Inferring the History of the Polyploid Silene aegaea (Caryophyllaceae) Using Plastid and Homoeologous Nuclear DNA Sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.     

利用RFLP分子标记确定导入小麦的鹅观草(R. kamoji)染色体的部分同源群归属

选用来自小麦族7个部分同源群的26个DNA探针对45个小麦-鹅观草衍生后代株系及鹅观草、中国春和扬麦5号亲本进行RFLP分析,结果表明16个小麦-鹅观草异附加系、异代换系或可能的易位系中所涉及鹅观草染色体分别属于第1、3、5、6、7部分同源群。小麦-鹅观草异染色体系中导入的成对鹅观草染色体能够较稳定地遗传给后代。K139、K141、K214、K218、K219、K224二体附加系所添加的鹅观草染色体属第1部分同源群,但K214和K218所添加的鹅观草染色体与K219、K224的添加的鹅观草染色体分别来自鹅观草不同的染色体组。K147端体添加系涉及鹅观草第1部分同源群染色体长臂,而K139、K141和K147所涉及的鹅观草染色体长臂分别来自鹅观草3个不同的染色体组。鹅观草U染色体与小麦第1部分同源群有同源关系,属第1部分同源群的鹅观草染色体尤其是其长臂与赤霉病抗性有关。鹅观草第1部分同源群与第6部分同源群染色体之间可能涉及重排。K203添加的2条鹅观草染色体分别与第1和6部分同源群同源。K166导入鹅观草染色体涉及第5部分同源群短臂。K177（2n=41,20Ⅱ I）中,所渗入的鹅观草染色质涉及第5（5L）、6（6S）、7（SL）部分同源群。鹅观草S、H和Y3个染色体组间具部分同源性。     

Differential Replication of DNA Sequences in Drosophila Chromosomes

During the DNA replications involved in polyploidization orpolytenization in Drosophila cells, not all DNA sequences arereplicated to the same extent. Cytological studies have demonstratedthat certain chromosome regions, such as the a-heterochromatin,are in some cells under-replicated and in other cells not replicatedat all. Similarly, such DNA fractions as the highly repeatedsatellite DNAs are also under- or non-replicated in polyploidand polytene cells. The genes for rRNA in polytene cells replicateone to three rounds less than the euchromatic DNA and are capableof differential synthesis to compensate for deficiencies. Thedifferential replication of DNA sequences indicates that thereis regulation of DNA replication at a level intermediate betweenthe replicon and the entire genome. Chromosomes of terminallydifferentiated polyploid or polytene cells have several domainsof DNA sequences, which in replication are controlled as units.This paper, which reviews the literature on differential replicationof DNA in Drosophila, discusses possible controls over thisprocess.     

A Possible Mechanism for Seed Coat Rupture by the Embryo in Germinating Wheat

The process of seed-coat splitting, terminating germination,in red wheats is described. We show that sequential expansionof the coleorhiza and coleoptile is required for germinativegrowth to be normal. Normal emergence requires the seed-coatsto be split by the expanding coleorhiza. We here describe agroup of cells associated with the coleorhizal tip forming apapilla-like projection which could aid this process. Embryo, germination, development, wheat, Triticum aestivum     

一种适于PCR扩增的小麦基因组DNA快速提取法

许多小麦分子生物学研究需要对大量的小麦样品进行PCR检测,因此,建立一种快速提取小麦基因组DNA的方法十分必要。根据国外报道的一种快速提取水稻和玉米基因组DNA的方法,我们对部分提取步骤进行变动后,在小麦上进行了尝试,长度为1．5kb的片段能得到稳定的扩增。该方法样品研磨在1.5ml的离心管内进行,后续操作不用酚、氯仿、CTAB、SDS和巯基乙醇,整个提取过程不需要使用通风橱,操作步骤简单,花费时间少,而且提取的小麦基因组DNA完整性好,量也较可观。一个DNA样品可供50～100次PCR反应使用,适用于小麦遗传多样性、分子标记辅助选择、转基因后代检测以及引物筛选、分子标记定位等多种研究。     

Nonstructural Chromosome Differentiation among Wheat Cultivars, with Special Reference to Differentiation of Chromosomes in Related Species

Wheat cultivar Chinese Spring (Triticum aestivum L. em. Thell.) was crossed with cultivars Hope, Cheyenne and Timstein. In all three hybrids, the frequencies of pollen mother cells (PMCs) with univalents at metaphase I (MI) were higher than those in the parental cultivars. No multivalents were observed in the hybrids, indicating that the cultivars do not differ by translocations. Thirty-one Chinese Spring telosomic lines were then crossed with substitution lines in which single chromosomes of the three cultivars were substituted for their Chinese Spring homologues. The telosomic lines were also crossed with Chinese Spring. Data were collected on the frequencies (% of PMCs) of pairing of the telesomes with their homologues at MI and the regularity of pairing of the remaining 20 pairs of Chinese Spring chromosomes in the monotelodisomics obtained from these crosses. The reduced MI pairing in the intercultivar hybrids was caused primarily by chromosome differentiation, rather than by specific genes. Because the differentiation involved a large part of the chromosome complement in each hybrid, it was concluded that it could not be caused by structural changes such as inversions or translocations. In each case, the differentiation appeared to be unevenly distributed among the three wheat genomes. It is proposed that the same kind of differentiation, although of greater magnitude, differentiates homoeologous chromosomes and is responsible, together with structural differentiation, for poor chromosome pairing in interspecific hybrids.     

A Sister-Strand Exchange Mechanism for Reca-Independent Deletion of Repeated DNA Sequences in Escherichia Coli

In the genomes of many organisms, deletions arise between tandemly repeated DNA sequences of lengths ranging from several kilobases to only a few nucleotides. Using a plasmid-based assay for deletion of a 787-bp tandem repeat, we have found that a recA-independent mechanism contributes substantially to the deletion process of even this large region of homology. No Escherichia coli recombination gene tested, including recA, had greater than a fivefold effect on deletion rates. The recA-independence of deletion formation is also observed with constructions present on the chromosome. RecA promotes synapsis and transfer of homologous DNA strands in vitro and is indispensable for intermolecular recombination events in vivo measured after conjugation. Because deletion formation in E. coli shows little or no dependence on recA, it has been assumed that homologous recombination contributes little to the deletion process. However, we have found recA-independent deletion products suggestive of reciprocal crossovers when branch migration in the cell is inhibited by a ruvA mutation. We propose a model for recA-independent crossovers between replicating sister strands, which can also explain deletion or amplification of repeated sequences. We suggest that this process may be initiated as post-replicational DNA repair; subsequent strand misalignment at repeated sequences leads to genetic rearrangements.     

BAC-Pool Sequencing and Analysis of Large Segments of A12 and D12 Homoeologous Chromosomes in Upland Cotton

Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.     

Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes)

Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.     

利用RFLP揭示普通小麦与野生二粒小麦间A和B组染色体的遗传分化

To investigate chromosome differentiation of genome A and B between common wheat and wild emmer wheat ( Triticum turgidum var. dicoccoides (Koern.) Bowden), the authors conducted a RFLP analysis of the two species using 153 genomic, cDNA and chromosome-specific probes. 75.8% of the probes had detected hybridization polymorphism in at least one of the five restriction enzymes. However, the polymorphic probes were unevenly distributed among different homoeologous groups, between different genomes and in different regions of a single chromosome. Homoeologous group 1 possessed the highest level of polymorphism (96.2%), followed by group 6 and 2 (84.6% and 82.1% respectively). In contrast, only 60%-67% of probes of the other four groups was polymorphic. In most groups the number of probes capable of detecting B chromosome polymorphism was slightly higher than that revealing A chromosome difference (totally 51.8% vs 43.1%). In a single chromosome, RFLP was predominant in the distal region (65.1%) and showed a decreasing trend from the proximal (46.2%) to the pericentric (42.4%) regions. The results suggest that there exists a substantial amount of DNA polymorphism between the A and B chromosomes of common wheat and those of wild emmer wheat, indicating that a considerable degree of genetic differentiation has taken place in the A and B genoms of two species during evolution from wild emmer to common wheat. The extent of the genetic differentiation may vary among different homoeologous groups, between A and B chromosomes and in different regions of individual chromosome.     

利用RFLP揭示普通小麦与野生二粒小麦间A和B组染色体的遗传分化

普通小麦是栽培二粒小麦（Ｔｒｉｔｉｃｕｍ ｔｕｒｇｉｄｕｍ ｖａｒ．ｄｉｃｏｃｃｕｍ Ｓｈｒａｎｋ ｅｘ Ｓｅｈｕｂｌｅｒ）与粗山羊草（Ｔ．ｔｏｕｓｃｈｉｉ Ｃｕｓｓ,）天然杂交并自然加倍的产物,而栽培二粒泪科是由野生二粒小麦（Ｔ．ｔｕｒｇｉｄｕｍ ｖａｒ．ｄｉｃｏｃｃｏｉｄｅｓ（Ｋｏｅｒｎ？）Ｂｏｗｄｅｎ）进行而来,从野生二粒注麦到普通泪科的进化过程中其遗传物质可能发生了许多变。以普通小麦－野生     

IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

A Simple and Rapid Aqueous Method for the Purification of DNA from Wheat Chloroplast

We report here a simple and rapid method for the purification of chloroplast DNA (ctDNA) from wheat (Triticum aestivum). It utilizes an aqueous procedure, which does not involve at any stage running of gradients. Due to use of DEPC which inactivates DNases activated by EDTA, the DNase action on crude chloroplast preparation containing ctDNA is avoided.     

Boom-Bust Turnovers of Megabase-Sized Centromeric DNA in Solanum Species: Rapid Evolution of DNA Sequences Associated with Centromeres

Centromeres are composed of long arrays of satellite repeats in most multicellular eukaryotes investigated to date. The satellite repeat–based centromeres are believed to have evolved from “neocentromeres” that originally contained only single- or low-copy sequences. However, the emergence and evolution of the satellite repeats in centromeres has been elusive. Potato (Solanum tuberosum) provides a model system for studying centromere evolution because each of its 12 centromeres contains distinct DNA sequences, allowing comparative analysis of homoeologous centromeres from related species. We conducted genome-wide analysis of the centromeric sequences in Solanum verrucosum, a wild species closely related to potato. Unambiguous homoeologous centromeric sequences were detected in only a single centromere (Cen9) between the two species. Four centromeres (Cen2, Cen4, Cen7, and Cen10) in S. verrucosum contained distinct satellite repeats that were amplified from retrotransposon-related sequences. Strikingly, the same four centromeres in potato contain either different satellite repeats (Cen2 and Cen7) or exclusively single- and low-copy sequences (Cen4 and Cen10). Our sequence comparison of five homoeologous centromeres in two Solanum species reveals rapid divergence of centromeric sequences among closely related species. We propose that centromeric satellite repeats undergo boom-bust cycles before a favorable repeat is fixed in the population.     

Repeated Polyploidy Drove Different Levels of Crossover Suppression between Homoeologous Chromosomes in Brassica napus Allohaploids

Allopolyploid species contain more than two sets of related chromosomes (homoeologs) that must be sorted during meiosis to ensure fertility. As polyploid species usually have multiple origins, one intriguing, yet largely underexplored, question is whether different mechanisms suppressing crossovers between homoeologs may coexist within the same polyphyletic species. We addressed this question using Brassica napus, a young polyphyletic allopolyploid species. We first analyzed the meiotic behavior of 363 allohaploids produced from 29 accessions, which represent a large part of B. napus genetic diversity. Two main clear-cut meiotic phenotypes were observed, encompassing a twofold difference in the number of univalents at metaphase I. We then sequenced two chloroplast intergenic regions to gain insight into the maternal origins of the same 29 accessions; only two plastid haplotypes were found, and these correlated with the dichotomy of meiotic phenotypes. Finally, we analyzed genetic diversity at the PrBn locus, which was shown to determine meiotic behavior in a segregating population of B. napus allohaploids. We observed that segregation of two alleles at PrBn could adequately explain a large part of the variation in meiotic behavior found among B. napus allohaploids. Overall, our results suggest that repeated polyploidy resulted in different levels of crossover suppression between homoeologs in B. napus allohaploids.     

A Rapid HCl/Toluidine Blue Squash Technic for Plant Chromosomes

Fresh or pretreated root tips are simultaneously fixed and hydrolysed in 5 N HC1 for 15 min at room temperature. After washing they are macerated on a slide in a drop of 0.05% toluidine blue made up in McIlvaine citric acid-Na₂HPO, buffer at pH 4.0. Pressure on the cover slip completes the squash preparation. It is made permanent by removing the cover slip on dry ice, air drying and mounting in Euparal.