Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 × Q165). The sequencing yielded 96 755 (IJ76-514) and 86 241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap 3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP – an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent – with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.     

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)_n trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5′ untranslated region. No primer pairs producing a polymorphic product were found in the 3′ untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.     

甘蔗细茎野生种云南不同生态类型的RAPD分析

利用25个随机引物对来自云南不同生态类型的82份甘蔗细茎野生种（Saccharnm spontanenm L．）和4份国外种材料进行RAPD标记,结果表明：云南甘蔗细茎野生种不同生态类型的遗传变异较大,具有丰富的遗传多样性,低续度类型的遗传多样性明显高于高纬度类型,在相同的纬度范围内,随着海拔的升高,其多态性逐渐减少,基于分子聚类分析,86份材料被划分为8个不同群体,表现出明显的地理分布的特点,结果初步证明了云南甘蔗细茎野生种可能起源于云南南部低海拔,低纬度地区,而后逐渐向高海拔,高纬度的西北和东北部演化,扩散,提出了云南南部可能是野生甘蔗起源中心之一的观点。     

Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene‐rich regions. Gene‐enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene‐enrichment strategy, we have compared assemblies using methyl‐filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA‐seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single‐nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.     

Comparative analysis reveals changes in transcriptomes of sugarcane upon infection by Leifsoniaxyli subsp. xyli

Ratoon stunting disease (RSD) caused by bacterium Leifsoniaxyli subsp. xyli (Lxx) is a devastating disease of sugarcane over a large part of the world. Genetic improvement for RSD‐resistant varieties is considered the most effective method to control the disease. However, genetic improvement of sugarcane is hindered by the limited information about the molecular mechanisms underlying Lxx pathogenicity and defence responses in sugarcane. In this study, genome‐wide gene expression profiling was used to compare RSD‐resistant (CP72‐2086) and RSD‐susceptible (GT11) genotypes at different infection time points in order to identify the candidate regulators for RSD resistance. A total of 14,494 differentially expressed genes (DEGs) were identified, indicating that dramatic changes had occurred in gene expression upon Lxx infection, especially in the susceptible genotype. Enrichment analysis showed that a large number of genes related to plant hormone signal transduction, phenylalanine metabolism, phenylpropanoid biosynthesis and starch and sucrose metabolism was responsible for sugarcane response to Lxx infection. Plant hormone signalling pathway genes were significantly differentially expressed at the early infection stage between the two genotypes. The resistant genotype chose the jasmonic acid‐ and ethylene‐dependent host‐defence pathways to resist Lxx infection, whereas the susceptible genotype preferred the salicylic acid‐dependent host‐defence pathways. These findings help unravel the molecular mechanisms of sugarcane plant–Lxx interactions and may pave the way for sugarcane breeding for disease resistance.     

Field-cage experiments on host fidelity in a pair of sympatric phytophagous ladybird beetles

Using field cages, we tested the hypothesis that two phytophagous ladybird beetles, Henosepilachna niponica (Lewis) and Henosepilachna yasutomii Katakura (Coleoptera: Coccinellidae: Epilachninae), are reproductively isolated from each other by their host fidelity alone. We planted host plants of the two species [the thistle Cirsium alpicola Nakai (Asteraceae) for H. niponica and the blue cohosh, Caulophyllum robustum Maxim. (Berberidaceae), for H. yasutomii] in 7 × 7 m cages (BT cages), and planted the two host plants and the beetles’ common food plant, the Japanese nightshade, Solanum japonense Nakai (Solanaceae), in other cages (BJT cages). We then released overwintered reproducing beetles into these cages and recorded their dispersal, the distribution of egg masses, and the incidence of copulations. In BT cages, the beetles moved almost exclusively between their own host plants, and all females laid eggs exclusively on their own host plants. In BJT cages, the beetles moved between their own host plants and the Japanese nightshade, resulting in occasional interspecific mating on the same nightshade plant. Females of both species laid eggs on their own host plants as well as on the Japanese nightshade. These results indicate that host fidelity functions as a strong barrier against gene flow between H. niponica and H. yasutomii. However, this barrier can easily be broken down if a third plant species bridges the beetle populations.     

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.     

甘蔗成熟期叶鞘纤维素酶活性对自然脱落的影响

采用脱叶性不同的4个甘蔗品种为材料,研究了在成熟期叶鞘纤维素酶活性对蔗叶脱落的影响。结果表明,纤维素酶活性影响甘蔗叶片脱落,在收获期叶鞘纤维素酶活性与植株脱落率均达到最高。不同叶位中,＋10叶位纤维素酶活性最高。相关性分析表明,4个甘蔗品种的＋10叶位叶鞘纤维素酶活性与脱落率呈正相关。     

11份割手密遗传多样性的SSR分析

利用10对多态性丰富的SSR引物,以国家甘蔗种质资源圃中保存的14份具有代表性的割手密为对照,对未收集过的云南地区11份割手密（Saccharum spontaneum L.）野生资源进行多样性分析。结果共扩增出233条DNA谱带,与对照相比,新采集材料的多态性条带为207条,其中14条为特有条带,多态性条带比率为0.89。遗传相似性系数和UPGMA聚类分析表明,新采集的材料并没有单独聚为一类,而是比较分散,在相似性系数为0.64处做切割线,参试材料可分为三个类群：第一类群主要由龙门割手密、河边村割手密和福建仙游1号组成;第二类群中包含19份材料,其中新采集的样品有上岗割手密、他拉割手密、安乐割手密、勐根割手密、芒美割手密、贺海割手密、回落割手密、里拉割手密和曼亨割手密,对照材料主要包含了云南、四川、越南、老挝、泰国地区的割手密,其共同特点是均分布在内陆地区;第三类群包括3个材料,分别是海南1号、海南92-2和广东化州割手密,其中不包含新采集的材料。而在相似性系数为0.654处作切割线又能将上述第二类群分为较细的三个亚群。由此可见,新采集的11份割手密资源具有丰富的遗传多样性,与已收集的资源相比,具有一定的差异性。说明依靠云南高山峡谷等立体气候特点,分布着遗传差异显著的割手密无性系。     

Territoriality and microhabitat selection in two intertidal New Zealand fish

Aspects of territoriality and microhabitat selection in Acanthoclinus fuscus and Forsterygion nigripenne robustum were studied in intra- and interspecific confrontation experiments under tidal and constant conditions. In both species aggressive responses were closely related to activity with maximal frequencies during the first 2 h of the experiments. Under tidal conditions activity was higher than at constant water level. Size predominantly affected contest outcome in both species. Initiation of the first bout and past experience were further important factors, whereas prior residence did not influence dominance relationships. Dominant individuals defended access to shelter and low tide pools as critical resources. A. fuscus preferred tubes matching the body size for cover, whereas F. n. robustum spent more time in caves and on open areas. Furthermore, A. fuscus preferred shallower water and was able to sustain long periods out of water. The different microhabitat preferences may reduce interspecific competition. Although A. fuscus did not win significantly more trials, higher aggressive tendencies were observed in this species.