Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in Upland cotton (Gossypium hirsutum L.)

MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited six A_t-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one D_t-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using A_t-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against a major pathogen.     

Finding needles in a genomic haystack: targeted capture identifies clear signatures of selection in a nonmodel plant species

Teasing apart neutral and adaptive genomic processes and identifying loci that are targets of selection can be difficult, particularly for nonmodel species that lack a reference genome. However, identifying such loci and the factors driving selection have the potential to greatly assist conservation and restoration practices, especially for the management of species in the face of contemporary and future climate change. Here, we focus on assessing adaptive genomic variation within a nonmodel plant species, the narrow‐leaf hopbush (Dodonaea viscosa ssp. angustissima), commonly used for restoration in Australia. We used a hybrid‐capture target enrichment approach to selectively sequence 970 genes across 17 populations along a latitudinal gradient from 30°S to 36°S. We analysed 8462 single‐nucleotide polymorphisms (SNPs) for F_ST outliers as well as associations with environmental variables. Using three different methods, we found 55 SNPs with significant correlations to temperature and water availability, and 38 SNPs to elevation. Genes containing SNPs identified as under environmental selection were diverse, including aquaporin and abscisic acid genes, as well as genes with ontologies relating to responses to environmental stressors such as water deprivation and salt stress. Redundancy analysis demonstrated that only a small proportion of the total genetic variance was explained by environmental variables. We demonstrate that selection has led to clines in allele frequencies in a number of functional genes, including those linked to leaf shape and stomatal variation, which have been previously observed to vary along the sampled environmental cline. Using our approach, gene regions subject to environmental selection can be readily identified for nonmodel organisms.     

In silico investigations on functional and haplotype tag SNPs associated with congenital long QT syndromes (LQTSs)

Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. It is still a major challenge to identify the functional SNPs in disease-related genes. In this review, the genetic variation that can alter the expression and the function of the genes, namely KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2, with the potential role for the development of congenital long QT syndrome (LQTS) was analyzed. Of the total of 3,309 SNPs in all five genes, 27 non-synonymous SNPs (nsSNPs) in the coding region and 44 SNPs in the 5′ and 3′ un-translated regions (UTR) were identified as functionally significant. SIFT and PolyPhen programs were used to analyze the nsSNPs and FastSNP; UTR scan programs were used to compute SNPs in the 5′ and 3′ untranslated regions. Of the five selected genes, KCNQ1 has the highest number of 26 haplotype blocks and 6 tag SNPs with a complete linkage disequilibrium value. The gene SCN5A has ten haplotype blocks and four tag SNPs. Both KCNE1 and KCNE2 genes have only one haplotype block and four tag SNPs. Four haplotype blocks and two tag SNPs were obtained for KCNH2 gene. Also, this review reports the copy number variations (CNVs), expressed sequence tags (ESTs) and genome survey sequences (GSS) of the selected genes. These computational methods are in good agreement with experimental works reported earlier concerning LQTS.     

Identification of Single Nucleotide Polymorphisms and Analysis of Linkage Disequilibrium in Different Bamboo Species Using the Candidate Gene Approach

Bamboos are one of the most beautiful and useful plants on Earth. The genetic background and population structure of bamboos are well known, which helps accelerate the process of artificial domestication of bamboo. Partial sequences of six genes involved in nitrogen use efficiency in 32 different bamboo species were analyzed for occurrence of single nucleotide polymorphisms (SNPs). The nucleotide diversity θw and total nucleotide polymorphisms π_T of the sequenced DNA regions was 0.05137 and 0.03332, respectively. Both πnonsyn /πsyn and Ka/Ks values were <1. The nucleotide sequences of these six genes were inferred to be relatively conserved, and the haplotype diversity was relatively high. The results of evolutionary neutrality tests showed that the six genes were in line with neutral evolution, and that the NRT2.1 and AMT2.1 gene sequences may have experienced negative selection. An inter-SNP recombination event at the NRT2.1 gene in the all pooled sample, of all 32 bamboo species was the lowest at 0.0645, whereas the AMT gene recombination events were all >0.1. Estimation and analysis of linkage disequilibrium of five genes revealed that with the increase in nucleotide sequence length, the degree of SNP linkage disequilibrium decreased rapidly. We inferred the population genetic structure of 32 bamboo species based on the SNP loci of six genes with frequencies >18%. 32 bamboo species were divided into five categories, which indicated that the combined population of all bamboo species had obvious multivariate characteristics and was heterogeneous; red (Group 1) and green (Group 2) were the main groups.     

Chromosomal Localization and Sequence Variation of 5S rRNA Gene in Five Capsicum Species

Chromosomal localization and sequence analysis of the 5S rRNA gene were carried out in five Capsicum species. Fluorescence in situ hybridization revealed that chromosomal location of the 5S rRNA gene was conserved in a single locus at a chromosome which was assigned to chromosome 1 by the synteny relationship with tomato. In sequence analysis, the repeating units of the 5S rRNA genes in the Capsicum species were variable in size from 278 bp to 300 bp. In sequence comparison of our results to the results with other Solanaceae plants as published by others, the coding region was highly conserved, but the spacer regions varied in size and sequence. T stretch regions, just after the end of the coding sequences, were more prominant in the Capsicum species than in two other plants. High G-C rich regions, which might have similar functions as that of the GC islands in the genes transcribed by RNA PolII, were observed after the T stretch region. Although we could not observe the TATA like sequences, an AT rich segment at -27 to -18 was detected in the 5S rRNA genes of the Capsicum species. Species relationship among the Capsicum species was also studied by the sequence comparison of the 5S rRNA genes. While C. chinense, C. frutescens, and C. annuum formed one lineage, C. baccatum was revealed to be an intermediate species between the former three species and C. pubescens.     

Ancient genome‐wide admixture extends beyond the current hybrid zone between Macaca fascicularis and M. mulatta

Macaca fascicularis and Macaca mulatta are two of the most commonly used laboratory macaques, yet their genetic differences at a genome‐wide level remain unclear. We analysed the multilocus DNA sequence data of 54 autosomal loci obtained from M. fascicularis samples from three different geographic origins and M. mulatta samples of Burmese origin. M. fascicularis shows high nucleotide diversity, four to five times higher than humans, and a strong geographic population structure between Indonesian‐Malaysian and Philippine macaques. The pattern of divergence and polymorphism between M. fascicularis and M. mulatta shows a footprint of genetic exchange not only within their current hybrid zone but also across a wider range for more than 1 million years. However, genetic admixture may not be a random event in the genome. Whereas randomly selected genic and intergenic regions have the same evolutionary dynamics between the species, some cytochrome oxidase P450 (CYP) genes (major chemical metabolizing genes and potential target genes for local adaptation) have a significantly larger species divergence than other genes. By surveying CYP3A5 gene sequences of more than a hundred macaques, we identified three nonsynonymous single nucleotide polymorphisms that were highly differentiated between the macaques. The mosaic pattern of species divergence in the genomes may be a consequence of genetic differentiation under ecological adaptation and may be a salient feature in the genomes of nascent species under parapatry.     

Tracking the progression of speciation: variable patterns of introgression across the genome provide insights on the species delimitation between progenitor–derivative spruces (Picea mariana × P. rubens)

The genic species concept implies that while most of the genome can be exchanged somewhat freely between species through introgression, some genomic regions remain impermeable to interspecific gene flow. Hence, interspecific differences can be maintained despite ongoing gene exchange within contact zones. This study assessed the heterogeneous patterns of introgression at gene loci across the hybrid zone of an incipient progenitor–derivative species pair, Picea mariana (black spruce) and Picea rubens (red spruce). The spruce taxa likely diverged in geographic isolation during the Pleistocene and came into secondary contact during late Holocene. A total of 300 SNPs distributed across the 12 linkage groups (LG) of black spruce were genotyped for 385 individual trees from 33 populations distributed across the allopatric zone of each species and within the zone of sympatry. An integrative framework combining three population genomic approaches was used to scan the genomes, revealing heterogeneous patterns of introgression. A total of 23 SNPs scattered over 10 LG were considered impermeable to introgression and putatively under diverging selection. These loci revealed the existence of impermeable genomic regions forming the species boundary and are thus indicative of ongoing speciation between these two genetic lineages. Another 238 SNPs reflected selectively neutral diffusion across the porous species barrier. Finally, 39 highly permeable SNPs suggested ancestral polymorphism along with balancing selection. The heterogeneous patterns of introgression across the genome indicated that the speciation process between black spruce and red spruce is young and incomplete, albeit some interspecific differences are maintained, allowing ongoing species divergence even in sympatry. The approach developed in this study can be used to track the progression of ongoing speciation processes.     

Comparative analysis of a transposon-rich Brassica oleracea BAC clone with its corresponding sequence in A. thaliana

We compared the sequence of a 96.7 Kb-long BAC clone (B19N3) from Brassica oleracea (broccoli) with its corresponding regions in Arabidopsis thaliana. B19N3 contains eight genes and 15 transposable elements (TEs). The first two genes in this clone, Bo1 and Bo2, have its corresponding region at the end of chromosome V of Arabidopsis (24 Mb). The third gene, Bo3, corresponds to an ortholog at the opposite end (2.6 Mb) of the same chromosome. The other five genes, Bo4 to Bo8 also have a corresponding region on the same chromosome but at 7.7 Mb . These five genes are colinear with those found in the corresponding region of Arabidopsis, which contains, however, 15 genes. Therefore, a cluster of 10 genes is missing in B. oleracea clone (B19N3). All five genes in common have the same order and orientation in the genomes of both species. Their 36 exons constituting the eight homologous genes have high conservation in size and sequence identity in both species. Among these, there is a major gene involved in aliphatic glucosinolate biosynthesis, BoGSL-ELONG (Bo4). Similar to A. thaliana, this gene, has a tandem duplicate, Bo5. A contig for this region was constructed by primer walking and BAC-end-sequencing, revealing general gene colinearity between both species. During the 20 million years separating A. thaliana from B. oleracea from a common ancestor both genomes have diverged by chromosomal rearrangements and differential TE activity. These events, in addition to changes in chromosome number are responsible for the evolution of the genomes of both species. In spite of these changes, both species conserve general colinearity for their corresponding genes.     

The geographical and environmental determinants of genetic diversity for four alpine conifers of the European Alps

Climate is one of the most important drivers of local adaptation in forest tree species. Standing levels of genetic diversity and structure within and among natural populations of forest trees are determined by the interplay between climatic heterogeneity and the balance between selection and gene flow. To investigate this interplay, single nucleotide polymorphisms (SNPs) were genotyped in 24 to 37 populations from four subalpine conifers, Abies alba Mill., Larix decidua Mill., Pinus cembra L. and Pinus mugo Turra, across their natural ranges in the Italian Alps and Apennines. Patterns of population structure were apparent using a Bayesian clustering program, STRUCTURE, which identified three to five genetic groups per species. Geographical correlates with these patterns, however, were only apparent for P. cembra. Multivariate environmental variables [i.e. principal components (PCs)] were subsequently tested for association with SNPs using a Bayesian generalized linear mixed model. The majority of the SNPs, ranging from six in L. decidua to 18 in P. mugo, were associated with PC1, corresponding to winter precipitation and seasonal minimum temperature. In A. alba, four SNPs were associated with PC2, corresponding to the seasonal minimum temperature. Functional annotation of those genes with the orthologs in Arabidopsis revealed several genes involved in abiotic stress response. This study provides a detailed assessment of population structure and its association with environment and geography in four coniferous species in the Italian mountains.     

Comparative Genome Structure,Secondary Metabolite,and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP–encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.     

Development of high‐density SNP genotyping arrays for white spruce (Picea glauca) and transferability to subtropical and nordic congeners

High‐density SNP genotyping arrays can be designed for any species given sufficient sequence information of high quality. Two high‐density SNP arrays relying on the Infinium iSelect technology (Illumina) were designed for use in the conifer white spruce (Picea glauca). One array contained 7338 segregating SNPs representative of 2814 genes of various molecular functional classes for main uses in genetic association and population genetics studies. The other one contained 9559 segregating SNPs representative of 9543 genes for main uses in population genetics, linkage mapping of the genome and genomic prediction. The SNPs assayed were discovered from various sources of gene resequencing data. SNPs predicted from high‐quality sequences derived from genomic DNA reached a genotyping success rate of 64.7%. Nonsingleton in silico SNPs (i.e. a sequence polymorphism present in at least two reads) predicted from expressed sequenced tags obtained with the Roche 454 technology and Illumina GAII analyser resulted in a similar genotyping success rate of 71.6% when the deepest alignment was used and the most favourable SNP probe per gene was selected. A variable proportion of these SNPs was shared by other nordic and subtropical spruce species from North America and Europe. The number of shared SNPs was inversely proportional to phylogenetic divergence and standing genetic variation in the recipient species, but positively related to allele frequency in P. glauca natural populations. These validated SNP resources should open up new avenues for population genetics and comparative genetic mapping at a genomic scale in spruce species.     

Allelic Variation in the Porcine MYF5 Gene Detected by PCR–SSCP

The MYF5 gene has been reported to be integral to muscle growth and development, and hence it has been considered as a candidate gene for meat selection programs in pig. To ascertain whether there was variation in the porcine MYF5 gene, we have developed a method of PCR–single-strand conformational polymorphism (PCR–SSCP) analysis. In this study, two coding regions of the MYF5 gene were investigated. Four unique SSCP patterns were detected in exon 1 and three patterns were identified in exon 3. Two SNPs detected in exon 1 led to a non-synonymous alanine/proline substitution. A nucleotide change in exon 3 did not affect the amino acid sequence. Five extended haplotypes were observed across the two regions. The variation detected in this study might underpin the development of gene markers for improved muscle growth in pig breeding.     

珍稀濒危飘带兜兰叶绿体全基因组种内变异研究(附录)

飘带兜兰(Paphiopedilum parishii)分布范围狭窄,仅在中国、缅甸、泰国以及老挝有少量分布。近年来,因生境破坏和人为滥采而导致飘带兜兰野生种群极度缩减。为开发种内多态性的分子标记用于保护生物学研究,该研究对飘带兜兰4个野生个体经测序、组装、注释获得的叶绿体基因组序列,与已公布的飘带兜兰2个个体的叶绿体全基因组序列进行比对,分析飘带兜兰叶绿体基因组的种内差异。结果表明:(1)飘带兜兰叶绿体基因组具有典型被子植物叶绿体基因组环状四分体结构,基因组长度为154 403～154 809 bp,共编码129个基因,包括78个蛋白质编码基因、39个tRNA基因、8个rRNA基因,以及4个假基因。(2)在飘带兜兰6个个体叶绿体基因组中检测到103～107个SSRs(simple sequence repeats)位点,其中21个SSR位点具有多态性。此外,在6个个体叶绿体基因组中还检测到60个长序列重复,包括17～21个正向重复、18～29个反向重复、9～16个回文重复、4～9个互补重复。(3)通过比较6个个体叶绿体基因组序列的核苷酸多样性,共发现70处变异,包括10个SNPs(single nucleotide polymorphism)、60个插入缺失(InDels)。其中,有3个SNP位点发生了非同义替换,导致编码功能基因的氨基酸发生改变; 19个插入缺失多态性较高,具有开发为分子标记的潜力。(4)通过计算核苷酸多样性值(P_i)共发现8个有变异的区域,P_i值为0～0.006 32,其中变异度较大的是rps3-rpl22、trnL-UAC-rpl32、rpoB-trnC-GCA以及ycf4,这些高变区可开发为分子标记用于评估飘带兜兰遗传多样性。(5)系统发生分析结果表明,飘带兜兰6个个体叶绿体基因组序列聚在一起,与长瓣兜兰互为姐妹群。综上表明,飘带兜兰叶绿体基因组的SSRs、长序列重复、SNPs、InDels以及核苷酸序列呈现了足够的种内多样性,可开发成分子标记用于该种的系统演化及保护生物学研究。     

The chloroplast genome of mulberry: complete nucleotide sequence, gene organization and comparative analysis

The complete nucleotide sequence of mulberry (Morus indica cv. K2) chloroplast genome (158,484 bp) has been determined using a combination of long PCR and shotgun-based approaches. This is the third angiosperm tree species whose plastome sequence has been completely deciphered. The circular double-stranded molecule comprises of two identical inverted repeats (25,678 bp each) separating a large and a small single-copy region of 87,386 bp and 19,742 bp, respectively. A total of 83 protein-coding genes including five genes duplicated in the inverted repeat regions, eight ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids, were assigned on the basis of homology to predicted genes from other chloroplast genomes. The mulberry plastome lacks the genes infA, sprA, and rpl21 and contains two pseudogenes ycf15 and ycf68. Comparative analysis, based on sequence similarity, both at the gene and genome level, indicates Morus to be closer to Cucumis and Lotus, phylogenetically. However, at genome level, inclusion of non-coding regions brings it closer to Eucalyptus, followed by Cucumis. This may reflect differential selection pressure operating on the genic and intergenic regions of the chloroplast genome.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Communicated by Y. Tsumura