The holocentric species Luzula elegans shows interplay between centromere and large‐scale genome organization

In higher plants, the large‐scale structure of monocentric chromosomes consists of distinguishable eu‐ and heterochromatic regions, the proportions and organization of which depend on a species' genome size. To determine whether the same interplay is maintained for holocentric chromosomes, we investigated the distribution of repetitive sequences and epigenetic marks in the woodrush Luzula elegans (3.81 Gbp/1C). Sixty‐one per cent of the L. elegans genome is characterized by highly repetitive DNA, with over 30 distinct sequence families encoding an exceptionally high diversity of satellite repeats. Over 33% of the genome is composed of the Angela clade of Ty1/copia LTR retrotransposons, which are uniformly dispersed along the chromosomes, while the satellite repeats occur as bands whose distribution appears to be biased towards the chromosome termini. No satellite showed an almost chromosome‐wide distribution pattern as expected for a holocentric chromosome and no typical centromere‐associated LTR retrotransposons were found either. No distinguishable large‐scale patterns of eu‐ and heterochromatin‐typical epigenetic marks or early/late DNA replicating domains were found along mitotic chromosomes, although super‐high‐resolution light microscopy revealed distinguishable interspersed units of various chromatin types. Our data suggest a correlation between the centromere and overall genome organization in species with holocentric chromosomes.     

Distribution and characterization of over 1000 T-DNA tags in rice genome

We generated T-DNA insertions throughout the rice genome for saturation mutagenesis. More than 1,000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene-rich regions. Few insertions (2.4%) were found in repetitive regions. T-DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5'- and 3'-regulatory regions outside the coding regions both at 500-bp size and in introns rather than in exons. Such distribution patterns and biases for T-DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T-DNA integration mechanism itself. Rice will require approximately the same number of T-DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T-DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).     

Obtaining the sequence of the rice genome and lessons learned along the way

Rice holds the record for the largest number of separate genome projects and for having the genome of two subspecies sequenced. This might be a short-lived record in the genomics era, but it highlights the significance of rice as a food staple and as a model plant for cereal species. Clearly, obtaining the genome sequence four times seems redundant, yet the rationale and motivation for each of these projects is valid; whether it is serving corporate shareholders or the general scientific community. Although the multiple projects resulted in some duplicated efforts, the value of data sharing was obvious and the winner in the end will be the global public.     

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.     

Physical maps and recombination frequency of six rice chromosomes

We constructed physical maps of rice chromosomes 1, 2, and 6-9 with P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones. These maps, with only 20 gaps, cover more than 97% of the predicted length of the six chromosomes. We submitted a total of 193 Mbp of non-overlapping sequences to public databases. We analyzed the DNA sequences of 1316 genetic markers and six centromere-specific repeats to facilitate characterization of chromosomal recombination frequency and of the genomic composition and structure of the centromeric regions. We found marked changes in the relative recombination rate along the length of each chromosome. Chromosomal recombination at the centromere core and surrounding regions on the six chromosomes was completely suppressed. These regions have a total physical length of about 23 Mbp, corresponding to 11.4% of the entire size of the six chromosomes. Chromosome 6 has the longest quiescent region, with about 5.6 Mbp, followed by chromosome 8, with quiescent region about half this size. Repetitive sequences accounted for at least 40% of the total genomic sequence on the partly sequenced centromeric region of chromosome 1. Rice CentO satellite DNA is arrayed in clusters and is closely associated with the presence of Centromeric Retrotransposon of Rice (CRR)- and RIce RetroElement 7 (RIRE7)-like retroelement sequences. We also detected relatively small coldspot regions outside the centromeric region; their repetitive content and gene density were similar to those of regions with normal recombination rates. Sequence analysis of these regions suggests that either the amount or the organization patterns of repetitive sequences may play a role in the inactivation of recombination.     

Cloning and Mapping of Telomere-Associated Sequences from Rice

We have isolated three telomere-associated sequences from riceusing cassette-ligation-mediated polymerase chain reaction (PCR).Each of the obtained clones hybridized to the terminal of oneor several rice chromosome arms. The telomeres recognized bythe clones displayed a high level of polymorphism between tworice varieties, Nipponbare (a japonica variety) and Kasalath(an indica variety). Variability in the chromosome termini wasalso detected among individual F₂ progeny plants, which werederived from a cross between the two rice varieties. One clonecontaining telomere-associated sequences was located to oneend of chromosome 5, and another clone to one end of chromosome11. For another clone, non-allelic segregation of polymorphichybridization bands was observed between japonica and indicarice; this clone was mapped to one end of chromosome 12 in japonicaand to one end of chromosome 11 in indica rice. This indicatesan exchange of termini between nonhomologous chromosomes.     

Repetitive sequences: cause for variation in genome size and chromosome morphology in the genus Oryza

Large variation in genome size as determined by the nuclear DNA content and the mitotic chromosome size among diploid rice species is revealed using flow cytometry and image analyses. Both the total chromosomal length (r_0.939) and the total chromosomal area (r_0.927) correlated well with the nuclear DNA content. Among all the species examined, Oryza australiensis (E genome) and O. brachyantha (F genome), respectively, were the largest and smallest in genome size. O. sativa (A genome) involving all the cultivated species showed the intermediate genome size between them. The distribution patterns of genome-specific repetitive DNA sequences were physically determined using fluorescence in situ hybridization (FISH). O. brachyantha had limited sites of the repetitive DNA sequences specific to the F genome. O. australiensis showed overall amplification of genome-specific DNA sequences throughout the chromosomes. The amplification of the repetitive DNA sequences causes the variation in the chromosome morphology and thus the genome size among diploid species in the genus Oryza.     

水稻几丁质酶基因克隆RCH8的DNA结构分析

用DNA外切核酸酶Ⅲ和S1核酸酶生成连续缺失突变体,用Sanger双脱氧链终止法对该克隆进行双向DNA顺序测定,测序全长2049个碱基,初步确定了1057bp的5'端上游顺序,966bp不含内含子的完整编码区和可能的TATAbox等。所编码的322个氨基酸包括N-端20个氨基酸的信号肽,其后40个氨基酸长度的含8个半胱氨酸的hevein结构域和一个催化结构域。     

Evolutionary Genomics of Weedy Rice in the USA

Red rice is an interfertiie, weedy form of cultivated rice (Oryza sativa L.) that competes aggressively with the cropin the southern US, reducing yields and contaminating harvests. No wild Oryza species occur In North America andthe weed has been proposed to have evolved through multiple mechanisms, including "de-domestication" of UScrop cultivars, accidental introduction of Asian weeds, and hybridization between US crops and Asian wild/weedyOryza strains. The phenotype of US red rice ranges from "crop mimics", which share some domestication traitswith the crop, to strains closely resembling Asian wild Oryza species. Assessments of genetic diversity haveindicated that many weed strains are closely related to Asian taxa (including indica and aus rice varieties, whichhave never been cultivated in the US, and the Asian crop progenitor O. rufipogon), whereas others show geneticsimilarity to the tropical japonica varieties cultivated in the southern US. Herein, we review what is known aboutthe evolutionary origins and genetic diversity of US red rice and describe an ongoing research project to furthercharacterize the evolutionary genomics of this aggressive weed.     

Genome-wide Investigation of Intron Length Polymorphisms and Their Potential as Molecular Markers in Rice (Oryza sativa L.)

Intron length polymorphisms (ILPs) have been used as geneticmarkers in some studies. However, a systematic investigationand large-scale exploitation of ILP markers has not been reported.In this study, we performed a genome-wide search of ILPs betweentwo subspecies (indica and japonica) in rice using the draftgenomic sequences of cultivars 93-11 (indica) and Nipponbare(japonica) and 32 127 full-length cDNA sequences of Nipponbareobtained from public databases. We identified 13 308 putativeILPs. Based on these putative ILPs, we developed 5811 candidateILP markers via electronic-PCR with primers designed in flankingexons. We further conducted experiment to verify the candidateILP markers. Out of 215 candidate ILP markers tested on 93-11,Nipponbare and their hybrid, we successfully exploited 173 codominantILP markers. Further analyses on 10 rice accessions showed thatthese ILP markers were widely applicable and most (71.1%) exhibitedsubspecies specificity. This feature suggests that ILPs wouldbe useful for the studies of genome evolution and inter-subspeciesheterosis and for cross-subspecies marker-assisted selectionin rice. In addition, by testing 51 pairs of the ILP primerson five Gramineae plants and three dicot plants, we found anotherdesirable characteristic of rice ILP markers that they havehigh transferability to other plants.     

Gene content and organization of the oat mitochondrial genome

The physical organization of the oat mitochondrial genome has been established. The master chromosome, one of the most complex described so far among higher plants, accounts for 596 kb and contains six direct repeats. Reiterated inverted repeats of 12 and 3 kb are also present and imply the possible existence of multiple isomeric forms. Fourteen genes coding for proteins, components of chain respiration and oxidative phosphorylation complexes, and of mitochondrial ribosomes have been detected together with rrn26, rrn18 and rrn5 genes and a set of 18 tRNA genes (ten genuine and eight cp-like). Some of them are clustered in a conserved form with respect to other monocots. Only the trnS (GGA) gene is silent. Received: 26 October 2000 / Accepted: 24 November 2000     

Sequence composition, organization, and evolution of the core Triticeae genome

We investigated the composition and the basis of genome expansion in the core Triticeae genome using Aegilops tauschii, the D-genome donor of bread wheat. We sequenced an unfiltered genomic shotgun (trs) and a methylation-filtration (tmf) library of A. tauschii, and analyzed wheat expressed sequence tags (ESTs) to estimate the expression of genes and transposable elements (TEs). The sampled D-genome sequences consisted of 91.6% repetitive elements, 2.5% known genes, and 5.9% low-copy sequences of unknown function. TEs constituted 68.2% of the D-genome compared with 50% in maize and 14% in rice. The DNA transposons constituted 13% of the D-genome compared with 2% in maize. TEs were methylated unevenly within and among elements and families, and most were transcribed which contributed to genome expansion in the core Triticeae genome. The copy number of a majority of repeat families increased gradually following polyploidization. Certain TE families occupied discrete chromosome territories. Nested insertions and illegitimate recombination occurred extensively between the TE families, and a majority of the TEs contained internal deletions. The GC content varied significantly among the three sequence sets examined ranging from 42% in tmf to 46% in trs and 52% in the EST. Based on enrichment of genic sequences, methylation-filtration offers one option, although not as efficient as in maize, for isolating gene-rich regions from the large genome of wheat.     

Performance of Hybrids between Weedy Rice and Insect-resistant Transgenic Rice under Field Experiments： Implication for Environmental Biosafety Assessment

Transgene escape from genetically modified (GM) rice Into weedy rice via gene flow may cause undesired environmental consequences. Estimating the field performance of crop-weed hybrids will facilitate our understanding of potential introgression of crop genes (including transgenes) into weedy rice populations, allowing for effective biosafety assessment. Comparative studies of three weedy rice strains and their hybrids with two GM rice lines containing different insect-resistance transgenes (CpTl or BtlCpTI) indicated an enhanced relative performance of the crop-weed hybrids, with taller plants, more tillers, panicles, and spikelets per plant, as well as higher 1000-seed weight, compared with the weedy rice parents, although the hybrids produced less filled seeds per plant than their weedy parents. Seeds from the F1 hybrids had higher germination rates and produced more seedlings than the weedy parents, which correlated positively with 1000-seed weight. The crop-weed hybrids demonstrated a generally enhanced relative performance than their weedy rice parents in our field experiments. These findings indicate that transgenes from GM rice can persist to and introgress into weedy rice populations through recurrent crop-to-weed gene flow with the aid of slightly increased relative fitness in F1 hybrids.     

Proteomic Analysis of the Response of Liangyoupeijiu （Super High-Yield Hybrid Rice） Seedlings to Cold Stress

Liangyoupeijiu is a super high-yield hybrid rice. Despite its advantages with respect to yield and grain quality, it is sensitive to cold, which keeps it from being widely cultivated. We subjected Liangyoupeijiu seedlings to 4 ℃ cold treatment, then extracted the leaf proteins. After 2-D gel electrophoresis separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, a series of differentially displayed proteins were identified. Some metabolism-associated proteins were found among the downregulated proteins, such as carbamoyl phosphate synthetase, transketolase 1, dihydrolipoamide dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase. The upregulated proteins included both stress-resistance proteins such as nucleoside diphosphate kinase I and proteins that are negative for rice growth, such as FtsH-like protein, plastid fusion and/or translocation factor （Pftf） and actin. Our results indicate that cold may inhibit Liangyoupeijiu growth through decreasing metabolic activity and damaging cell structure.     

DNA sequence organization and transcription of the chicken genome

A new approach has been used to examine DNA sequence organization in the chicken genome. The interspersion pattern was determined by studying the fraction of labelled DNA fragments of different lengths that hybridized to an excess of short chicken repeated DNA sequences. The results indicate that chicken DNA has a pattern of sequence organization quite different than the standard ‘Xenopus’ or ‘Drosophila’ patterns. Two classes of unique sequences are found. One, 34% of the genome, consists of unique sequences approx. 4 kb long interspersed with repeated sequences. The second, non-interspersed fraction, 38% of the genome, consists of unique sequences found in long tracts, a minimum of approx. 22 kb in length. In an attempt to determine whether a relationship exists between DNA sequence organization and the distribution of structural genes we have isolated chicken DNA sequences belonging to different interspersion classes and tested each for the presence of structural genes by hybridization to excess poly(A)⁺ mRNA. Sequences complementary to poly(A)⁺ mRNA can be found with approximately the same frequency in both the non-interspersed fraction of the genome and a repeat-contiguous fraction enriched for interspersed sequences.     

籼型光敏核不育水稻育性可转换性的基因定位和基因互作研究

本研究以来源于农垦58S的灿型光敏核不育系培矮64S（短日条件下育性难转换）和8902S（短日条件下育性蝗转换）及其F1,F2群体为材料,通过短日不同光温和不同生态条件4种处理,利用RFLP分子标记研究了影响光敏偿育水稻在短日条件下的育性可转换性的遗传,基因定位和基因互作,主要结果表明：影响光敏不育水稻的育性可转换性表现为微效基因的作用,定位了7个控制光敏核不育水稻的育性可转换性QTL,即S2,S3a,S3b,S5,S8和S10,揭示了基因互作真实存在于光敏核不育水稻中,基因互作形式和互作类型对光敏核不育水稻的育性可转换性的影响表现多种多样,不同类型的基因互作所解释的遗传变异处于2．15％－10．07％之间。     

粳稻SRAP分子标记遗传群的构建与分析

用超级稻品种‘沈农606’和普通粳稻‘丽江新团黑谷’为亲本杂交获得的102份F_2代单株,通过SRAP分子标记遗传分析,构建了包含14个连锁群,由129个多态性位点组成的水稻连锁图谱,此图谱覆盖基因组长度1671.5 cM,平均图距13.0 cM。连锁群上有17.2%的多态性位点表现偏分离,偏分离标记在连锁群上存在热点区域。     

Rice transgene flow: its patterns,model and risk management

Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10¹ and 10³ higher than that to O. rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30 years in 993 major rice producing counties of southern China. The MTD_0.1% for rice cultivars is basically ≤5 m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long‐term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F₁ hybrids of transgenic rice/O. rufipogon gradually disappeared within 3–5 years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O. rufipogon may possess a strong mechanism of exclusiveness for self‐protection. (iv) The flowering time isolation and a 2‐m‐high cloth‐screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice.     

Rice reproductive development stage thermal time and calendar day intervals for six US rice cultivars in the Grand Prairie,Arkansas, over 4 years

The rice crop's reproductive developmental timing in days and thermal time is needed for effective modelling, research interpretation and management of the crop. To obtain these data, a field study was conducted at Stuttgart, Arkansas, USA in 2007, 2008, 2009 and 2010. The study utilised data collected from randomised complete block design field experiments with three replications and six rice lines in each of the years. Averaged across years and cultivars, the degree‐day‐10 (DD10) intervals (thermal time units with a base temperature of 10°C) for Reproductive Stages R3, R4, R5, R6, R7 and R8 were 21, 30, 19, 48, 70 and 189°C‐day, respectively. The average intervals in calendar days for R3, R4, R5, R6, R7 and R8 were 2.3, 3.3, 2.3, 6.0, 4.5 and 26.7 days, respectively. For R4 and R5, cultivar rankings differed over the 4 years with cultivar differences being mostly small, non‐significant or inconsistent. For R6, the cultivar Cypress had either the longest or among the longest intervals. For R7, the medium grains had the longest or among the longest intervals. For R3 and R8, cultivar differences were significant with no significant year by cultivar interactions. For the R3 intervals, the primary difference was between Bengal and the five other lines. For R8, the intervals in both days and DD10 were least for Cypress, followed by Wells, followed by LaGrue and XL723 followed by the medium grains Bengal and Jupiter which had the longest intervals for R8. Consequently, the R3 interval could be generalised to five of the six lines in the study while R4, R5, R6 and R7 intervals could be generally applied with some caution. The R8 intervals were different among lines and grain types. These differences should not be ignored. The extremely short R8 interval for Cypress is likely associated with its high head rice yields across a range of environments compared to other long‐grain rice cultivars and hybrids in the USA The utilisation of the rice reproductive growth stage intervals can potentially improve analysis and interpretation of field plot research, model predictions and management of the rice crop.