A combination of genome-wide association study and selection signature analysis dissects the genetic architecture underlying bone traits in chickens

The bones of chicken play an important role in supporting and protecting the body. The growth and development of bones have a substantial influence on the health and production performance in chickens. However, genetic architecture underlying chicken bone traits is not well understood. The objectives of this study are to dissect the genetic basis of bone traits in chickens and to identify valuable genes and genetic markers for chicken breeding. We performed a combination of genome-wide association study (GWAS) and selection signature analysis (fixation index values and nucleotide diversity ratios) in an F₂ crossbred experimental population with different genetic backgrounds (broiler × layer) to identify candidate genes and significant variants related to femur, shank, keel length, chest width, metatarsal claw weight, metatarsal length, and metatarsal circumference. A total of 545 individuals were genotyped based on the whole genome re-sequencing method (26 F₀ individuals were re-sequenced at 10 × coverage; 519 F₂ individuals were re-sequenced at 3 × coverage). A total of 2 028 112 single-nucleotide polymorphisms (SNPs) remained to carry out analysis after quality control and imputation. The integration of GWAS and selection signature analysis indicated that all significant SNPs responsible for bone traits were mainly localized on chicken chromosomes 1, 4, and 27. Finally, we identified 21 positional candidate genes that might regulate chicken bone growth and development, including LRCH1, RB1, FNDC3A, MLNR, CAB39L, FOXO1, LHFP, TRPC4, POSTN, SMAD9, RBPJ, PPARGC1A, SLIT2, NCAPG, NKX3-2, CPZ, SPOP, NGFR, SOST, ZNF652, and HOXB3. Additionally, an array of uncharacterized genes was identified. The findings provide an in-depth understanding of the genetic architecture of chicken bone traits and offer a molecular basis for applying genomics in practical chicken breeding.     

Whole‐genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep

The identification of genes influencing fitness is central to our understanding of the genetic basis of adaptation and how it shapes phenotypic variation in wild populations. Here, we used whole‐genome resequencing of wild Rocky Mountain bighorn sheep (Ovis canadensis) to >50‐fold coverage to identify 2.8 million single nucleotide polymorphisms (SNPs) and genomic regions bearing signatures of directional selection (i.e. selective sweeps). A comparison of SNP diversity between the X chromosome and the autosomes indicated that bighorn males had a dramatically reduced long‐term effective population size compared to females. This probably reflects a long history of intense sexual selection mediated by male–male competition for mates. Selective sweep scans based on heterozygosity and nucleotide diversity revealed evidence for a selective sweep shared across multiple populations at RXFP2, a gene that strongly affects horn size in domestic ungulates. The massive horns carried by bighorn rams appear to have evolved in part via strong positive selection at RXFP2. We identified evidence for selection within individual populations at genes affecting early body growth and cellular response to hypoxia; however, these must be interpreted more cautiously as genetic drift is strong within local populations and may have caused false positives. These results represent a rare example of strong genomic signatures of selection identified at genes with known function in wild populations of a nonmodel species. Our results also showcase the value of reference genome assemblies from agricultural or model species for studies of the genomic basis of adaptation in closely related wild taxa.     

Detection of selection signatures in dairy and beef cattle using high-density genomic information

Background

Artificial selection for economically important traits in cattle is expected to have left distinctive selection signatures on the genome. Access to high-density genotypes facilitates the accurate identification of genomic regions that have undergone positive selection. These findings help to better elucidate the mechanisms of selection and to identify candidate genes of interest to breeding programs.

Results

Information on 705 243 autosomal single nucleotide polymorphisms (SNPs) in 3122 dairy and beef male animals from seven cattle breeds (Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental) were used to detect selection signatures by applying two complementary methods, integrated haplotype score (iHS) and global fixation index (F_ST). To control for false positive results, we used false discovery rate (FDR) adjustment to calculate adjusted iHS within each breed and the genome-wide significance level was about 0.003. Using the iHS method, 83, 92, 91, 101, 85, 101 and 86 significant genomic regions were detected for Angus, Belgian Blue, Charolais, Hereford, Holstein-Friesian, Limousin and Simmental cattle, respectively. None of these regions was common to all seven breeds. Using the F_ST approach, 704 individual SNPs were detected across breeds. Annotation of the regions of the genome that showed selection signatures revealed several interesting candidate genes i.e. DGAT1, ABCG2, MSTN, CAPN3, FABP3, CHCHD7, PLAG1, JAZF1, PRKG2, ACTC1, TBC1D1, GHR, BMP2, TSG1, LYN, KIT and MC1R that play a role in milk production, reproduction, body size, muscle formation or coat color. Fifty-seven common candidate genes were found by both the iHS and global F_ST methods across the seven breeds. Moreover, many novel genomic regions and genes were detected within the regions that showed selection signatures; for some candidate genes, signatures of positive selection exist in the human genome. Multilevel bioinformatic analyses of the detected candidate genes suggested that the PPAR pathway may have been subjected to positive selection.

Conclusions

This study provides a high-resolution bovine genomic map of positive selection signatures that are either specific to one breed or common to a subset of the seven breeds analyzed. Our results will contribute to the detection of functional candidate genes that have undergone positive selection in future studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0127-3) contains supplementary material, which is available to authorized users.     

Genome-wide patterns of copy number variation in the diversified chicken genomes using next-generation sequencing

Background

Copy number variation (CNV) is important and widespread in the genome, and is a major cause of disease and phenotypic diversity. Herein, we performed a genome-wide CNV analysis in 12 diversified chicken genomes based on whole genome sequencing.

Results

A total of 8,840 CNV regions (CNVRs) covering 98.2 Mb and representing 9.4% of the chicken genome were identified, ranging in size from 1.1 to 268.8 kb with an average of 11.1 kb. Sequencing-based predictions were confirmed at a high validation rate by two independent approaches, including array comparative genomic hybridization (aCGH) and quantitative PCR (qPCR). The Pearson’s correlation coefficients between sequencing and aCGH results ranged from 0.435 to 0.755, and qPCR experiments revealed a positive validation rate of 91.71% and a false negative rate of 22.43%. In total, 2,214 (25.0%) predicted CNVRs span 2,216 (36.4%) RefSeq genes associated with specific biological functions. Besides two previously reported copy number variable genes EDN3 and PRLR, we also found some promising genes with potential in phenotypic variation. Two genes, FZD6 and LIMS1, related to disease susceptibility/resistance are covered by CNVRs. The highly duplicated SOCS2 may lead to higher bone mineral density. Entire or partial duplication of some genes like POPDC3 may have great economic importance in poultry breeding.

Conclusions

Our results based on extensive genetic diversity provide a more refined chicken CNV map and genome-wide gene copy number estimates, and warrant future CNV association studies for important traits in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-962) contains supplementary material, which is available to authorized users.     

Whole‐genome SNP data unravel population structure and signatures of selection for black plumage of indigenous chicken breeds from Jiangxi province,China

Ten indigenous chicken breeds were originally distributed in Jiangxi Province, China, and they define a critical component of Chinese chicken genetic resources. We have investigated the population genetics of seven Jiangxi chicken breeds using 600K chicken BeadChip SNP data. To provide a genome‐wide perspective for the population structure of all 10 Jiangxi chicken breeds, we herein genotyped 78 additional individuals from the seven breeds and 63 chickens from three uninvestigated breeds—Yugan Black (YG), Nancheng Black (NC) and Wanzai Yellow using 55K chicken SNP arrays. We then explored merged data of 17 101 SNPs from 235 individuals to infer the population structure of the 10 breeds. We showed that NC and YG are two regional populations of the same breed, as individuals from the two populations clustered together to form a branch separate from the other breeds in the neighbor‐joining tree, they always grouped together in multidimensional principal component analyses and they displayed an identical pattern of ancestral lineage composition. Hence, NC and YG should be considered a single breed in the state‐supported conservation scheme. Moreover, we conducted a genome scan for signatures of selection for black plumage. bayescan and hapflk analyses of two contrasting groups (three black‐feathered breeds vs. six non‐black‐feathered breeds) consistently detected 25 putative regions under selection. Nine pigmentation‐ associated genes (DCT, SLC24A5, SLC30A4, MYO5A, CYP19A1, NADK2, SLC45A2, GNAQ and DCP2) reside within these regions, and these genes are interesting candidates for black plumage and provide a starting point for further identification of causative mutations for black feathers in chicken.     

Genome-Wide Detection of Selective Signature in Chinese Holstein

Selective signatures in whole genome can help us understand the mechanisms of selection and target causal variants for breeding program. In present study, we performed Extended Haplotype Homozygosity (EHH) tests to identify significant core regions harboring such signals in Chinese Holstein, and then verified the biological significance of these identified regions based on commonly-used bioinformatics analyses. Results showed a total of 125 significant regions in entire genome containing some of important functional genes such as LEP, ABCG2, CSN1S1, CSN3 and TNF based on the Gene Ontology database. Some of these annotated genes involved in the core regions overlapped with those identified in our previous GWAS as well as those involved in a recently constructed candidate gene database for cattle, further indicating these genes under positive selection maybe underlie milk production traits and other important traits in Chinese Holstein. Furthermore, in the enrichment analyses for the second level GO terms and pathways, we observed some significant terms over represented in these identified regions as compared to the entire bovine genome. This indicates that some functional genes associated with milk production traits, as reflected by GO terms, could be clustered in core regions, which provided promising evidence for the exploitability of the core regions identified by EHH tests. Findings in our study could help detect functional candidate genes under positive selection for further genetic and breeding research in Chinese Holstein.     

Genome-wide detection of selective signatures in Simmental cattle

Artificial selection has greatly improved the beef production performance and changed its genetic basis. High-density SNP markers provide a way to track these changes and use selective signatures to search for the genes associated with artificial selection. In this study, we performed extended haplotype homozygosity (EHH) tests based on Illumina BovineSNP50 (54 K) Chip data from 942 Simmental cattle to identify significant core regions containing selective signatures, then verified the biological significance of these identified regions based on some commonly used bioinformatics analyses. A total of 224 regions over the whole genome in Simmental cattle showing the highest significance and containing some important functional genes, such as GHSR, TG and CANCNA2D1 were chosen. We also observed some significant terms in the enrichment analyses of second GO terms and KEGG pathways, indicating that these genes are associated with economically relevant cattle traits. This is the first detection of selection signature in Simmental cattle. Our findings significantly expand the selection signature map of the cattle genome, and identify functional candidate genes under positive selection for future genetic research.     

Genome-wide association study of body weight in Wenshang Barred chicken based on the SLAF-seq technology

Chicken body weight (BW) is an economically important trait, and many studies have been conducted on genetic selection for BW. However, previous studies have detected functional chromosome mutations or regions using gene chips. The present study used the specific-locus amplified fragment sequencing (SLAF-seq) technology to perform a genome-wide association study (GWAS) on purebred Wengshang Barred chicken. A total of 1,286,715 single-nucleotide polymorphisms (SNPs) were detected, and 175,211 SNPs were selected as candidate SNPs for genome-wide association analysis using TASSEL general linear models. Six SNP markers reached genome-wide significance. Of these, rs732048524, rs735522839, rs738991545, and rs15837818 were significantly associated with body weight at 28 days (BW28), while rs314086457 and rs315694878 were significantly associated with BW120. These SNPs are close to seven genes (PRSS23, ME3, FAM181B, NABP1, SDPR, TSSK6L2, and RBBP8). Moreover, 24 BW-associated SNPs reached “suggestive” genome-wide significance. Of these, 6, 13, 1, and 4 SNPs were associated with BW28, BW56, BW80, and BW120, respectively. These results would enrich the studies on BW and promote the use of Chinese chicken, especially the Wenshang Barred chicken.     

Insights into the Effects of Long-Term Artificial Selection on Seed Size in Maize

Grain produced from cereal crops is a primary source of human food and animal feed worldwide. To understand the genetic basis of seed-size variation, a grain yield component, we conducted a genome-wide scan to detect evidence of selection in the maize Krug Yellow Dent long-term divergent seed-size selection experiment. Previous studies have documented significant phenotypic divergence between the populations. Allele frequency estimates for ∼3 million single nucleotide polymorphisms (SNPs) in the base population and selected populations were estimated from pooled whole-genome resequencing of 48 individuals per population. Using F_ST values across sliding windows, 94 divergent regions with a median of six genes per region were identified. Additionally, 2729 SNPs that reached fixation in both selected populations with opposing fixed alleles were identified, many of which clustered in two regions of the genome. Copy-number variation was highly prevalent between the selected populations, with 532 total regions identified on the basis of read-depth variation and comparative genome hybridization. Regions important for seed weight in natural variation were identified in the maize nested association mapping population. However, the number of regions that overlapped with the long-term selection experiment did not exceed that expected by chance, possibly indicating unique sources of variation between the two populations. The results of this study provide insights into the genetic elements underlying seed-size variation in maize and could also have applications for other cereal crops.     

A genome-wide detection of copy number variation using SNP genotyping arrays in Beijing-You chickens

Copy number variation (CNV) has been recently examined in many species and is recognized as being a source of genetic variability, especially for disease-related phenotypes. In this study, the PennCNV software, a genome-wide CNV detection system based on the 60 K SNP BeadChip was used on a total sample size of 1,310 Beijing-You chickens (a Chinese local breed). After quality control, 137 high confidence CNVRs covering 27.31 Mb of the chicken genome and corresponding to 2.61 % of the whole chicken genome. Within these regions, 131 known genes or coding sequences were involved. Q-PCR was applied to verify some of the genes related to disease development. Results showed that copy number of genes such as, phosphatidylinositol-5-phosphate 4-kinase II alpha, PHD finger protein 14, RHACD8 (a CD8α- like messenger RNA), MHC B-G, zinc finger protein, sarcosine dehydrogenase and ficolin 2 varied between individual chickens, which also supports the reliability of chip-detection of the CNVs. As one source of genomic variation, CNVs may provide new insight into the relationship between the genome and phenotypic characteristics.     

Genomic Analysis of Natural Selection and Phenotypic Variation in High-Altitude Mongolians

Deedu (DU) Mongolians, who migrated from the Mongolian steppes to the Qinghai-Tibetan Plateau approximately 500 years ago, are challenged by environmental conditions similar to native Tibetan highlanders. Identification of adaptive genetic factors in this population could provide insight into coordinated physiological responses to this environment. Here we examine genomic and phenotypic variation in this unique population and present the first complete analysis of a Mongolian whole-genome sequence. High-density SNP array data demonstrate that DU Mongolians share genetic ancestry with other Mongolian as well as Tibetan populations, specifically in genomic regions related with adaptation to high altitude. Several selection candidate genes identified in DU Mongolians are shared with other Asian groups (e.g., EDAR), neighboring Tibetan populations (including high-altitude candidates EPAS1, PKLR, and CYP2E1), as well as genes previously hypothesized to be associated with metabolic adaptation (e.g., PPARG). Hemoglobin concentration, a trait associated with high-altitude adaptation in Tibetans, is at an intermediate level in DU Mongolians compared to Tibetans and Han Chinese at comparable altitude. Whole-genome sequence from a DU Mongolian (Tianjiao1) shows that about 2% of the genomic variants, including more than 300 protein-coding changes, are specific to this individual. Our analyses of DU Mongolians and the first Mongolian genome provide valuable insight into genetic adaptation to extreme environments.     

Human genomic regions with exceptionally high levels of population differentiation identified from 911 whole-genome sequences

Background

Population differentiation has proved to be effective for identifying loci under geographically localized positive selection, and has the potential to identify loci subject to balancing selection. We have previously investigated the pattern of genetic differentiation among human populations at 36.8 million genomic variants to identify sites in the genome showing high frequency differences. Here, we extend this dataset to include additional variants, survey sites with low levels of differentiation, and evaluate the extent to which highly differentiated sites are likely to result from selective or other processes.

Results

We demonstrate that while sites with low differentiation represent sampling effects rather than balancing selection, sites showing extremely high population differentiation are enriched for positive selection events and that one half may be the result of classic selective sweeps. Among these, we rediscover known examples, where we actually identify the established functional SNP, and discover novel examples including the genes ABCA12, CALD1 and ZNF804, which we speculate may be linked to adaptations in skin, calcium metabolism and defense, respectively.

Conclusions

We identify known and many novel candidate regions for geographically restricted positive selection, and suggest several directions for further research.     

A Chromosome-level Genome Assembly of Wild Castor Provides New Insights into its Adaptive Evolution in Tropical Desert

Wild castor grows in the high-altitude tropical desert of the African Plateau,a region known for high ultraviolet radiation,strong light,and extremely dry condition.To investigate the potential genetic basis of adaptation to both highland and tropical deserts,we generated a chromosome-level genome sequence assembly of the wild castor accession WT05,with a genome size of 316 Mb,a scaffold N50 of 31.93 Mb,and a contig N50 of 8.96 Mb,respectively.Compared with cultivated castor and other Euphorbiac...     

Assessing signatures of selection through variation in linkage disequilibrium between taurine and indicine cattle

Background

Signatures of selection are regions in the genome that have been preferentially increased in frequency and fixed in a population because of their functional importance in specific processes. These regions can be detected because of their lower genetic variability and specific regional linkage disequilibrium (LD) patterns.

Methods

By comparing the differences in regional LD variation between dairy and beef cattle types, and between indicine and taurine subspecies, we aim at finding signatures of selection for production and adaptation in cattle breeds. The VarLD method was applied to compare the LD variation in the autosomal genome between breeds, including Angus and Brown Swiss, representing taurine breeds, and Nelore and Gir, representing indicine breeds. Genomic regions containing the top 0.01 and 0.1 percentile of signals were characterized using the UMD3.1 Bos taurus genome assembly to identify genes in those regions and compared with previously reported selection signatures and regions with copy number variation.

Results

For all comparisons, the top 0.01 and 0.1 percentile included 26 and 165 signals and 17 and 125 genes, respectively, including TECRL, BT.23182 or FPPS, CAST, MYOM1, UVRAG and DNAJA1.

Conclusions

The VarLD method is a powerful tool to identify differences in linkage disequilibrium between cattle populations and putative signatures of selection with potential adaptive and productive importance.     

Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.     

Genome analysis and signature discovery for diving and sensory properties of the endangered Chinese alligator

Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.     

Impact of host demography and evolutionary history on endosymbiont molecular evolution: A test in carpenter ants (genus Camponotus) and their Blochmannia endosymbionts

Obligate endosymbioses are tight associations between symbionts and the hosts they live inside. Hosts and their associated obligate endosymbionts generally exhibit codiversification, which has been documented in taxonomically diverse insect lineages. Host demography (e.g., effective population sizes) may impact the demography of endosymbionts, which may lead to an association between host demography and the patterns and processes of endosymbiont molecular evolution. Here, we used whole‐genome sequencing data for carpenter ants (Genus Camponotus; subgenera Camponotus and Tanaemyrmex) and their Blochmannia endosymbionts as our study system to address whether Camponotus demography shapes Blochmannia molecular evolution. Using whole‐genome phylogenomics, we confirmed previous work identifying codiversification between carpenter ants and their Blochmannia endosymbionts. We found that Blochmannia genes have evolved at a pace ~30× faster than that of their hosts'' molecular evolution and that these rates are positively associated with host rates of molecular evolution. Using multiple tests for selection in Blochmannia genes, we found signatures of positive selection and shifts in selection strength across the phylogeny. Host demography was associated with Blochmannia shifts toward increased selection strengths, but not associated with Blochmannia selection relaxation, positive selection, genetic drift rates, or genome size evolution. Mixed support for relationships between host effective population sizes and Blochmannia molecular evolution suggests weak or uncoupled relationships between host demography and Blochmannia population genomic processes. Finally, we found that Blochmannia genome size evolution was associated with genome‐wide estimates of genetic drift and number of genes with relaxed selection pressures.     

Identification and functional characterization of copy number variations in diverse chicken breeds

Background

The detection and functional characterization of genomic structural variations are important for understanding the landscape of genetic variation in the chicken. A recently recognized aspect of genomic structural variation, called copy number variation (CNV), is gaining interest in chicken genomic studies. The aim of the present study was to investigate the pattern and functional characterization of CNVs in five characteristic chicken breeds, which will be important for future studies associating phenotype with chicken genome architecture.

Results

Using a commercial 385 K array-based comparative genomic hybridization (aCGH) genome array, we performed CNV discovery using 10 chicken samples from four local Chinese breeds and the French breed Houdan chicken. The female Anka broiler was used as a reference. A total of 281 copy number variation regions (CNVR) were identified, covering 12.8 Mb of polymorphic sequences or 1.07% of the entire chicken genome. The functional annotation of CNVRs indicated that these regions completely or partially overlapped with 231 genes and 1032 quantitative traits loci, suggesting these CNVs have important functions and might be promising resources for exploring differences among various breeds. In addition, we employed quantitative PCR (qPCR) to further validate several copy number variable genes, such as prolactin receptor, endothelin 3 (EDN3), suppressor of cytokine signaling 2, CD8a molecule, with important functions, and the results suggested that EDN3 might be a molecular marker for the selection of dark skin color in poultry production. Moreover, we also identified a new CNVR (chr24: 3484617–3512275), encoding the sortilin-related receptor gene, with copy number changes in only black-bone chicken.

Conclusions

Here, we report a genome-wide analysis of the CNVs in five chicken breeds using aCGH. The association between EDN3 and melanoblast proliferation was further confirmed using qPCR. These results provide additional information for understanding genomic variation and related phenotypic characteristics.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-934) contains supplementary material, which is available to authorized users.