一种单核苷酸多态性的单倍型分析技术

运用多步PCR和测序技术,完成基因组中相距较远的单核苷酸多态位点的单倍型构建。通过设计2条等位基因特异性引物,扩增大片段DNA(10kb左右),以此大片段DNA作为下一轮PCR反应的模板,再在该片段中设计待检测区域的PCR引物,进行第2轮PCR。对PCR产物进行测序分析,确定其多态位点处的等位基因。结合第1轮PCR中的等位基因特异性引物,即可确定该大片段DNA中不同单核苷酸多态性构成的单倍型。以脂蛋白脂酶基因为例,应用其启动子区以及第4外显子区的等位基因特异性引物扩增约16kb的DNA片段,然后检测位于该片段中第2、3外显子的多态性。在￡-尸￡-基因第2内含子中发现了 13557G→A多态性。经分析确定出-421G／ 13557G／ 15222A、-421A／ 13557G／ 15222A、-421G／ 13557G／ 15222G、-421G／ 13557A／ 15222A等4种单倍型。等位基因特异性PCR结合小片段测序是一种快捷高效的对相距较远的多个SNP进行单倍型构建的新策略。     

Neorhodomela enomotoi sp. nov. (Ceramiales, Rhodophyta) from Japan

The red alga Neorhodomela enomotoi Masuda et Kogame (Rhodomelaceae, Ceramiales) is described as a new species from Japan. It is characterized by the following combination of features: (i) thalli reddish-brown and slightly rigid; (ii) first-order branches mostly indeterminately branched; (iii) adventitious branches infrequent, determinately branched and formed chiefly in the axils of lateral branches; (iv) vegetative trichoblasts abundant; (v) tetrasporangia produced on paniculate ultimate and penultimate branches; and (vi) cystocarps flask-shaped (urceolate). This species has been found growing only in the upper subtidal zone in the warm temperate waters of Japan.     

A rearranged chamigrene derivative and its potential biogenetic precursor from a new species of the marine red algal genus Laurencia (rhodomelaceae)

The structures of a novel rearranged sesquiterpenoid and a biogenetically-related chamigrene derivative have been determined by combined spectral and chemical methods. These sesquiterpenoids were components of an undescribed Laurencia species, and each was toxic toward the damselfish Pomacentrus coeruleus.     

Haplotype variation of Green Revolution gene Rht-D1 during wheat domestication and improvement

Green Revolution made a substantial contribution to wheat yields worldwide in the 1960 s and 1970 s. It is of great importance to analyze the haplotype variation of Rht-D1, the Green Revolution gene, during wheat(Triticum aestivum L.)domestication and breeding to understand its evolution and function in wheat breeding history. In this study, the Rht-D1 and its flanking regions were sequenced and single nucleotide polymorphisms were detected based on a panel of 45 accessions of Aegilops tauschii, 51 accessions of landraces and 80 accessions of commercial varieties. Genetic diversity in the wild accessions was much higher than that in the varieties and higher than that reported previously. Seven haplotypes(Hapl I to Hapl VII) of Rht-D1 were identified and their evolutionary relationships were proposed. In addition to the well-known Green Revolution allele Rht-D1b, Hapl VII(an allele Rht-D1k) was identified in early breeding varieties, which reduced plant height by 16%. The results suggested that Rht-D1k had been used in breeding before the Green Revolution and made a great contribution to wheat production worldwide.Based on the breeding history and molecular evidence, we proposed that the wheat Green Revolution in China and International Maize and Wheat Improvement Center(CIMMYT)occurred independently.     

Estimation of the number of SNP genetic markers required for parentage verification

Formulae were developed to compute exclusion probabilities for parentage confirmation for any number of diallelic markers under the assumption that the minor allele frequency (MAF) varied among markers, but has a uniform distribution. Three scenarios were analysed: a progeny with (1) a single putative parent; (2) two putative parents; and (3) one actual parent and one putative parent. Exclusion probabilities were computed for minimum values for the MAFs of 0.1, 0.2 and 0.3, and required either one or at least two conflicts for exclusion. The numbers of markers required to obtain 99% exclusion probabilities based on a single conflict for the three minimum MAFs were 54, 45 and 39 for scenario 1; 17, 16 and 15 for scenario 2; and 28, 25 and 24 for scenario 3. The requirement of at least two conflicts for exclusion increased the number of markers required by approximately 45% for all three scenarios and all three minimum MAFs. The results obtained by the analytical formulae were very close to results obtained by simulation and to values in the literature for specific marker sets.     

Hybridization studies in Bostrychia. 1: B. radicans (Rhodomelaceae, Rhodophyta) from Pacific and Atlantic North America

Bostrychia radicans(Montagne) Montagne is a pantropical/temperate red alga associated with mangroves and saltmarsh plants. Collections were made from a similar north-south geographic distribution along both the Pacific and Atlantic coasts of North America. Hybridization studies were performed with cultured isolates to assess the extent of interfertility and reproductive isolation along these two coastlines. All male and female gametophytes derived from single tetrasporophytes were intercompatible. Almost all isolates extending over 1500 km of coast line from northern Pacific Mexico are compatible, forming cystocarps that released viable carpospores. Even isolates which morphologically would be placed in two species [B. radicans and B. moritziana(Sender ex Kützing) J. Agardh], based on the presence or absence of monosiphonous branches, were capable of hybridizing. Crosses of isolates from the Atlantic USA showed a greater amount of incompatibility. Certain isolates were not compatible with any other isolates including isolates collected in close proximity (North Carolina isolates), while other isolates from the same locality were compatible (South Carolina). An isolate from South Carolina formed tetrasporophytes with isolates from Pacific Mexico but tetraspores were not viable. Certain incompatible crosses formed ‘pseudocystocarps’ but viable carposporophytes did not develop. Generalizations about reproductive isolation within a species must also consider differences between populations from different biogeographic regions that may reflect different paleoclimatological histories, founder effects and unique dispersal events.     

Using blocks of linked single nucleotide polymorphisms as highly polymorphic genetic markers for parentage analysis

Single nucleotide polymorphisms (SNPs) are plentiful in most genomes and amenable to high throughput genotyping, but they are not yet popular for parentage or paternity analysis. The markers are bi-allelic, so individually they contain little information about parentage, and in nonmodel organisms the process of identifying large numbers of unlinked SNPs can be daunting. We explore the possibility of using blocks of between three and 26 linked SNPs as highly polymorphic molecular markers for reconstructing male genotypes in polyandrous organisms with moderate (five offspring) to large (25 offspring) clutches of offspring. Haplotypes are inferred for each block of linked SNPs using the programs Haplore and Phase 2.1. Each multi-SNP haplotype is then treated as a separate allele, producing a highly polymorphic, 'microsatellite-like' marker. A simulation study is performed using haplotype frequencies derived from empirical data sets from Drosophila melanogaster and Mus musculus populations. We find that the markers produced are competitive with microsatellite loci in terms of single parent exclusion probabilities, particularly when using six or more linked SNPs to form a haplotype. These markers contain only modest rates of missing data and genotyping or phasing errors and thus should be seriously considered as molecular markers for parentage analysis, particularly when the study is interested in the functional significance of polymorphisms across the genome.