 affected putative candidate genes, 24 genes showed effect in the upstream/downstream of the genic region and two genes showed effect in the genes. Validation of these 16 candidate Indels in other FW‐ and SMD‐resistant and FW‐ and SMD‐susceptible genotypes revealed a significant association of five Indels (three for FW and two for SMD resistance). Comparative analysis of Indel‐seq with other genetic mapping approaches highlighted the importance of the approach in identification of significant genomic regions associated with target traits. Therefore, the Indel‐seq approach can be used for quick and precise identification of candidate genomic regions for any target traits in any crop species.     

Single nucleotide polymorphisms in the REG‐CTNNA2 region of chromosome 2 and NEIL3 associated with impulsivity in a Native American sample

Impulsivity is a multi‐faceted construct that, while characterized by a set of correlated dimensions, is centered around a core definition that involves acting suddenly in an unplanned manner without consideration for the consequences of such behavior. Several psychiatric disorders include impulsivity as a criterion, and thus it has been suggested that it may link a number of different behavioral disorders, including substance abuse. Native Americans (NA) experience some of the highest rates of substance abuse of all the US ethnic groups. The described analyses used data from a low‐coverage whole genome sequence scan to conduct a genome‐wide association study (GWAS) of an impulsivity phenotype in an American Indian community sample (n = 658). Demographic and clinical information were obtained using a semi‐structured interview. Impulsivity was assessed using a scale derived from the Maudsley personality inventory that combines both novelty seeking and lack of planning items. The impulsivity score was tested for association with each variant adjusted for demographic variables, and corrected for ancestry and kinship, using emmax . Simulations were conducted to calculate empirical P‐values. Genome‐wide significant findings were observed for a variant 50‐kb upstream from catenin cadherin‐associated protein, alpha 2 (CTNNA2), a neuronal‐specific catenin, in the REG gene cluster. A meta‐analysis of GWAS had previously identified common variants in CTNNA2 as being associated with excitement seeking. A second locus upstream of nei endonuclease VIII‐like 3 (NEIL3) on chromosome 4 also achieved genome‐wide significance. The association between sequence variants in these regions suggests their potential roles in the genetic regulation of this phenotype in this population.     

Whole‐exome sequencing identifies a novel mutation of GPD1L (R189X) associated with familial conduction disease and sudden death

Cardiac conduction disease (CCD) is a serious disorder and the leading cause of mortality worldwide. It is characterized by arrhythmia, syncope or even sudden cardiac death caused by the dysfunction of cardiac voltage‐gated channel. Previous study has demonstrated that mutations in genes encoding voltage‐gated channel and related proteins were the crucial genetic lesion of CCD. In this study, we employed whole‐exome sequencing to explore the potential causative genes in a Chinese family with ventricular tachycardia and syncope. A novel nonsense mutation (c.565C>T/p.R189X) of glycerol‐3‐phosphate dehydrogenase‐like (GPD1L) was identified and co‐segregated with the affected family members. GPD1L is a crucial interacting protein of SCN5A, a gene encoded sodium channel α‐subunit Na_v1.5 and mainly associated with Brugada syndrome (BrS). The novel mutation (c.565C>T/p.R189X) may result in a premature stop codon at position 189 in exon 4 of the GPD1L gene and lead to functional haploinsufficiency of GPD1L due to mRNA carrying this mutation will be degraded by nonsense‐mediated mRNA decay, which has been confirmed by Western blot in HEK293 cells transfected HIS‐GPD1L plasmid. The levels of GPD1L decreasing may disturb the function of Na_v1.5 and induce arrhythmia and syncope in the end. In conclusion, our study not only further supported the important role of GPD1L in CCD, but also expanded the spectrum of GPD1L mutations and will contribute to the genetic diagnosis and counselling of families with CCD.