Next-generation sequencing （NGS） technologies provide enormous power and new potential to access the complex polyploid genomes and transcriptomes of major crops， including cotton. This article summarises the applications of NGS in cotton genomic studies， including genome sequencing and resequencing， transcriptomic analysis， small RNA sequencing and miRNA identification， and identification of single nucleotide polymorphisms. NGS has rapidly accelerated genomic studies in cotton and will further expand our understanding of the evolution and polyploidisation of cotton at the species level as well as our understanding of the biology of the development of the unique seed trichomes that characterise the commercial textile fibres produced by some members of the Gossypium genus. Realisation of substantial impacts on applied cotton genetics and breeding will largely depend on the formulation of novel applications of NGS and the development of handy bioinformatic tools for dealing with and interpretation of the vast amounts of data generated by this technology．

There are many non-coding small RNAs in plants and animals, which regulate gene expression via direct cleavage of target mRNAs or via inhibition of translation at the posttranscriptional level. In this paper, recent studies on microRNA (miRNA)mediated phased siRNAs (phasiRNA) and endogenous target mimics (eTMs) were reviewed: 1) phasiRNAs can be generated both in coding and noncoding loci, and some require miRNA-mediated cleavage for their biogenesis. Pattern and evolutionary mechanisms of miRNA-mediated phasiRNAs were discussed. 2) Genomewide identification and application of eTMs as miRNA decoy targets were recommended, including the use of artificial target mimics to validate the functions of miRNAs and employing bioinformatics methods to identify eTMs in the wholegenome level.

One of the key objectives in genomics studies is to understand genetic architecture of complex traits and diseases. Quantitative trait locus (QTL) mapping and genome-wide association study (GWAS) mapping have been using to dissect genetic architecture of complex traits and diseases that assists in genetic breeding and drug discovery. In this paper, we reviewed QTL mapping and GWAS mapping methodologies and softwares for complex traits or diseases analysis. PLINK, TASSEL, SNPassoc, GenABEL and ProbABEL are most popular softwares providing many useful functions for GWAS mapping. PLINK is the highest popular opensource whole genome association analysis toolset, which implements a number of functions for SNP data analysis. TASSEL is another popular software, implements Q+K composite approach for association mapping. SNPassoc, GenABEL and ProbABEL are popular open source R packages using for association mapping. We have briefly described above popular softwares for GWAS mapping and have described implemented functions in QTXNetwork software for GWAS mapping of quantitative trait with markers (QTLs), SNPs (QTSs), transcripts (QTTs), proteins (QTPs), and metabolites (QTMs). We also describe some other popular softwares such as Windows QTL Cartographer, QTL Express, Map Manager QTX, R/qtl and QTLNetwork for QTL mapping.

Currently， developing genetic linkage map mostly use the derivedpopulations from crossing of two homogenous parents， which only covers limited genetic diversity and is inappropriate for some species， such as tobacco with lower diversity in genome. It is very general that there are no sufficient polymorphic markers to construct linkage map and ineffective to conduct marker-assisted selection （MAS） and quantitative trait locus (QTL) mapping based on lower density linkage map. This study proposed a method for developing genetic linkage map based on a four-way cross population. Computer simulation was conducted to investigate the feasibility and effectiveness of the method and a supporting program was designed. The main procedures and features of the proposed method were summarized as follows: 1） estimating genetic distance of any paired markers based on maximum likelihood method; 2） splitting all markers into different groups （linkage group） by cluster analysis based on genetic distance of markers; 3） for each linkage group， two end markers were first determined， then the marker order could be determined by inserting other markers in appropriate position by distance analysis of any three neighboring markers. Monte Carlo simulation showed that the proposed method is feasible， effective， and applicable in other derived populations from crossing of two homogenous parents.

Complete chloroplast genome sequence is very useful for studying the evolution of species. To get chloroplast genome sequences， purification of the chloroplast or PCR amplification prior to sequencing is commonly involved in conventional approaches. Advances in DNA sequencing technology provide new opportunities to obtain chloroplast genome sequence from the wholegenome highthroughput sequencing data without purification of the chloroplast. In this study， we finished the complete chloroplast genome sequence of Dongxiang wild rice based on highthroughput sequencing data from their fresh green leaves. The chloroplast genome was 134 537 bp in size， and had a typical quadripartite structure with the large single copy （LSC， 80 585 bp） and small single copy （SSC， 12 346 bp） regions separated by two copies of an inverted repeat （IR， 20 803 bp each） region. One hundred and fiftytwo chloroplast genes were successfully annotated. A phylogenetic tree was constructed based on the chloroplast genomes of Dongxiang wild rice， Indica， Japonica and 10 other genera of grasses using the neighborjoining method. The result showed that Dongxiang wild rice had a closer relationship with Bambusa oldhamii and Panicoideae. Furthermore， the SNPs of 22 rice accessions were identified using the chloroplast genome of Dongxiang wild rice as a reference sequence and a phylogenetic tree was constructed based on these SNPs. The result illustrated that Indica had a closer relationship with wild rice-I， while Japonica was closer to wild rice-III， suggesting that Indica and Japonica were domesticated independently from different wild rice populations．

Camellia sinensis cv. Longjing 43 is a domestic variety of tea species and an important economic crop in China. In this study, we developed a rapid method to get the chloroplast (cp) genome and sequenced the entire cp genome sequence of C. sinensis cv. Longjing 43. The C. sinensis cv. Longjing 43 cp genome was 157 085 bp in length, which contained a large singlecopy (LSC, 86 642 bp) region, a small single-copy (SSC, 18 283 bp) region, and two inverted repeat (IR, each with a size of 26 080 bp) regions. With the cp genome of Korean C. sinensis cultivar as a reference, 134 chloroplast genes were successfully annotated. There were 15 genes with non-synonymous mutations in the coding region and more than 100 polymorphic sites in the non-coding region, which could be the DNA markers for the determination of different C. sinensis varieties. We also investigated the relationship of 12 C. sinensis varieties in China based on several cp genomic regions, which contain many variant sites. The result showed that these varieties were divided into two groups with Lingyunbaimaocha in one group and the other 11 in another group. Among the other 11 varieties, the Longjingchangye, Longjingyuanye, Longjingguazi, and Zhongcha 102 had a closer relationship and were formed into one cluster with 100% support rate, demonstrating the reliability of the method that used the cp genome sequences to investigate the genetic relationships.

The ratio of FEV1 (forced expiratory volume in one second) to FVC (forced vital capacity) is an index for pulmonary obstruction measurement and one of the most significant predictors for chronic obstructive pulmonary disease (COPD), which is a heritable multi-factorial disease. We present genome-wide association study (GWAS) to map the genetic architecture of this trait and investigate the networks between the external factors (smoking and gender) and genetic factors. By using a mixed linear model and a conditional model, we conducted GWAS in a cohort suffered COPD from the U.S. National Heart, Lung and Blood Institute. Among 561 467 single nucleotide polymorphisms, we found 12 significant quantitative trait SNPs (QTSs) fitted the full model. And for each of them, we demonstrated the mechanisms and relationship between pulmonary function and genes detected. STIM2 and MRE11A (PEW-value<1×10-5) showed unambiguous evidence of association with COPD. APOL3 (PEW-value<1×10-5) was influenced by different genders in different ways and previous studies also implicated its associations with smoking behavior. The variation of genes MRE11A and DNAJC15 was related to lung adenocarcinoma, which is a serious complication of COPD. The significant epistasis effects of these genes suggested the possibility of multiple functional polymorphisms. These associations offer mechanistic insight into pulmonary function regulation and networks between genetics factors and environmental factors, which indicate potential ways for interventions to COPD and many other respiratory diseases.

Obesity has been reported as an increasingly prevalent and highly heritable health problem， resulting in increased risk for several common diseases. Despite the consensus view， that epistasis and gene-environment interactions have a prominent role in its pathogenesis， they are largely ignored in the current genome-wide association study （GWAS）. A new approach based on a linear mixed model was conducted for GWAS to detect plausible genes and potential interactions among genes， impacts of smoking and gender on body mass index （BMI）. We conducted analysis based on database of genotype and phenotype （dbGaP） from chronic obstructive pulmonary disease （COPD） study， and identified 20 genes and two pairs of epistasis associated with BMI， some of which were smoking-influencing and gender-specific. Bioinformatics analysis revealed a complex gene network among the identified genes connected with BMI and other related diseases. These findings highlight that personalized measures including lifestyle modifications such as smoking is essential for prevention and treatment of obesity.

The dualism of genetic predisposition and gender influences has long been a hot topic in the development of chronic obstructive pulmonary disease (COPD), and smoking is considered a primary risk factor for this lung disease. This paper aimed to detect susceptibility genes for COPD with the data downloaded from dbGaP. A linear mixed model was employed to conduct association-mapping QTSs (quantitative trait single-nucleotide polymorphisms) because of its effectiveness in unbiased estimation of random effects with unbalanced data and in controlling population stratification. The primary focus of the study is to identify genetic risk factors that determine susceptibility for COPD and COPD-related phenotypes with the goal of providing insight into clinically relevant COPD subtypes. By comparing the conditional model excluding the cofactor smoking with the full model, we can detect related QTSs, which will reveal the gene expression on COPD caused or suppressed by smoking. As a result, there are significant genes with high heritability: TNS1 and DGKH were not caused by smoking, MACROD2 and CNIH3 were due to smoking, and LINC00426, METTL4 and GSDMC were suppressed by smoking.

Plant germplasm is an important gene resource for plant breeding and genetic improvement of traits. Core collection， as a representative subset of germplasm， provides an effective entry to investigate genetic diversity and screen potential breeding material. Most agronomic traits of tobacco often exhibit strong genetic variation which can be decomposed into genotypic and genotype × environment interaction variations. A tobacco collection of 805 accessions were evaluated in two locations， Fuquan and Jinsha City of Guizhou Province， China. The genetic variations of 12 agronomic traits were analyzed and the genotypic values of each accession were predicted by a mixed linear model approach. Significant genotypic variations were detected for 11 traits， as well as genotype × environment interaction for 10 traits. Based on the predicted genotypic values， the combination strategies of sampling and clustering methods in the procedure of stepwise clustering for constructing core collection were screened in terms of magnitude of genetic variation captured by sampled subsets. Finally， the subset （S1C3_10）， sampled by a centroid method combined with preferred sampling at 10% proportion， was determined to be a core collection of the tobacco germplasm. The representative and validation of the core collection was examined visually by the accession distribution pattern in the plot established by first two principal components， as well as by the correlation coefficients in terms of magnitude and significance between the core and the initial collections. The results showed that the initial collection was well represented by the core collection， and some potential breeding materials for high yield breeding program of tobacco， such as Y177 and Y178， were included in the core collection. In sum， the constructed core collection will facilitate tobacco breeder to access to whole resources and promote the utilization of genetic diversity in future breeding program of tobacco in China．

Programmed cell death （PCD） is crucial for plant growth and development. It maintains plant normal physiologic metabolism by eliminating damaged， diseased and old cells. Numerous studies have demonstrated that cytoplasmic male sterility （CMS） in plant was associated with programmed cell death （PCD）. The proper timing of PCD is very important for floral development. Based on RNA-Seq （RNA sequencing） result， we found that programmed cell death 5 gene pcd5， cytochrome oxidase gene cox1 and malate dehydrogenase gene mdh were all down-regulated in CMS-C line of maize. In this study， real-time fluorescence quantitative polymerase chain reaction （RT-qPCR） was done to further validate the expression of pcd5，cox1 and mdh genes. These analyses will contribute to better understand CMS-C mechanisms. The CMS-C line （C48-2） and its maintainer line （N48-2） were planted in the same research field of Sichuan Agricultural University. Total RNA of anther was extracted from N48-2 and C48-2 at uninucleate stages using Trizol kit. The RNA was reversely transcribed into a cDNA rst strand using PrimeScript RT reagent kit （TaKaRa）， and the genomic DNA was completely removed from total RNA before reverse transcription. The genomic DNA was extracted from leaf using cetyltrimethyl ammonium bromide （CTAB） method. To verify the expression levels of pcd5， cox1 and mdh genes， the RT-qPCR was performed using a commercial kit （AccuPower 2X Greenstar qPCR Master Mix， Bioneer）. Specific primers were designed in conserved region of coding sequence （CDS）， and the 18S and β-actin were assigned as internal control genes for RT-qPCR. PCR and RT-PCR amplification of pcd5 were performed with TAR HS DNA polymerase （TaKaRa） using specific primer. To verify the results of pyrosequencing， high resolution melting curve （HRM） was performed using a commercial kit （AccuPower 2X Greenstar qPCR Master Mix， Bioneer）. HRM primers were designed in the mutant region based on RT-qPCR. All reactions were performed in triplicate． Gene expression analysis showed that the pcd5， cox1 and mdh genes were down-regulated in C48-2 in comparison to N48-2， and the result was consistent with RNA-Seq result. The results of PCR revealed that the coding region of genomic DNA had no difference between C48-2 and N48-2. An insertion mutation was detected in C48-2 by RT-qPCR and pyrosequencing， which was also proved by HRM. As a consequence， the mutant pcd5 transcript may be associated with the down-regulated in C48-2. In conclusion， the differential expression of pcd5， cox1 and mdh genes may interfere with the programmed cell death， and the abortion of CMS-C microspores might be closely related to the cell death. The findings provide an improved sight for investigating the relationship of PCD and pollen abortion in maize

The mismatch repair (MMR) is a major pathway in DNA repair system，which is critical for maintaining genome stability and DNA replication fidelity，as it is responsible for the recognizing and repairing erroneous insertions, deletions and mismatch of bases newly arising during DNA replication and genetic recombination, as well as during the repair of some forms of DNA damage. The major components in the MMR system include MutS, MutH, and MutL in Escherichia coli. In eukaryotes, homologues of MutS and MutL have been found，but not of MutH. Based on homology， several MutS homologues have also been identified and cloned from Arabidopsis thaliana. Amongst these homlogues MSH2 complexes with MSH6, MSH3 or MSH7 form MutSα, MutSβ and MutSγ heterodimers to recognize different types of mutations, respectively. Mutation or disruption of MMR gene causes significantly increased frequencies of point mutations and microsatellite instability, thus to enhance genetic diversity for plant breeding. Rice is an important food crop and a prominent molecular model species for monocotyledonous plants. Some MMR genes have been annotated in Rice Annotation Project Database, one of which is Os09g24220, a homologue to Msh6 (At4g02070) in the MMR system of A. thaliana. However, no information is available for the Os09g24220 gene function and its mutator phenotype in rice. We reasoned that, if the disrupted Os09g24220 gene can enhance genetic diversity, offspring exhibiting mutations in agronomic traits could be generated using this approach. Herein, we mainly conducted molecular characterization of Tos17 insertion mutants of the Os09g24220 gene and analyzed their agronomic traits, to provide a foundation for the function studies of the Os09g24220 gene and exploitation of the mutant in rice mutation breeding. Three insertion mutants, NF9010, NF7784 and ND6011 with the Tos17 insertion at the 1st, 8th exons and 3′-UTR (untranslated region), respectively, were characterized by triple-primer PCR and RT-PCR. The Tos17 insertion in Os09g24220 gene was confirmed and all three insertion mutants were homozygous. In both NF9010 and NF7784 mutant seedlings, partial Os09g24220 mRNA transcripts were detected with the primer sets that amplified upstream region of the insertion site, but it was not detectable with the primer sets at the downstream region or across the insertion site, indicating that NF9010 and NF7784 lack full-length, functional Os09g24220 mRNA despite the existence of truncated transcripts. However, all Os09g24220 transcripts were detected with primer sets that amplified the upstream, internal and downstream fragments of whole functional region in ND6011 mutant, but no transcript was detected with the primer set across the insertion site, suggesting that ND6011 has the functional mRNA, but not full-length mRNA. Agronomic traits, such as plant height, number of productive panicles, panicle length, seed-setting rate and mass of 1 000 grains, of the insertion mutants were checked and compared with those of their wild type, Nipponbare. Results indicated that at least one trait for each mutant changed significantly. The mutant ND6011 just showed significantly (P<0.05) decreased seed-setting rate, but both NF7784 and NF9010 displayed significant (P<0.01) reduction in ear length and seed-setting rate, moreover plant height of NF9010 was also significantly lowered (P<0.05). In conclusion, some agronomic traits of Tos17 insertion mutants of the Os09g24220 gene changed significantly in rice plants, showing the mutator phenotype. Furthermore, different insertion mutants displayed different mutator phenotypes, which are related to effects on Os09g24220 gene function of different Tos17 insertion sites. These findings provide foundations for investigating the function of the Os09g24220 gene in DNA repair and exploiting such mutants in induced mutation breeding in rice.

Cymbidium ensifolium is one of Cymbidium genus, having elegant shape, beautiful appearance and fragrant aroma. Because of these features, this species gets with extremely high ornamental value. Owing to the lack of its genomic resource, the development and application of molecular marker is still limited. With the development of RNA-Seq technology, the transcriptomic data gradually accumulate and become a useful resource to explore marker with low cost and high efficiency.
Here, the transcriptome in C. ensifoliumwas subjected to RNA-seq. Illumina sequencing was performed at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer‘s instructions (Illumina, San Diego, CA). Highquality reads were assembled de novo using Trinity with optimized Kmer length of 25. The program Msatcommander was used to analyze microsatellite (as called simple sequence repeat, SSR) frequencies. The minimum numbers of repeats for SSR detection were as follows: six repeats for di-SSRs; and four for tri-, tetra-, penta- and hexa-SSRs. Single nucleotide polymorphisms (SNPs) were detected and filtered using SAMtools and VarScan. The open reading frame (ORF) and untranslated region (UTR) within the isogene were identified using Trinity software. Isogenes containing SSR and SNP were annotated on the basis of BLAST similarity searches.
All high-quality reads were assembled into 101 423isogenes, with total residues of 139 385 689.The isogenes averaged 1 374bp, ranging from 351 bp to 17 260 bp, and 70 583 isogenes, accounted for 69.60%, were about 600 bp. In total, 17 793SSRs and 16 676 SNPs were identified within transcriptomic database. The density of SSR and SNP was 1.28 SSRs/10 kb and 1.20 SSRs/10 kb, respectively. Among these SSRs, tri-SSR was the most types, followed by diSSR, except mono-SSR. Di-SSR and tri-SSR accounted for 20.46% and 21.98% in all SSRs, respectively. The location of SSR was also estimated. The estimated locations were obtained for 7 936 SSRs, but sequence information could not be determined for the remaining 6 586SSR regions as it extended over both estimated coding and non-coding regions. We found that most tri-SSRs and hexa-SSRs occurred more frequently in coding regions. In contrast, di-SSR, tetra-SSR, and penta-SSR, were more likely to appear in UTR rather than coding regions. Among these SNPs, C/T was the most common base substitution, followed by A/G. The two kinds of substitutions, C/T and A/G, accounted for 30.80% and 28.81% in all SNPs, respectively.
The number of isogenes containing SSR and SNP was 13 768 and 7 519, respectively. These isogenes were annotated by Clusters of Orthologous Groups (COG), Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. A large number of them were annotated with crucial genes that were associated with important biological functions. There were 1 748SSR and 1 932 SNP isogenes assigned into 23 COG classifications, respectively. There were 4 994 SSR and 4 819SNP isogenes classified into 80 and 78 GO terms, respectively. There were 2 107SSR and 2 188 SNP isogenes involved in 300 and 308 KEGG pathways, respectively.
The numerous SSRs and SNPs identified in this study will contribute to marker development. The annotation of isogenes containing SSR and SNP will help in constructing genetic maps and exploring the associations between these markers and the interesting traits. The map will in turn accelerate research on genomics and functional genomics of C. ensifolium.