黄河上游青海沙蜥种组的系统地理学研究

沙蜥属（Phrynocephalus）的卵胎生类群主要分布在我国青藏高原，包括南疆沙蜥（P. forsythii）、西藏沙蜥（P. theobaldi）、红尾沙蜥（P. erythrurus）、贵德沙蜥（P. putjatia）和青海沙蜥（P. vlangalii）。其卵胎生生殖方式适应了高寒生境，与青藏高原隆升有关。纵观前人的研究，上述几种卵胎生沙蜥的分类、系统发育关系以及生物地理都还存在疑问。本文研究了分布在若尔盖湿地的青海沙蜥红原亚种（P. v hongyuanensis）以及分布在黄河上游其它地区青海沙蜥种组的地理分布格局，并探讨了其形成机制。 青海沙蜥在黄河上游主要分布于若尔盖湿地以及青海湖周边地区。若尔盖湿地青海沙蜥红原亚种的生境由于沼泽的形成被切割成不连续的斑块，通过遗传分析可以推测这种特殊生境对它们遗传结构的影响。其次，贵德沙蜥、青海沙蜥的青海湖周边各居群以及若尔盖湿地居群之间的系统地理格局还未见报道。因此本文以居群为单位，将它们作为一个复合体，通过系统地理研究，可以了解其种群遗传结构，据此分析相关的地质历史事件对其分布的影响。主要结果如下： 1. 若尔盖湿地青海沙蜥红原亚种的种群遗传结构： 共研究了三个地理单元（红原（HY）、辖曼（XM）、玛曲（MQ））的7个采集点的72个个体。所有ND4-tRNALeu序列比对得到785 bp的片断，定义了9种单倍型。结果显示总的核苷酸多样性较低，单倍型多样性较高。分子变异分析（AMOVA）显示3个单元间差异显著（P<0.01），遗传变异主要存在于地理单元间，占62.61%。除MQ单元，XM各居群与HY居群混杂在一起，单倍型网络图没有显示出单倍型和地理位置的对应关系。XM单元单倍型的不配对分布（Mismatch distribution）为明显左移的单峰，且Fu’s Fs test为负值，表明XM单元可能经历了近期种群扩张，有足够的时间积累单倍型的多态性，还不足以大幅提高核苷酸多样性，这是其单倍型多样性较高和核苷酸多样性较低的原因。MQ单元遗传多样性低而与其他单元显著分化，推测这与3万年前黄河在若尔盖玛曲之间贯通有关。近期沼泽的形成对XMb居群的隔离时间短，使得其遗传多样性低但还不足以形成大的遗传差异。无论黄河的贯通还是沼泽的形成其隔离形成的时间都不长，其作用改变了单倍型出现的频率，也出现了一些特有单倍型，但共享单倍型还广泛存在，还不足以使得不同居群之间形成较大的遗传距离。 2. 黄河上游青海沙蜥种组的分布格局与地史过程的关系： 黄河上游青海沙蜥种组包括贵德沙蜥、青海沙蜥指名亚种的青海湖周边各居群、青海沙蜥红原亚种若尔盖湿地居群、以及青海湖以西的部分居群（序列由Genbank下载获得），总计22个居群189个样品。所有ND4-tRNALeu序列比对得到703个位点，定义了39种单倍型。以南疆沙蜥为外群构建的贝叶斯树以及MP法构建的无根树，都分为A、B两大组。其中A包括若尔盖湿地居群以及玛多居群（A1）、青海湖以西的居群和兴海居群（A2）、西藏沙蜥；B包括青海湖以南的居群和天祝居群（B1）、青海湖以东北的居群（B2）。单倍型网络图分别对应了系统发育树上的各支。按照系统发育结果分组进行分子变异分析，得到组间变异占88.63％，各组间差异显著（P=0.000）。种群遗传结构分析得到，A1和B2可能经历了近期的种群扩张，前者扩张时间约为0.105－0.189 Ma B.P.（million years before present），后者为0.057－0.102 Ma B.P.，可能与末次间冰期的气候变暖有关。A2和B1对应的两个地理单元都具有较强的种群遗传结构，较为稳定。 青海沙蜥种组A、B两大支之间遗传距离大，分化明显，分化大约发生在4.29－2.38 Ma B.P.，推测青藏运动的A幕运动后复杂的地形变化可能是它们产生分化的原因。B1和B2分化大约发生在1.73－0.96 Ma B.P.，这与湟水流域构造运动发生的时间相符。在早、中更新世时期，B1支内部各居群可能有交流，中更新世末共和盆地出现的抬升以及河流溯源改道等事件可能是引起这支内部多个单倍型丢失的原因。A1、A2支的分化可能与倒数第三次冰期降临之后气候变冷、阿尼玛卿山的大冰帽有关。 The viviparous group of genus Phrynocephalus is mainly distributed in the Qinghai –Tibetan Plateau, including P. forsythii、P. theobaldi、P. erythrurus、P. putjatia and P. vlangalii. These species are adapted well to the cold clime there, and the origin of this group was the result of a vicariance event associated with the uplifting of the Qinghai -Tibetan Plateau. Although many works have been done, there are still several questions about classification、phylogenetic relationships and the biogeography of this group. The phylogeographic pattern of the P. vlangalii complex on the upper reaches of the Yellow River and the P. v. hongyuanensis in Zoige Wetland were studied in this thesis. On the upper reaches of the Yellow River, P. vlangalii complex are distributed in Zoige Wetland and the southeast and northeast region of Kuku-noor Lake. Because of the forming of the wetland in Zoige, the habitats for sand lizards are divided into many discontinuous ones, and it is necessary to analyze genetic structure in these unique habitats. The phylogeographic patter among P. putjatia、populations of P. vlangalii in the southeast region of Kuku-noor Lake and populations of P. vlangalii in Zoige Wetland hasn’t been studied yet, and the complicated geological events of the Plateau may play an important role in the populations’ diversity and species forming there. So these populations were gathered as a complex, and phylogeographic analysis were used to clarify these doubts. According to the two topics above, this thesis has two parts of results as follows: 1. Three geographic units of P. vlangalii hongyuanensis in Zoige Wetland were defined, and they were Xiaman （XM）、Hongyuan （HY） and Maqu （MQ）. 785bp fragments of the mtDNA ND4-tRNAleu were determined from 72 samples and nine haplotypes were identified. As a whole, the nucleotide diversity was low，but the haplotype diversity was high. Analysis of molecular variance （AMOVA） showed that the three units were distinctly different（P<0.01），and 62.61% of the total genetic diversity was attributable to variation among units. There were 3 haplotypes shared among XM and HY，and no geographic clustering was observed except MQ from the TCS network. The results from the mismatch distribution analysis and Fu’s Fs test implied that there might be a recent population expansion in the XM unit, and this may be the reason why XM had a high haplotype diversity but a low nucleotide diversity. We estimate that the MQ and XMb have lower diversities because of some very recent geographic events, such as the formation of the Yellow river’s upriver and the Zoige Wetland. Although they are distinctly different, not enough time has passed for them to have diverged a great genetic distance. 2. 189 samples in 22 populations of P. vlangalii complex were collected, including P. putjatia、populations of P. vlangalii in the southeast and northeast region of Kuku-noor Lake、 populations of P. vlangalii in Zoige Wetland and the data from Genbank. 703bp ND4-tRNALeu sequences identified 39 haplotypes. P. forsythii was selected as outgroup, and both the Bayesian tree and the MP unrooted tree were divided into two groups（A、B）. A included populations in Zoige Wetland and Xinghai（A1）、populations in the west of Kuku-noor Lake（A2）、P. theobaldi, and B included populations in the southeast of Kuku-noor Lake and Tianzhu（B1）、populations in the northeast of Kuku-noor Lake（B2）. The haplotype network agreed with these groups. AMOVA showed that these five groups were distinctly different（P<0.01）, and 88.63% of the total genetic diversity was attributable to variation among groups. There might be recent population expansion in A1 and A2, which corresponded to the dry climate of the last interglacial period. The expansion times were 0.189-0.105 Ma B.P. and 0.102-0.057 Ma B.P., respectively. A2 and B1 had strong genetic structure. The large genetic distance between A and B showed that they had been separated from each other for a long time（about 4.29-2.38 Ma B.P.）, and it corresponded to the A phase of Qingzang Movement. The diversity between B1 and B2 at 1.73-0.96 Ma B.P. may be caused by the geological event in Huangshui valley. In early Pleistocene, populations in B1 may have gene flow because of geographic linkage, and later the uplift of the Plateau and the change of river route there made a few haplotypes lost. A1 and A2 were divided into two parts by A’nyemaqen Mountains at 0.66-0.37 Ma B.P., which maybe corresponded to glaciations at about 0.7 Ma B.P.

PHYLOGEOGRAPHIC PATTERNS INDICATE TRANSATLANTIC MIGRATION FROM EUROPE TO NORTH AMERICA IN THE RED SEAWEED CHONDRUS CRISPUS (GIGARTINALES, RHODOPHYTA)1

Inferring how the Pleistocene climate oscillations have repopulated the extant population structure of Chondrus crispus Stackh. in the North Atlantic Ocean is important both for our understanding of the glacial episode promoting diversification and for the conservation and development of marine organisms. C. crispus is an ecologically and commercially important red seaweed with broad distributions in the North Atlantic. Here, we employed both partial mtDNA Cox1 and nrDNA internal transcribed spacer region 2 (ITS2) sequences to explore the genetic structure of 17 C. crispus populations from this area. Twenty-eight and 30 haplotypes were inferred from these two markers, respectively. Analysis of molecular variance (AMOVA) and of the population statistic Theta(ST) not only revealed significant genetic structure within C. crispus populations but also detected significant levels of genetic subdivision among and within populations in the North Atlantic. On the basis of high haplotype diversity and the presence of endemic haplotypes, we postulate that C. crispus had survived in Pleistocene glacial refugia in the northeast Atlantic, such as the English Channel and the northwestern Iberian Peninsula. We also hypothesize that C. crispus from the English Channel refugium repopulated most of northeastern Europe and recolonized northeastern North America in the Late Pleistocene. The observed phylogeographic pattern of C. crispus populations is in agreement with a scenario in which severe Quaternary glaciations influenced the genetic structure of North Atlantic marine organisms with contiguous population expansion and locally restricted gene flow coupled with a transatlantic dispersal in the Late Pleistocene.

Lack of genetic differentiation in the shrimp Penaeus chinensis in the northwestern pacific

Genetic differentiation of the shrimp Penaeus chinensis in the Yellow Sea and Bohai Sea was investigated using the mitochondrial control region (CR). RFLP of a partial CR segment (613 bp) shows that 106 out of 122 (86.9%) individuals from six sampling localities along the coast of northern China and the west coast of the Korean Peninsula share the same haplotype, and the haplotype frequencies among localities are not significantly different. The findings are further confirmed by sequencing the complete CR. Divergence of the complete CR (992 bp) is less than 1.6% in 14 individuals from the six localities. F-statistics based on RFLP data and the TCS network of sequencing data suggest little genetic differentiation of P. chinensis in the Yellow Sea and Bohai Sea. Mismatch analysis suggests a rapid expansion of P. chinensis population to the Yellow Sea and the Bohai Sea, which probably occurred with the rapid rise in sea level after the last glacial maximum. Despite the lack of genetic heterogeneity, we propose that P. chinensis populations in this region should be treated as separate management units, as fishery management programs have to be applied on a local basis by different governments.

高原鼢鼠线粒体谱系地理学和遗传多样性

高原鼢鼠是一类地下独居啮齿动物,为青藏高原特有种之一.为研究该物种的谱系地理学和遗传多样性,本文测定了采自青藏高原东部3个地理种群8个小种群共37个个体的线粒体D-loop区序列变异.在长度为627 bp的序列中,共发现50个变异位点,定义了26种单倍型.该物种的单倍型多样性(Haplotype diversity,H)较高和核苷酸多样性(Nucleotide diversity,πn)较低.谱系分析得到3个稳定的分支,分别与采集的地理种群相吻合:同一地理种群内单倍型之间遗传差异小,而不同地理来源的单倍型之间存在较大区别.距离隔离分析表明高原鼢鼠的遗传分化与地理距离呈正相关.AMOVA分析同样表明地理种群之间存在显著差异:地理种群间变异占遗传变异的80.45%.高原鼢鼠的这种遗传结构特点可能主要是由于第四纪气候变迁、该物种稳定的地下生活环境和有限的迁移能力造成的

基于叶绿体DNA trnT-trnF序列研究祁连圆柏的谱系地理学

由于青藏高原的地貌效应,第四纪冰期气候的反复变化应对现今该地区生物的地理分布及其居群遗传结构产生重大影响.本文对这一地区特有分布物种祁连圆柏Juniperus przewalskii Kom.整个分布区内20居群392个个体的trnT-trnF序列变化进行了研究;共发现3种单倍型(haplotype),构成两种地理区域:高原台面上的居群主要固定HapA,而HapA、HapB和HapC在高原边缘居群均有分布.所有居群总的遗传多样性HT=0.511,GST=0.847.在低海拔的高原边缘,Hap A、Hap B和Hap C高频率固定在不同的居群中,表明可能存在多个不同的避难所,居群反复缩小和扩张的瓶颈效应造成了遗传多样性的丢失.而边缘的一个居群含有两种相似单倍型频率则可能是冰期后迁移融合而成或者该居群在冰期经受的瓶颈作用更弱.高原台面东部间断分布的居群只固定Hap A,表明它们可能经历了冰期后共同的回迁过程和由此产生的奠基者效应.我们的研究结果表明祁连圆柏在冰期可能存在多个避难所,瓶颈效应和奠基者效应造成了这些居群现在的遗传多样性分布式样.

Potential refugium on the Qinghai-Tibet Plateau revealed by the chloroplast DNA phylogeography of the alpine species Metagentiana striata (Gentianaceae)

Metagentiana striata is an alpine annual herbaceous plant endemic to the east of the Qinghai-Tibet (Q-T) Plateau and adjacent areas. The phylogeography of M. striata was studied by sequencing the chloroplast DNA (cpDNA) trnS-trnG intergenic spacer. Ten haplotypes were identified from an investigation of 232 individuals of M. striata from 14 populations covering the entire geographical range of this species. The level of differentiation amongst populations was very high (G(ST) = 0.746; N-ST = 0.774) and a significant phylogeographical structure was observed (P < 0.05). An analysis of molecular variance found a high variation amongst populations (76%), with F-ST = 0.762 (highly significant, P < 0.001), indicating that little gene flow occurred amongst the different regions; this was explained by the isolation of populations by high mountains along the Q-T Plateau and adjacent areas (N-m = 0.156). Only one ancestral haplotype (A) was common and widespread throughout the distributional range of M. striata. The populations of the Hengduan Mountains region of the south-eastern Q-T Plateau showed high diversity and uniqueness of haplotypes. It is suggested that this region was the potential refugium of M. striata during the Quaternary glaciation, and that interglacial and postglacial range expansion occurred from this refugium. This scenario was in good agreement with the results of nested clade analysis, which inferred that the current spatial distribution of cpDNA haplotypes and populations resulted from range expansion, together with past allopatric fragmentation events. (c) 2008 The Linnean Society of London.

Molecular evidence for double maternal origins of the diploid hybrid Hippophae goniocarpa (Elaeagnaceae)

Homoploid hybrid plant species are rare, and the mechanisms of their speciation are largely unknown, especially for homoploid hybrid tree species. Two contrasting hypotheses have been proposed to explain the origin of Hippophae goniocarpa: (1) it is a diploid hybrid originating from H. rhamnoides ssp. sinensis x H. neurocarpa ssp. neurocarpa, and (2) it originated via marginal differentiation from H. rhamnoides ssp. sinensis. Regardless of which of these hypotheses is true (if either), previous studies have suggested that H. rhamnoides ssp. sinensis is the only maternal donor for this hybrid species. In this study, we aim to elucidate the maternal composition of H. goniocarpa and to test the two hypotheses. For this purpose, we sequenced the maternal chloroplast DNA trnL-F region of 75 individuals representing H. goniocarpa, H. rhamnoides ssp. sinensis, and H. neurocarpa ssp. neurocarpa in two co-occurring sites of the taxa. Seven haplotypes were identified from three taxonomic units, and their phylogenetic relationships were further constructed by means of maximum parsimony, maximum likelihood, and network analyses. These seven haplotypes clustered into two distinct, highly divergent lineages. Two haplotypes from one lineage were found in H. rhamnoides ssp. sinensis, and five (representing the other lineage) in H. neurocarpa ssp. neurocarpa. Hippophae goniocarpa shared four common haplotypes from both lineages, but the haplotypes detected from the two populations differed to some extent, and in each case were identical to local haplotypes of the putative parental species. Thus, both H. rhamnoides ssp. sinensis and H. neurocarpa ssp. neurocarpa appear to have together contributed to the maternal establishment of H. goniocarpa. These results clearly demonstrate that the marginal origin hypothesis should be rejected, and support the hybrid origin hypothesis. Hippophae goniocarpa exhibits a sympatric distribution with its two parent species, without occupying new niches or displaying complete ecological isolation. However, this species has effectively developed reproductive isolation from its sympatric parent species. Our preliminary results suggest that H. goniocarpa may provide a useful model system for studying diploid hybrid speciation in trees. (c) 2008 The Linnean Society of London.

Mitochondrial and chloroplast phylogeography of Picea crassifolia Kom. (Pinaceae) in the Qinghai-Tibetan Plateau and adjacent highlands

The disjunct distribution of forests in the Qinghai-Tibetan Plateau (QTP) and adjacent Helan Shan and Daqing Shan highlands provides an excellent model to examine vegetation shifts, glacial refugia and gene flow of key species in this complex landscape region in response to past climatic oscillations and human disturbance. In this study, we examined maternally inherited mitochondrial DNA (nad1 intron b/c and nad5 intron 1) and paternally inherited chloroplast DNA (trnC-trnD) sequence variation within a dominant forest species, Picea crassifolia Kom. We recovered nine mitotypes and two chlorotypes in a survey of 442 individuals from 32 populations sampled throughout the species' range. Significant mitochondrial DNA population subdivision was detected (G(ST) = 0.512; N-ST = 0.679), suggesting low levels of recurrent gene flow through seeds among populations and significant phylogeographical structure (N-ST > GST, P < 0.05). Plateau haplotypes differed in sequence from those in the adjacent highlands, suggesting a long period of allopatric fragmentation between the species in the two regions and the presence of independent refugia in each region during Quaternary glaciations. On the QTP platform, all but one of the disjunct populations surveyed were fixed for the same mitotype, while most populations at the plateau edge contained more than one haplotype with the mitotype that was fixed in plateau platform populations always present at high frequency. This distribution pattern suggests that present-day disjunct populations on the QTP platform experienced a common recolonization history. The same phylogeographical pattern, however, was not detected for paternally inherited chloroplast DNA haplotypes. Two chlorotypes were distributed throughout the range of the species with little geographical population differentiation (G(ST) = N-ST = 0.093). This provides evidence for highly efficient pollen-mediated gene flow among isolated forest patches, both within and between the QTP and adjacent highland populations. A lack of isolation to pollen-mediated gene flow between forests on the QTP and adjacent highlands is surprising given that the Tengger Desert has been a geographical barrier between these two regions for approximately the last 1.8 million years.

Phylogeography of the Qinghai-Tibetan Plateau endemic Juniperus przewalskii (Cupressaceae) inferred from chloroplast DNA sequence variation

The vegetation of the northeast Qinghai-Tibetan Plateau is dominated by alpine meadow and desert-steppe with sparse forests scattered within it. To obtain a better understanding of the phylogeography of one constituent species of the forests in this region, we examined chloroplast trnT-trnF and trnS-trnG sequence variation within Juniperus przewalskii, a key endemic tree species. Sequence data were obtained from 392 trees in 20 populations covering the entire distribution range of the species. Six cpDNA haplotypes were identified. Significant population subdivision was detected (G(ST) = 0.772, N-ST = 0.834), suggesting low levels of recurrent gene flow among populations and significant phylogeographic structure (N-ST > G(ST), P < 0.05). Eight of the nine disjunct populations surveyed on the high-elevation northeast plateau were fixed for a single haplotype (A), while the remaining, more westerly population, contained the same haplotype at high frequency together with two low frequency haplotypes (C and F). In contrast, most populations that occurred at lower altitudes at the plateau edge were fixed or nearly fixed for one of two haplotypes, A or E. However, two plateau edge populations had haplotype compositions different from the rest. In one, four haplotypes (A, B, D and E) were present at approximately equivalent frequencies, which might reflect a larger refugium in the area of this population during the last glacial period. Phylogenetic analysis indicated that the most widely distributed haplotype A is not ancestral to other haplotypes. The contrasting phylogeographic structures of the haplotype-rich plateau edge area and the almost haplotype-uniform plateau platform region indicate that the plateau platform was recolonized by J. przewalskii during the most recent postglacial period. This is supported by the findings of a nested clade analysis, which inferred that postglacial range expansion from the plateau edge followed by recent fragmentation is largely responsible for the present-day spatial distribution of cpDNA haplotypes within the species.

Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.)

Sk?t, L., Humphreys, J., Humphreys, M. O., Thorogood, D., Gallagher, J. A., Sanderson, R., Armstead, I. P., Thomas, I. D. (2007). Association of candidate genes with flowering time and water-soluble carbohydrate content in Lolium perenne (L.). Genetics, 177 (1), 535-547. Sponsorship: BBSRC RAE2008

Mapping the gene for autosomal dominant restless legs syndrome in an Irish family

Restless Legs Syndrome (RLS) is a common neurological disorder affecting nearly 15% of the general population. Ironically, RLS can be described as the most common condition one has never heard of. It is usually characterised by uncomfortable, unpleasant sensations in the lower limbs inducing an uncontrollable desire to move the legs. RLS exhibits a circadian pattern with symptoms present predominantly in the evening or at night, thus leading to sleep disruption and daytime somnolence. RLS is generally classified into primary (idiopathic) and secondary (symptomatic) forms. Primary RLS includes sporadic and familial cases of which the age of onset is usually less than 45 years and progresses slowly with a female to male ratio of 2:1. Secondary forms often occur as a complication of another health condition, such as iron deficiency or thyroid dysfunction. The age of onset is usually over 45 years, with an equal male to female ratio and more rapid progression. Ekbom described the familial component of the disorder in 1945 and since then many studies have been published on the familial forms of the disorder. Molecular genetic studies have so far identified ten loci (5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p). No specific gene within these loci has been identified thus far. Association mapping has highlighted a further five areas of interest. RLS6 has been found to be associated with SNPs in the BTBD9 gene. Four other variants were found within intronic and intergenic regions of MEIS1, MAP2K5/LBXCOR1, PTPRD and NOS1. The pathophysiology of RLS is complex and remains to be fully elucidated. Conditions associated with secondary RLS, such as pregnancy or end-stage renal disease, are characterised by iron deficiency, which suggests that disturbed iron homeostasis plays a role. Dopaminergic dysfunction in subcortical systems also appears to play a central role. An ongoing study within the Department of Pathology (University College Cork) is investigating the genetic characteristics of RLS in Irish families. A three generation RLS pedigree RLS3002 consisting of 11 affected and 7 unaffected living family members was recruited. The family had been examined for four of the known loci (5q, 12q, 14p and 9p) (Abdulrahim 2008). The aim of this study was to continue examining this Irish RLS pedigree for possible linkage to the previously described loci and associated regions. Using informative microsatellite markers linkage was excluded to the loci on 5q, 12q, 14p, 9p, 20p, 16p, 19p, 4q, 17p and also within the regions reported to be associated with RLS. This suggested the presence of a new unidentified locus. A genome-wide scan was performed using two microsatellite marker screening sets (Research Genetics Inc. Mapping set and the Applied Biosystems Linkage mapping set version 2.5). Linkage analysis was conducted under an autosomal dominant model with a penetrance of 95% and an allele frequency of 0.01. A maximum LOD score of 3.59 at θ=0.00 for marker D19S878 indicated significant linkage on chromosome 19p. Haplotype analysis defined a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to 2.5 Mb. There are approximately 100 genes annotated within the critical region. Sequencing of two candidate genes, KLF16 and GAMT, selected on the assumed pathophysiology of RLS, did not identify any sequence variant. This study provides evidence of a novel RLS locus in an Irish pedigree, thus supporting the picture of RLS as a genetically heterogeneous trait.

Aspects of the biology of Mya arenaria and Ensis spp. (Mollusca; Bivalvia) in the Irish Sea and adjacent areas

This study was undertaken to investigate the general biology, including the reproductive cycle and health status, of two clam taxa in Irish waters, with particular reference to the Irish Sea area. Monthly samples of the soft shell clam, Mya arenaria, were collected from Bannow Bay, Co. Wexford, Ireland, for sixteen months, and of the razor clam, Ensis spp. from the Skerries region (Irish Sea) between June 2010 and September 2011. In 2010, M. arenaria in Bannow Bay matured over the summer months, with both sexes either ripe or spawning by August. The gonads of both sexes of E. siliqua developed over autumn and winter 2010, with the first spawning individuals being recorded in January 2011. Two unusually cold winters, followed by a warmer than average spring, appear to have affected M. arenaria and E. siliqua gametogenesis at these sites. It was noted that wet weight of E. siliqua dropped significantly in the summer of both 2010 and 2011, after spawning, which may impact on the economic viability of fishing during this period. Additional samples of M. arenaria were collected at Flaxfort (Ireland), and Ensis spp. at Oxwich (Wales), and the pathology of all clams was examined using both histological and molecular methods. No pathogenic conditions were observed in M. arenaria while Prokaryote inclusions, trematode parasites, Nematopsis spp. and inflammatory pathologies were observed at low incidences in razor clams from Ireland but not from Wales; the first time these conditions have been reported in Ensis spp. in northern European waters. Mya arenaria from sites in Europe and eastern and western North America were investigated for genetic variation using both mitochondrial (cytochrome oxidase I (COI) and 16S ribosomal RNA genes) and nuclear markers (10 microsatellite loci). Both mitochondrial CO1 and all nuclear markers showed reduced levels of variation in certain European samples, with significant differences in haplotype and allelic composition between most samples, particularly those from the two different continents, but with the same common haplotypes or alleles throughout the range. The appearance of certain unique rare haplotypes and microsatellite alleles in the European samples suggest a complicated origin involving North American colonization but also possible southern European Pleistocene refugia. Specimens of Ensis spp. were obtained from five coastal areas around Ireland and Wales and species-specific PCR primers were used to amplify the internal transcribed spacer region 1 (ITS1) and the mitochondrial DNA CO1 gene and all but 15 razor clams were identified as Ensis siliqua. Future investigations should focus on continued monitoring of reproductive biology and pathology of the two clam taxa (in particular, to assess the influence of environmental change), and on genetics of southern European M. arenaria and sequencing the CO1 gene in Ensis individuals to clarify species identity

Molecular basis of complete C4 deficiency. A study of three patients.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes, C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes and is also remarkable by the high frequency of the null alleles, C4A*Q0 and C4B*Q0. Complete C4 deficiency is exceptional because this condition appears only in homozygotes for the very rare double-null haplotype C4AQ0,BQ0. This condition in most cases gives rise to systemic lupus erythematosus and an increased susceptibility to infections. The molecular basis for complete C4 deficiency has not yet been established. Therefore we studied the DNA of three previously described C4 deficient patients belonging to unrelated families by restriction fragment length polymorphism analysis using C4 and 21-OH probes. These studies revealed a deletion of the C4B and 21-OHA genes in two patients and no deletion at all in the third patient. Therefore, complete C4 deficiency as a result of homozygosity for the C4AQ0, BQ0 haplotype is not a consequence of a deletion of the C4 genes. The molecular basis of this genetic abnormality is certainly very complex and may vary also from one case to another.

Impact of gene variants on sex-specific regulation of human Scavenger receptor class B type 1 (SR-BI) expression in liver and association with lipid levels in a population-based study.

BACKGROUND: Several studies have noted that genetic variants of SCARB1, a lipoprotein receptor involved in reverse cholesterol transport, are associated with serum lipid levels in a sex-dependent fashion. However, the mechanism underlying this gene by sex interaction has not been explored. METHODS: We utilized both epidemiological and molecular methods to study how estrogen and gene variants interact to influence SCARB1 expression and lipid levels. Interaction between 35 SCARB1 haplotype-tagged polymorphisms and endogenous estradiol levels was assessed in 498 postmenopausal Caucasian women from the population-based Rancho Bernardo Study. We further examined associated variants with overall and SCARB1 splice variant (SR-BI and SR-BII) expression in 91 human liver tissues using quantitative real-time PCR. RESULTS: Several variants on a haplotype block spanning intron 11 to intron 12 of SCARB1 showed significant gene by estradiol interaction affecting serum lipid levels, the strongest for rs838895 with HDL-cholesterol (p=9.2x10(-4)) and triglycerides (p=1.3x10(-3)) and the triglyceride:HDL cholesterol ratio (p=2.7x10(-4)). These same variants were associated with expression of the SR-BI isoform in a sex-specific fashion, with the strongest association found among liver tissue from 52 young women<45 years old (p=0.002). CONCLUSIONS: Estrogen and SCARB1 genotype may act synergistically to regulate expression of SCARB1 isoforms and impact serum levels of HDL cholesterol and triglycerides. This work highlights the importance of considering sex-dependent effects of gene variants on serum lipid levels.

Assessment of LD matrix measures for the analysis of biological pathway association.

Complex diseases will have multiple functional sites, and it will be invaluable to understand the cross-locus interaction in terms of linkage disequilibrium (LD) between those sites (epistasis) in addition to the haplotype-LD effects. We investigated the statistical properties of a class of matrix-based statistics to assess this epistasis. These statistical methods include two LD contrast tests (Zaykin et al., 2006) and partial least squares regression (Wang et al., 2008). To estimate Type 1 error rates and power, we simulated multiple two-variant disease models using the SIMLA software package. SIMLA allows for the joint action of up to two disease genes in the simulated data with all possible multiplicative interaction effects between them. Our goal was to detect an interaction between multiple disease-causing variants by means of their linkage disequilibrium (LD) patterns with other markers. We measured the effects of marginal disease effect size, haplotype LD, disease prevalence and minor allele frequency have on cross-locus interaction (epistasis). In the setting of strong allele effects and strong interaction, the correlation between the two disease genes was weak (r=0.2). In a complex system with multiple correlations (both marginal and interaction), it was difficult to determine the source of a significant result. Despite these complications, the partial least squares and modified LD contrast methods maintained adequate power to detect the epistatic effects; however, for many of the analyses we often could not separate interaction from a strong marginal effect. While we did not exhaust the entire parameter space of possible models, we do provide guidance on the effects that population parameters have on cross-locus interaction.