家蚕地方品种线粒体基因组A+T丰富区的序列及分子进化分析

为了研究家蚕线粒体基因组A+T丰富区的结构和家蚕品种的进化,用PCR方法扩增了12个家蚕(Bombyxmori)地方品种线粒体基因组A+T丰富区及其侧翼序列,分离纯化后克隆到pMD18-T载体进行测序。序列分析表明,克隆片段长度约1.1 kb,基因排列顺序与C108线粒体相同,依次为12S rRNA基因3′端、A+T丰富区、tRNAMet、tRNAIle、tRNAGln和ND2基因5′端,在tRNAGln和ND2基因之间有47 bp的非编码区。以日本野桑蚕(Bombyxmandarina)为外群,用Phylip软件包构建了基于12个家蚕品种线粒体基因组A+T丰富区序列的NJ进化树。结果显示,甘肃种单独聚为一群,其进化早于其它11个品种聚成的类群,说明甘肃种是供试家蚕品种中进化最早的品种。这一结果在分子水平上为黄河流域是家蚕品种的发祥地之一提供了证据,也进一步支持了家蚕品种的中国起源说。对A+T丰富区及其侧翼基因的结构分析表明,家蚕线粒体12S rRNA和ND2基因都十分保守,14个品种统计只分别发生1个和2个碱基转换;A+T丰富区中(A+T)比例高达94.9%以上,第27 nt开始有1个T-串结构,长度为16~19 bp不等;同时还根据3个tRNA基因的核苷酸序列推定了其二级结构。     

多枝赖草高分子量核DNA的有效制备

以多枝赖草Leymus multicaulis黄化苗幼嫩叶片为材料,在液氮中研磨成粉末,加入冰冷的细胞核抽提缓冲液,经一系列钢制细胞筛过滤,离心分离细胞核,相差显微镜镜检后,用低熔点琼脂糖包埋,蛋白酶K原位裂解,经脉冲场电泳分离,用1%低熔点琼脂糖回收获得了较纯净的分子量大于2 Mb的核DNA,Southern杂交显示没有叶绿体和线粒体DNA的污染.利用该方法制备的高分子量核DNA,可用于后续可转化人工染色体(TAC)文库的构建和基因组分析.     

Canine oral melanoma

Melanoma is the most common oral malignancy in the dog. Oral and/or mucosal melanoma has been routinely considered an extremely malignant tumor with a high degree of local invasiveness and high metastatic propensity. Primary tumor size has been found to be extremely prognostic. The World Health Organization staging scheme for dogs with oral melanoma is based on size, with stage I = <2-cm-diameter tumor, stage II = 2- to <4-cm-diameter tumor, stage III = > or = 4cm tumor and/or lymph node metastasis, and stage IV = distant metastasis. Median survival times for dogs with oral melanoma treated with surgery are approximately 17 to 18, 5 to 6, and 3 months with stage I, II, and III disease, respectively. Significant negative prognostic factors include stage, size, evidence of metastasis, and a variety of histologic criteria. Standardized treatments such as surgery, coarse-fractionation radiation therapy, and chemotherapy have afforded minimal to modest stage-dependent clinical benefits and death is usually due to systemic metastasis. Numerous immunotherapeutic strategies have been employed to date with limited clinical efficacy; however, the use of xenogeneic DNA vaccines may represent a leap forward in clinical efficacy. Oral melanoma is a spontaneous syngeneic cancer occurring in outbred, immunocompetent dogs and appears to be a more clinically faithful therapeutic model for human melanoma; further use of canine melanoma as a therapeutic model for human melanoma is strongly encouraged. In addition, the development of an expanded but clinically relevant staging system incorporating the aforementioned prognostic factors is also strongly encouraged.     

Capsicum tovarii, a new member of the Capsicum baccatum complex

Interspecific hybrid performance and meiotic chromosome behavior of F₁ hybrids were studied to elucidate the genetic relationship between C. tovarii and the other Capsicum species. C. tovarii was hybridized, as a female and a male parent, to C. annuum, C. chinense, C. frutescens, C. chacoense, C. galapogense, C. baccatum, C. praetermissum, C. cardenasii, C. eximium and C. pubescens. When the hybridization of C. baccatum × C. tovarii was performed, F₁, F₂ and backcross progenies were successfully obtained. In addition, a successful hybridization of C. praetermissum × C. tovarii was also obtained. A cytological investigation of F₁ hybrids of C. baccatum × C. tovarii revealed that most meiotic chromosomes paired as bivalents. However, multivalents, chromosome bridges, and chromosome lags were observed. These results suggest that C. baccatum differs from C. tovarii by at least a chromosomal reciprocal translocation. Crosses of C. tovarii to C. chinense and C. frutescens produced plump seeds, but none of the seeds germinated. Hybridizations of C. tovarii to C. pubescens, C. eximium and C. cardenasii did not produce seed. These hybridization results indicate that C. tovarii is genetically more closely related to the C. baccatum complex than to the C. annuum complex or the C. pubescens complex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Feulgen-DNA densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex

The chromosomes of sexual diploid plants of Paspalum simplex were colchicine-doubled and the plant obtained were crossed with their aposporous natural tetraploid counterparts to generate a F1 population segregating for apomixis. Analysis of the DNA content during megagametogenesis indicated that although the nuclei of nucellus and developing embryo sacs were in both the G1 and G2 phases, polar nuclei and egg cells of mature embryo sacs tended to remain in the G1 phase. Because both meiotic and aposporous mature embryo sacs are of the 8-nucleated-type in P. simplex and are barely distinguishable, nuclear DNA content of polar nuclei was used to distinguish apomictic and sexual phenotypes and confirmation obtained by progeny testing. This revised version was published online in August 2006 with corrections to the Cover Date.     

DNA amplification fingerprinting and marker screening for pseudo-testcross mapping of flowering dogwood ( Cornus florida L.)

DNA amplification fingerprinting (DAF) with arbitrary oligonucleotide primers was used to study genetic relationships between cultivars of flowering dogwood (Cornus florida L.), evaluate extent of plant hybridization, and generate markers in pseudo-testcross mapping at the intraspecific level. Modified Taguchi optimization methods defined a robust DAF system based on high annealing temperature (48–52 °C) and primer concentration (typically 8 μM) that was used to study genetic diversity of representative dogwood cultivars and hybrids. Phenetic analysis using cluster and numerical methods showed that: (1) cultivars were relatively conserved at the genetic level; (2) their hybridization could be identified in the F1 progeny in the absence of phenotypic or physiological markers; (3) several cultivars grouped according to their recorded ancestry; and (4) dogwood anthracnose-resistant lines originally selected in Catoctin Mountain Park (Maryland) grouped separately from those of southern origin. The DAF protocol was also tested in pseudo-testcross mapping of dogwood at the intraspecific level. A preliminary screening of parents ‘Pink Sachet’ and ‘Fragrant Cloud’ and 7 F1 segregants with 22 octamer primers produced 703 amplified loci, 30 and 39 of which were male and female markers segregating at 1:1 ratios with 98.6% confidence levels in pseudo-testcross configuration. Overall results show that DAF generated markers very efficiently (3 per primer) despite the close relatedness of parental dogwood cultivars. This study constitutes the basis for a future genetic linkage mapping and marker-assisted selection (MAS) effort initially targeted to control important fungal diseases in dogwood. This revised version was published online in July 2006 with corrections to the Cover Date.     

四种竹子的RAPD指纹图谱的初步研究

本实验以RAPD技术对考顺竹、凤尾竹、绿竹、白绿竹四个品种用20种已知序列的10nt引物进行PCR反应扩增。其中有17种可扩增出DNA条带,构成RAPD指纹图谱。各种引物PCR扩增的DNA条带数目在0—6条之间,大小在0．3—2．0kb之间。各种竹子的17种引物扩增的DNA条带总数在23—44条之间。四种竹子两两之间的相似系数在36—73％之间。     

RFLP analysis of a PCR amplified region of chloroplast DNA in eggplant and related Solanum species

RFLP analysis of a PCR amplified 3.2-kbp region of cpDNA bounded by the conserved sequences in rbc L and ORF 106 was performed in eggplant (Solanum melongena), its related Solanum species, S. incanum, S. virginianum (= S. surattense), S. torvum, S. aethiopicum (= S. gilo), S. aethiopicum (= S. integrifolium), S. violaceum (= S. indicum), S. violaceum (= S. sanitwongsei) and S. mammosum and the reciprocal hybrids between S. aethiopicum (= S. integrifolium) and S. melongena 'Uttara'. The target region of cpDNA was amplified correctly by PCR. The amplified products were digested with each of 10 restriction enzymes (Alu I, Ase I, BamH I, Hinf I, Msp I, Rsa I, ScrF I, Sty I, Taq I and Xba I). Variations of restriction patterns among the species were recognized after digesting the amplified products with each of the seven restriction enzymes, Taq I, Alu I, Rsa I, Sty I, Ase I, Hinf I and Xba I. The restriction patterns divided the examined nine species into the following five clusters, 1) S. melongena and S. incanum, 2) S. virginianum (= S. surattense), 3) S. torvum, 4) S. aethiopicum (= S. gilo), S. aethiopicum (= S. integrifolium), S. violaceum (= S. indicum) and S. violaceum (= S. sanitwongsei) and 5) S. mammosum. The restriction pattern with Alu I in each of the reciprocal hybrids between S. melongena 'Uttara' and S. aethiopicum (= S. integrifolium) was identical with that of seed parent. The present study demonstrated the availability of the PCR-RFLP analysis of cpDNA for assessing taxonomic relationships and identifying cytoplasmic parentage of interspecific hybrids in eggplant and related Solanum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low level of polymorphism detected by SSR probes in bread wheat

In-gel hybridization patterns were studied in a set of nine diverse bread wheat ( Triticum aestivum L. em. Thell) genotypes using 23 simple sequence repeat (SSR) probes in combination with 14 different restriction enzymes. Multilocus fingerprints due to SSR probes, shown earlier to be characteristic of a majority of plant genomes, were not obtained and only a very low level of polymorphism was detected when using as many as 142 probe-enzyme combinations. The hybridization of a prominent solitary high molecular weight fragment (> 23 kb) with a number of SSR probes suggested the presence of these SSRs (microsatellites) within the long stretches of repeated DNA sequences. This indicates that the genome of bread wheat differs from that of other plants in the organization and distribution of SSRs and that SSR probes detect very little polymorphism.     

Genetic diversity of fast-and slow-growing soybean rhizobia determined by random amplified polymorphic DNA analysis

The genetic relationships among six strains of rhizobia, including three strains of Rhizobium fredii and three strains of Bradyrhizobium japonicum, was determined using random amplified polymorphic DNA (RAPD) technique. In this study, 46 arbitrary 10mer primers were employed for RAPD, generating a total of 251 informative fragments. A dendrogram of phylogenetic relationships among the six strains was constructed. The results indicated that geographical distribution may affect phylogeny, as there were closer relationships among the four Taiwanese strains, SB138, SB562, SB368 and SB651, than between these strains and USDA192, which originated from mainland China. The strain USDA110, obtained from the United States, was used in the parsimony analysis. The greatest similarity (55.6%), existed between two strains of B. japonicum, SB562 and SB138, which both, and the lowest R. fredii (44.4%) between two strains of R. fredii, SB368 and USDA192. We also found a RAPD marker specific to the four Taiwanese SB strains used in the study. The RAPD technique is a potential tool for the identification of the genetics and systematics of different populations. Received: 23 January 1997