Structural Variation Analysis of Mutated Nannochloropsis oceanica Caused by Zeocin Through Genome Re-Sequencing

Zeocin can cause double strand breaks of DNA and thus may be employed as a mutagen. In this study, two strains of Nannochloropsis oceanica, the wild and the Zeocin-tolerant strains, were re-sequenced to verify such function of Zeocin, The results showed that Zeocin can mutate the N. oceanica genome and cause the structural variation. Zeocin either swept away or selected the alleles of genes functioning in ubiquitin-mediated proteolysis, alpha-linolenic acid metabolism, ascorbate and aldarate metabolism, ribosome biogenesis, and circadian rhythm, indicating that N. oceanica may have adjusted its metabolic performances for protein, carbohydrate, and lipid, and changed its ribosome biosynthesis and living rhythm to survive in Zeocin containing medium. In addition, Zeocin caused mutation may have influenced the expression of a set of tanscription factors. It was concluded that Zeocin effectively caused the structural variation of the genome of N. oceanica, and forced the microalgae to select out the alleles of a set of genes around these variations in order to adapt to Zeocin containing medium. Further studies on the genetic basis of the phenotypic adaptation of this haploid and asexual microalga and the application of Zeocin to its genetic improvement are very important.

QTL mapping by whole genome re-sequencing and analysis of candidate genes for salt tolerance in linseed(Linum usitatissmum L.)

Soil salinization is detrimental to the growth and development of flax and ultimately leads to a decrease in yield.However,the molecular mechanism of linseed response to salt stress is still unclear.In this study,a salt-tolerant(ST)linseed variety STS and a salt-sensitive(SS)variety DYM were selected as experiment materials.Bulk segregation analysis and whole-genome resequencing technologies were performed to map salt tolerance quantitative trait loci(QTL).A total of 38,625 QTL loci were identified.Fifteen genes(which were not annotated in the reference genome)were identified within a 2.597 Mb region in chromosome 1.Two salt tolerance candidate genes Lus.o.m.scaffold91.141 and Lus.o.m.Scaffold1.14 encoding WD40 and cytochrome P450 were identified by predicting protein functions.Previous studies showed that WD40 and cytochrome P450 could significantly improve plant salt stress tolerance.In this paper,results showed that Lus.o.m.scaffold91.141 and Lus.o.m.Scaffold1.14 might be involved in response to salt stress in lineseed.The fine mapping and functional analysis of these genes provide a molecular breeding basis for the genetic improvement of high salt-tolerant linseed varieties.

Comparative genomic study of a wild soybean by whole genome re-sequencing

Wild soybean resources, which are progenitor of cultivated soybean with selected agronomic characters, have rich genetic diversity. Here we used genome re-sequencing technology to analyze genetic variations between the wild soybean ＇ED059’ and cultivar ＇Tianlong 2＇. In genome level, 3,214,319 and 1,519,765 single nucleotide polymorphisms （SNPs）, 553,141 and 314,430 insertion/deletion polymorphisms （InDels）, and 471,063 and 334,412 structural variations （SVs） were identified between ＇ED0595＇ and ＇Tianlong 2＇ respec-tively based on soybean （Glycine max L. Merr） reference genome. Base on gene annotation of reference genome, 68,830 （2.14%） and 34,570 （2.27%） non-synonymous SNPs, 8,478 and 4,826 frameshift substitution were detected in CDS regions of ＇ED0595＇ and ＇Tianlong 2＇. ＇ED059’ harbored much more specific genetic variations of jasmonic acid （JA）, salicylic acid （SA） and ethylene （ET） biosynthesis and signal pathway genes than those in ＇Tianlong 2＇ indicating its unique strong insect defense activity. This work provides important information allowing better understanding of the soybean genome and being helpful for dissecting the genetic basis of important traits such as insect defense in soybean.

Whole genome re-sequencing reveals evolutionary patterns of sacred lotus (Nelumbo nucifera)

Sacred lotus(Nelumbo nucifera or lotus) is an important aquatic plant in horticulture and ecosystems. As a foundation for exploring genomic variation and evolution among different germplasms, we re-sequenced 19 individuals from three cultivated temperate lotus subgroups(rhizome,seed and flower lotus), one wild temperate lotus subgroup(wild lotus), one tropical lotus group(Thai lotus) and an outgroup(Nelumbo lutea). Through genetic diversity and polymorphism analysis by non-missing SNP sites widely distributed in the whole genome, we confirmed that wild and Thai lotus exhibited greater differentiation with a higher genomic diversity compared to cultivated lotus. Rhizome lotus had the lowest genomic diversity and a closer relationship to wild lotus, whereas the genomes of seed and flower lotus were admixed. Genes in energy metabolism process and plant immunity evolved rapidly in lotus, reflecting local adaptation.We established that candidate genes in genomic regions with significant differentiation associated with temperate and tropical lotus divergence always exhibited highly divergent expression pattern. Together, this study comprehensive and credible interpretates important patterns of genetic diversity and relationships, gene evolution, and genomic signature from ecotypic differentiation of sacred lotus.

Development of SNP parentage assignment techniques in the yellowfin seabream Acanthopagrus latus

Acanthopagrus latus is an essential aquaculture species on the south coast of China.However,there is a lack of systematic breeding of A.latus,which considerably limits the sustainable development of A.latus.As a result,genetic improvements are urgently needed to breed new strains of A.latus with rapid growth and strong resistance to disease.During selective breeding,it is necessary to estimate the genetic parameters of the target trait,which in turn depends on an accurate disentangled pedigree for the selective population.Therefore,it is necessary to establish the parentage assignment technique for A.latus.In this study,95 individuals selected from their parents and their 14 families were used as experimental material.SNPs were developed by genome resequencing,and highly polymorphic SNPs were screened on the basis of optimized filtering parameters.A total of 14392738 SNPs were discovered and 205 SNPs were selected for parentage assignment using the CERVUS software.In the model where the gender of the parents is known,the assignment success rate is 98.61%for the male parent,97.22%for the female parent,and 95.83%for the parent pair.In the model where the gender of the parents is unknown,the assignment success rate is 100%for a single parent and 90.28%for the parent pair.The results of this study were expected to serve as a reference for the breeding of new varieties of A.latus.

Construction of EMS-Induced Peanut Mutant Libraries and Identification of Pod-Related Traits Mutant Lines

Peanut(Arachis hypogaea L.)is an oil and economic crop of vital importance,and peanut pod is the key organ influencing the yield and processing quality.Hence,the Pod-related traits(PRTs)are considered as important agronomic traits in peanut breeding.To broaden the variability of PRTs in current peanut germplasms,three elite peanut cultivars were used to construct Ethyl methane sulfonate(EMS)-induced mutant libraries in this study.The optimal EMS treatment conditions for the three peanut varieties were determined.It was found that the median lethal dose(LD50)of EMS treatment varied greatly among different genotypes.Finally,the EMS-induced peanut mutant libraries were constructed and a total of 124 mutant lines for PRTs were identified and evaluated.Furthermore,“M-8070”,one of the mutant lines for pod constriction,was re-sequenced via high-throughput sequencing technology.The genome-wide variations between“M-8070”and its wild parent“Fuhua 8”(FH 8)were detected.2994 EMS-induced single nucleotide polymorphisms(SNPs)and 1188 insertion-deletions(InDels)between“M-8070”and its wild parent were identified.The predominant SNP mutation type was C/G to T/A transitions,while the predominant InDel mutation type was“1-bp”.We analyzed the distribution of identified mutations and annotated their functions.Most of the mutations(91.68%of the SNPs and 77.69%of the InDels)were located in the intergenic region.72 SNPs were identified in the exonic region,leading to 27 synonymous,43 nonsynonymous and 2 stop-gain variation for gene structure.13 Indels were identified in the exonic region,leading to 4 frame-shift,8 non-frame-shift and 1 stop-gain variations of genes.These mutations may lead to the phenotypic variation of“M-8070”.Our study provided valuable resources for peanut improvement and functional genomic research.

Imputation from SNP chip to sequence: a case study in a Chinese indigenous chicken population

Background: Genome-wide association studies and genomic predictions are thought to be optimized by using whole-genome sequence(WGS) data. However, sequencing thousands of individuals of interest is expensive.Imputation from SNP panels to WGS data is an attractive and less expensive approach to obtain WGS data. The aims of this study were to investigate the accuracy of imputation and to provide insight into the design and execution of genotype imputation.Results: We genotyped 450 chickens with a 600 K SNP array, and sequenced 24 key individuals by whole genome re-sequencing. Accuracy of imputation from putative 60 K and 600 K array data to WGS data was 0.620 and 0.812 for Beagle, and 0.810 and 0.914 for FImpute, respectively. By increasing the sequencing cost from 24 X to 144 X, the imputation accuracy increased from 0.525 to 0.698 for Beagle and from 0.654 to 0.823 for FImpute. With fixed sequence depth(12 X), increasing the number of sequenced animals from 1 to 24, improved accuracy from 0.421 to0.897 for FImpute and from 0.396 to 0.777 for Beagle. Using optimally selected key individuals resulted in a higher imputation accuracy compared with using randomly selected individuals as a reference population for resequencing. With fixed reference population size(24), imputation accuracy increased from 0.654 to 0.875 for FImpute and from 0.512 to 0.762 for Beagle as the sequencing depth increased from 1 X to 12 X. With a given total cost of genotyping, accuracy increased with the size of the reference population for FImpute, but the pattern was not valid for Beagle, which showed the highest accuracy at six fold coverage for the scenarios used in this study.Conclusions: In conclusion, we comprehensively investigated the impacts of several key factors on genotype imputation. Generally, increasing sequencing cost gave a higher imputation accuracy. But with a fixed sequencing cost, the optimal imputation enhance the performance of WGP and GWAS. An optimal imputation strategy should take size of reference population, imputation algorithms, marker density, and population structure of the target population and methods to select key individuals into consideration comprehensively. This work sheds additional light on how to design and execute genotype imputation for livestock populations.

Genomic dissection of widely planted soybean cultivars leads to a new breeding strategy of crops in the post-genomic era

Soybeans specially the widely planted cultivars have been dramatically improved in agronomic performance and is well adapted to local planting environments after long-time domestication and breeding.Uncovering the unique genomic features of popular cultivars will help to understand how soybean genomes have been modified through breeding.We re-sequenced 134 soybean cultivars that were released and most widely planted over the last century in China.Phylogenetic analyses established that these cultivars comprise two geographically distinct sub-populations:Northeast China (NE) versus the HuangHuai-Hai River Valley and South China (HS).A total of 309 selective regions were identified as being impacted by geographical origins.The HS sub-population exhibited higher genetic diversity and linkage disequilibrium decayed more rapidly compared to the NE sub-population.To study the association between phenotypic differences and geographical origins,we recorded the vegetative period under different growing conditions for two years,and found that clustering based on the phenotypic data was closely correlated with cultivar geographical origin.By iteratively calculating accumulated genetic diversity,we established a platform panel of cultivars and have proposed a novel breeding strategy named "Potalaization"for selecting and utilizing the platform cultivars that represent the most genetically diversity and the highest available agronomic performance as the "plateau"for accumulating elite loci and traits,breeding novel widely adapted cultivars,and upgrading breeding technology.In addition to providing new genomic information for the soybean research community,the "Potalaization"strategy that we devised will also be practical for integrating the conventional and molecular breeding programs of crops in the post-genomic era.

bHLH genes polymorphisms and their association with growth traits in the Pacifi c oyster Crassostrea gigas

The basic helix-loop-helix(bHLH)genes encode a large superfamily of transcription factors in the Pacific oyster(Crassostrea gigas),and play a very important role in regulation of growth and development.To investigate the oyster growth traits and the associations with bHLH genes variations,we analyzed the gene polymorphisms-traits association in a wild population,and confirmed the results in another independent wild population by targeted gene re-sequencing and SNPshot analysis.After screening the single nucleotide polymorphisms(SNPs)in three candidate genes of the bHLH family(88 bHLH genes in two wild oyster populations in total),we identified the allele CgLoblHLH4-T/G located in the exon of the CgLoblHLH4 gene.This allele is a non-synonymous mutation(Phe/Leu)with an extremely significant association with shell width(P<0.01)and allele G is beneficial to shell width.This SNP on the CgLoblHLH4 gene might have a potential value as a genetic marker of growth traits that could be used in breeding in C.gigas in the future.

Identification and characterization of long-InDels through whole genome resequencing to facilitate fine-mapping of a QTL for plant height in soybean(Glycine max L.Merr.)

With the development of sequencing technology, insertions-deletions(InDels) have been increasingly reported to be involved in the genetic determination of agronomical traits. However, most studies have focused on the identification and application of short-InDels(1–15 bp) for genetic analysis. The objective of this study was to deeply deploy long-InDels(>15 bp) for the genetic analysis of important agronomic traits in soybean. A total of 13 573 polymorphic long-InDels were identified between parents Zhongpin 03-5373(ZP) and Zhonghuang 13(ZH), which were unevenly distributed on 20 chromosomes of soybean, varying from 321 in Chr11 to 1 246 in Chr18. Consistent with the distribution pattern of annotated genes, the average density of long-InDels in arm regions was significantly higher than that in pericentromeric regions at the P=0.01 level. A total of 2 704(19.9% of total) long-InDels were located in genic regions, including 319 large-effect long-InDels, which resulted in truncated or elongated protein sequences. A previously identified QTL(qP H16) underlying plant height was further analyzed, and it was found that 26 out of 35(74.3%) long-InDel markers located in the qPH16 region showed clear polymorphisms between parents ZP and ZH. Seven markers, including three long-InDels and four previously reported SNP markers, were used to genotype 242 recombinant inbred lines derived from ZP×ZH. As a result, the qPH16 locus was narrowed from a 960-kb region to a 477.55-kb region, containing 65 annotated genes. Therefore, these long-InDels are a complementary genetic resource of SNPs and short-InDels for plant height and can facilitate genetic studies and molecular assisted selection breeding in soybean.

QTL analysis for plant height and fine mapping of two environmentally stable QTLs with major effects in soybean

Plant height is an important agronomic trait, which is governed by multiple genes with major or minor effects. Of numerous QTLs for plant height reported in soybean, most are in large genomic regions, which results in a still unknown molecular mechanism for plant height. Increasing the density of molecular markers in genetic maps will significantly improve the efficiency and accuracy of QTL mapping. This study constructed a high-density genetic map using 4 011 recombination bin markers developed from whole genome re-sequencing of 241 recombinant inbred lines(RILs) and their bi-parents, Zhonghuang 13(ZH) and Zhongpin 03-5373(ZP). The total genetic distance of this bin map was 3 139.15 cM,with an average interval of 0.78 cM between adjacent bin markers. Comparative genomic analysis indicated that this genetic map showed a high collinearity with the soybean reference genome. Based on this bin map, nine QTLs for plant height were detected across six environments, including three novel loci(qPH-b_11, qPH-b_17 and qPH-b_18). Of them, two environmentally stable QTLs qPH-b_13 and qPH-b_19-1 played a major role in plant height, which explained 10.56-32.7% of the phenotypic variance. They were fine-mapped to 440.12 and 237.06 kb region, covering 54 and 28 annotated genes, respectively. Via the function of homologous genes in Arabidopsis and expression analysis, two genes of them were preferentially predicted as candidate genes for further study.

Unveiling potential sex-determining genes and sex-specific markers in autotetraploid Carassius auratus

Autotetraploid Carassius auratus is a stable hereditary autotetraploid fish resulting from the hybridization of Carassius auratus red var.(RCC,♀)×Megalobrama amblycephala(BSB,♂),containing four sets of RCC chromosomes.However,the molecular mechanism underlying the determination of sex in this species remains largely unknown.Currently,there lacks a full understanding of the molecular mechanisms governing sex determination and specific molecular markers to differentiate sex in this species.In this study,25,801,677 SNPs(Singlenucleotide polymorphism)and 6,210,306 Indels(insertion-deletion)were obtained from whole-genome resequencing of 100 individuals(including 50 female and 50 male).Further identification confirmed the candidate chromosomes as Chr46B,with the sex-determining region located at Chr46B:22,500,000‒22,800,000 bp.Based on the male-specific insertion(26 bp)within the candidate sex-determining region,a pair of sex-specific molecular markers has been identified.In addition,based on the screening of candidate sex-determining region genes and RT-qPCR validation analysis,ADAM10,AQP9 and tc1a were identified as candidate sex-determining genes.These findings provide a robust foundation for investigating sex determination mechanisms in fish,the evolution of sex chromosomes,and the development of monosex populations.

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 （ARCS） 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 Csa7Go5143o 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.

An exon skipping in a SEPALLATA-Like gene is associated with perturbed floral and fruits development in cucumber

Summary We isolated a mutant showing perturbations in the development of male and female floral organs and fruits. Analysis of the single nucleotide polyTnorphisms from bulked F2 pools identified the causative variant occurring in Csa4G126690. Csa4G126690 shows high homol- ogy to Arobidopsis 5EPALLATA2 （SEP2） thus being desig- nated CsSEP2. The causative variant was located on the splicing site of CsSEP2, resulting in the skipping of exon 6 and abolishment of the transcriptional activity. Our data suggest that CsSEP2 is involved in the floral organ and fruits development by conferring transcriptional activity.

Genome-wide analysis reveals selection for Chinese Rongchang pigs

Livestock have undergone domestication and consequently strong selective pressure on genes or genomic regions that control desirable traits. To identify selection signatures in the genome of Chinese Rongchang pigs, we generated a total of about 170 Gb of DNA sequence data with about 6.4-fold coverage for each of six female individuals. By combining these data with the publically available genome data of 10 Asian wild boars,we identified 449 protein-coding genes with selection signatures in Rongchang pigs, which are mainly involved in growth and hormone binding, nervous system development, and drug metabolism. The accelerated evolution of these genes may contribute to the dramatic phenotypic differences between Rongchang pigs and Chinese wild boars. This study illustrated how domestication and subsequent artificial selection have shaped patterns of genetic variation in Rongchang pigs and provides valuable genetic resources that can enhance the use of pigs in agricultural production and biomedical studies.

Getting started in mapping-by-sequencing

Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2ooo platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulateand process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed ＂mapping-by-sequencing＇, and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila mela- nogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.