用SSR标记检测杂交籼稻三系亲本的遗传差异

随机选用分布于水稻(Oryza sativa L.)12条染色体上的110对引物,共筛选出26对具有多态性的SSR引物,对24份杂交籼稻三系亲本材料进行遗传多样性和亲缘关系分析.全部24份亲本中共检测出116对等位基因,每个SSR引物可检测到的等位基因数目为2～7个不等,平均每对引物可以检测到4.4个等位基因.多态信息量(PIC)的变动范围为0.068 9～0.803 6,平均PIC值为0.603 4;期望杂合度(He)的变动范围为0.081 6～0.829 8,平均值为0.616 9;多样性指数(1)变幅为0.173 2～1.791 3,平均为1.160 0.对供试亲本材料根据遗传距离进行UPGMA聚类分析,结果表明,SSR微卫星标记可以将24份亲本材料分为3个大群,各组亲本间的遗传差异较大.通过分子标记辅助育种,可以有效地鉴定供试亲本材料之间的亲缘关系,分析预测新种质材料的遗传差异,可作为选育强优势组合杂交水稻的重要参考指标之一.图2表3参24     

Isozyme, ISSR and RAPD profiling of genotypes in marvel grass (Dichanthium annulatum)

Genetic analysis of 30 accessions of marvel grass (Dichanthium annulatum Forsk.), a tropical range grass collected from grasslands and open fields of drier regions, was carried out with the objectives of identifying unique materials that could be used in developing the core germplasm for such regions as well as to explore gene (s) for drought tolerance. Five inter-simple sequence repeat (ISSR) primers [(CA)4, (AGAC), (GACA) 4; 27 random amplified polymorphic DNA (RAPD) and four enzyme systems were employed in the present study. In total, ISSR yielded 61 (52 polymorphic), RAPD 269 (253 polymorphic) and enzyme 55 isozymes (44 polymorphic) bands. The average polymorphic information content (PIC) and marker index (MI) across all polymorphic bands of 3 markers systems ranged from 0.419 to 0.480 and 4.34 to 5.25 respectively Dendrogram analysis revealed three main clusters with all three markers. Four enzymes namely esterase (EST), polyphenoloxidase (PPO), peroxidase (PRX) and superoxide dismutase (SOD) revealed 55 alleles from a total of 16 enzyme-coding loci. Of these, 14 loci and 44 alleles were polymorphic. The mean number of alleles per locus was 3.43. Mean heterozygosity observed among the polymorphic loci ranged from 0.406 (SOD) to 0.836 (EST) and accession wise from 0.679 (1G3108) to 0.743 (IGKMD-10). Though there was intermixing of few accessions of one agro-climatic region to another largely groupings of accessions were with their regions of collections. Bootstrap analysis at 1000 iterations also showed large numbers of nodes (11 to 17) having strong clustering (> 50 bootstrap values) in all three marker systems. The accessions of the arid and drier regions forming one cluster are assigned as distinct core collection of Dichanthium and can be targeted for isolation of gene (s) for drought tolerance. Variations in isozyme allele numbers and high PIC (0.48) and MI (4.98) as observed with ISSR markers indicated their usefulness for germplasm characterization.     

Analysis of genetic structure of blacklip abalone (Haliotis rubra) populations using RAPD, minisatellite and microsatellite markers

 We investigated the utility of three polymerase chain-reaction (PCR)-based DNA molecular markers in analysing genetic structure of the populations of the blacklip abalone Haliotis rubra (Leach) of Victoria, Australia. The DNA markers included 84 randomly amplified polymorphic DNA (RAPD) bands amplified using six random primers, two minisatellites, GHR (putative growth-hormone-gene-repeat) and MIPR (putative mollusca-insulin-like peptide-gene-repeat), and three microsatellites, RUBGT1 [containing (GT)_n repeats], RUBCA1 [containing (CA)_n repeats] and RUBGACA1 [containing (GACA)_n repeats]. All three types of DNA markers revealed significant subdivision in the H. rubra populations along the coastline. This is postulated as being related to the abalone's relatively short pelagic period and limited dispersion. Further analysis revealed that a Point Cook population sampled from within the semi-enclosed Port Phillip Bay was distinct from two other central zone populations (Apollo Bay and Cape Schanck). The genotypes of microsatellites indicated excessive homozygotes across all the populations at all three microsatellite loci, and possible causes such as larval recruitment pattern and asynchronous spawning are discussed. The excessive homozygotes recorded for the three microsatellite loci contrast with those observed in the minisatellite loci GHR and MIPR, the heterozygosities of which were at Hardy–Weinberg equilibrium. Received: 17 March 1999 / Accepted: 24 November 1999     

用SSR标记检测同源四倍体与二倍体水稻的遗传差异

随机选用分布于水稻(Oryza stativa L.)12条染色体上的15对SSR(simplej sequence repeats)引物，对18种中科院成都生物所培育的四倍体水稻和9种大面积种植的二倍体水稻进行了SSR多态性分析．11对具多态性的引物共检测到33条多态性条带，平均每对引物检测到3个等位基因．研究结果发现，所用同源四倍体水稻的基因组与二倍体水稻基因组大部分相同，只是在某些位点上具有差异，并筛选出部分SSR标记来区分二倍体与其同源四倍体．本研究还对二倍体与同源四倍体之间遗传差异的原因进行了初步的探讨．图2表2参12     

Use of EST database markers from M. truncatula in the transferability to other forage legumes

In general tropical forage legumes lack microsatellites or simple sequence repeat (SSR) markers. Development of genic SSR markers from expressed sequence tagged (EST) database is an alternate and efficient approach to generate the standard DNA markers for genome analysis of such crop species. In the present paper a total of 816 EST-SSRs containing perfect repeats of mono (33.5%), di (14.7%), tri (39.3%), tetra (2.7%), penta (0.7%) and hexa (0.4%) nucleotides were identified from 1,87,763 ESTs of Medicago truncatula. Along with, 70 (8.5%) SSRs of a compound type were also observed. Seven primer pairs of tri repeats were tested for cross transferability in 19 accessions of forage legumes comprising 11 genera. At two different annealing temperatures (55 and 60 degreesC) all primer pairs except AJ410087 reacted with many accessions of forage legumes. Atotal of 51 alleles were detected with six M. truncatula EST-SSRs primer-pairs against DNAfrom 19 accessions representing 11 genera where number of alleles ranged from 2 to 13. The cross-transferability of these EST-SSRs was 40.6% at 55 degreesC and 32.3% at 60 degreesC annealing temperature. 24 alleles of the total 50 (48%) at 55 degreesC and 27 of 51 (53%) at 60 degreesC were polymorphic among the accessions. These 27 polymorphic amplicons identified could be used as DNA markers. This study demonstrates the developed SSR markers from M. truncatula ESTs as a valuable genetic markers and also proposes the possibility of transferring these markers between species of different genera of the legumes of forage importance. It was evident from the results obtained with a set of Desmanthus virgatus accessions where SequentialAgglomerative Hierarchical and Nested (SAHN) cluster analysis based on Dice similarity and Unweighted Pair Group Method with Arithmetic mean Algorithm (UPGMA) revealed significant variability (24 to 74%) among the accessions. High bootstrap values (>30) supported the nodes generated by dendrogram analysis of accessions.     

Fluorescence spectroscopic studies of the effect of granular activated carbon adsorption on structural properties of dissolved organic matter fractions

This work investigated the effect of granular activated carbon adsorption (GACA) on fluorescence characteristics of dissolved organic matter (DOM) in secondary effluent, by means of excitation–emission matrix (EEM) spectra, the fluorescence regional integration (FRI) method, synchronous spectra, the fluorescence index defined as the ratio of fluorescence emission intensity at wavelength 450 nm to that at 500 nm at excitation (λ_ex)=370 nm, and the wavelength that corresponds to the position of the normalized emission band at its half intensity (λ_0.5). DOM in the secondary effluent from the North Wastewater Treatment Plant (Shenyang, China) was fractionated using XAD resins into 5 fractions: hydrophobic acid (HPO–A), hydrophobic neutral (HPO–N), transphilic acid (TPI–A), transphilic neutral (TPI–N) and hydrophilic fraction (HPI). Results showed that fluorescent materials in HPO–N and TPI–N were less readily removed than those in the other fractions by GACA. The relative content of fluorescent materials in HPO–A, TPI–A and HPI decreased whereas that in HPO–N and TPI–N increased as a consequence of GACA. Polycyclic aromatics in all DOM fractions were preferentially absorbed by GACA, in comparison with bulk DOM expressed as DOC. On the other hand, the adsorption of aromatic amino acids and humic acid-like fluorophores exhibiting fluorescence peaks in synchronous spectra by GACA seemed to be dependent on the acid/neutral properties of DOM fractions. All five fractions had decreased fluorescence indices as a result of GACA. GACA led to a decreased λ_0.5 value for HPO–A, increased λ_0.5 values for HPO–N, TPI–A and HPI, and a consistent λ_0.5 value for TPI–N.     

Stress-induced rapid release of antibacterials by scleractinian corals

Mechanical stress on the coral Pocillopora damicornis caused the release of material that killed the coral pathogen Vibrio coralliilyticus. The bactericidal material was released into the surrounding seawater rapidly, reaching a maximum killing activity within 1 min of the stress. The coral antibacterial activity, referred to as CAA, was retained following filter sterilization and storage at –20°C. Exposure of V. coralliilyticus to CAA for 30 s, 1 min and 6 min resulted in the death of 82%, 89% and 99% of the bacteria, respectively. Release of CAA following mechanical stress was also observed with four other coral species tested. P. damicornis CAA was bactericidal to a wide variety of Gram-negative and Gram-positive bacteria. This is the first report that hard corals rapidly release fast-acting bactericidal material following mechanical stress. The release of CAA was demonstrated with both aquarium corals and corals taken directly from the sea. It is suggested that CAA is part of hard corals host defense system against infection, the natural stimulation for release of CAAs being the bite of a predator. Previous failures to detect antibacterial activity in hard corals can be attributed to a lack of understanding of the sensitive mechanism by which they are released.Communicated by M. Kühl, Helsingør     

11个猕猴桃品种间的遗传多样性分析

利用随机扩增多态性DNA标记(RAPD)技术对采自四川省猕猴桃科研基地的11个猕猴桃品种进行了遗传多样性分析.通过引物筛选,从15个RAPD引物中扩增出135条带,其中100条为多态带,占74.07%.UPGMA聚类分析结果揭示了各品种间的亲缘关系.RAPD标记说明猕猴桃品种间遗传距离与地理分布有一定关系,地理位置较近的材料能聚在一起.分子标记可用于猕猴桃种质资源的分类、鉴定及良种选育.图2表2参15     

柚类资源及其近缘种SSR标记的分子评价

采用31对SSR引物对122份柚类种质资源及其近缘种的遗传多样性进行了研究.31对SSR引物可以检测到335个等位基因变异,平均每个位点可检测到9.85个等位基因.每个位点多态性信息指数(PIC)平均值为0.7085.从这些引物中有效地筛选出21对高信息量SSR引物,与31对引物在122份柚类资源间遗传关系达到极显著相关(r=0.99104).柚类资源及其近缘种等位基因平均数(A)、平均杂合位点百分比(P)、SSR表型杂合度(H0)分别为2.8、83.54%、0.524.用UPGMA方法将122份研究材料分成7个组群,110个柚类品种在相似系数0.712时可细分成18个亚组,分类结果有利于今后有目的地利用这些丰富育种材料的遗传背景.图3表2参21     

小麦高分子量谷蛋白亚基基因的PCR及AS-PCR标记

选用高分子量麦谷蛋白亚基(HMW-GS)在Glu-D1位点已知的 26个普通小麦品种,包括 15个 (TriticumaestivumL. )川育系列品种及其 11个亲本,根据其亚基Dx5和Dy10基因的特异序列设计引物.通过对Dx5基因的PCR扩增及对Dy10基因的AS-PCR扩增,结果表明,所设计的引物对Dx5和Dy10都能扩增出特异条带,而携有Dx5基因的材料同时也携有Dy10基因.说明用所设计的引物可快速有效的鉴定出普通小麦中是否带有优质的 5 10亚基.图 4参 15     

林木外生菌根真菌彩色豆马勃巢式PCR检测

为探讨林木外生菌根真菌的分子检测方法,利用真菌通用引物ITS1-F/ITS4-B扩增了外生菌根真菌彩色豆马勃的核糖体DNA内转录间隔区并进行了序列测定.通过序列比较,设计了一对彩色豆马勃特异性引物PtF/PtR.利用该对特异性引物与ITS1-F/ITS4-B组合进行巢式PCR,能从供试的彩色豆马勃10个菌株中特异性地扩增出1条347 bp的条带,而供试的其他6个参比菌株未出现扩增产物.经分析,该巢式PCR检测技术的灵敏度可达到10 fg的DNA,是常规PCR检测的1 000倍.利用该技术从马尾松苗木菌根中检测到目的外生菌根真菌.这表明采用本研究设计的特异性引物,利用巢式PCR技术可以灵敏、准确地从林木外生菌根中检测出彩色豆马勃.     

Identification of species-specific RAPD markers in genus Cenchrus

Cenchrus is an important component of major grass cover of world. Similar to the other major tropical grasses most of the species in genus Cenchrus are also apomictic in nature hence correct and precise identification of accessions and species are problematic and dubious. In the present study 187 decamer oligonucleotide primers were tested for PCR-based DNA amplification of six prominent species of genus Cenchrus. Of these, 32 potential repetitive and polymorphic primers were tested for identification of species-specific markers for C. ciliaris, C. setigerus, C. pennisetiformis, C. prieurri, C. biflorus and C. myosuroides. These primers yielded 51 unique RAPD markers either specific to a species (37) or shared by two or more species (14). Maximum markers were shared between C. ciliaris and C. setigerus confirming theirmore closeness to each other Primers like OPF09, OPF11, OPR15, OPAJ11, OPQ10 and OPAK20 generated strong intense bands can be used on priority in identifying the species from their natural habitat for the development of species-specific core germplasm. Due to apomictic nature this is the prime method of developing cultivars, as morphological characters are largely unable to distinguish them. The level of variation observed clearly suggest RAPD as an appropriate marker for genetic studies and in identifying the lines with species-specific markers for Cenchrus germplasm management activity and also maintaining identity and purity for proprietary reasons.     

The utility of the 16S gene in investigating cryptic speciation within the Brachionus plicatilis species complex

Recent reports indicate an extensive amount of molecular evolution separating cryptic taxa as well as significant population structure at a microgeographical scale. Appropriate molecular markers are particularly suitable for distinguishing cryptic biological species. In this study, we examine the phylogenetic utility of 16S rRNA in elucidating the evolutionary relationships within the recently described euryhaline Brachionus plicatilis species complex. In addition, we assess the applicability of this marker in the genetic identification and monitoring of rotifer populations. We have sequenced a 378-bp fragment of the mitochondrial 16S rRNA gene in laboratory reference strains, hatchery clones as well as collections from a wild population of the subsaline Lake Koroneia (Northern Greece). Also, restriction fragment length polymorphism (RFLP) analysis was performed with eight restriction endonucleases. Rotifer samples are distinguished into six genetically divergent lineages. Average sequence divergence between lineages is 0.1038. The evolutionary relationships and divergence time-scales revealed with the 16S sequence data are in agreement with previous analyses using different mitochondrial and nuclear markers. The 16S region appears to have several advantages over other regions of the genome regarding use of species-specific primers, ease of amplification from single specimens and undiluted informational content over both recent and more ancient separations. It has also exhibited maximum discriminatory power (100% success) between lineages during RFLP analysis. The 16S assayed region has proven especially informative and consistent in detecting, supporting and establishing the lineage status within the B. plicatilis species complex both from a phylogenetic perspective and as an identification tool.     

Geographic assignment of seabirds to their origin: combining morphologic, genetic, and biogeochemical analyses.

Longline fisheries, oil spills, and offshore wind farms are some of the major threats increasing seabird mortality at sea, but the impact of these threats on specific populations has been difficult to determine so far. We tested the use of molecular markers, morphometric measures, and stable isotope (delta15N and delta13C) and trace element concentrations in the first primary feather (grown at the end of the breeding period) to assign the geographic origin of Calonectris shearwaters. Overall, we sampled birds from three taxa: 13 Mediterranean Cory's Shearwater (Calonectris diomedea diomedea) breeding sites, 10 Atlantic Cory's Shearwater (Calonectris diomedea borealis) breeding sites, and one Cape Verde Shearwater (C. edwardsii) breeding site. Assignment rates were investigated at three spatial scales: breeding colony, breeding archipelago, and taxa levels. Genetic analyses based on the mitochondrial control region (198 birds from 21 breeding colonies) correctly assigned 100% of birds to the three main taxa but failed in detecting geographic structuring at lower scales. Discriminant analyses based on trace elements composition achieved the best rate of correct assignment to colony (77.5%). Body measurements or stable isotopes mainly succeeded in assigning individuals among taxa (87.9% and 89.9%, respectively) but failed at the colony level (27.1% and 38.0%, respectively). Combining all three approaches (morphometrics, isotopes, and trace elements on 186 birds from 15 breeding colonies) substantially improved correct classifications (86.0%, 90.7%, and 100% among colonies, archipelagos, and taxa, respectively). Validations using two independent data sets and jackknife cross-validation confirmed the robustness of the combined approach in the colony assignment (62.5%, 58.8%, and 69.8% for each validation test, respectively). A preliminary application of the discriminant model based on stable isotope delta15N and delta13C values and trace elements (219 birds from 17 breeding sites) showed that 41 Cory's Shearwaters caught by western Mediterranean long-liners came mainly from breeding colonies in Menorca (48.8%), Ibiza (14.6%), and Crete (31.7%). Our findings show that combining analyses of trace elements and stable isotopes on feathers can achieve high rates of correct geographic assignment of birds in the marine environment, opening new prospects for the study of seabird mortality at sea.     

Delayed maturation of multiple signals in a migratory songbird

Many animals use multiple signals in sexual communication, but our understanding of the interactions between multiple signals, particularly in inexperienced breeders, is limited. In birds, delayed plumage maturation (DPM) is well documented; young birds appear duller than adults, despite reaching sexual maturity. Age-related changes in song structure are also common in songbirds, though the extent to which songs of yearling males differ from those of adults (delayed song maturation, DSM) and its prevalence in species with DPM is unknown. We tested for DSM in the mate-attraction (repeat) song of a species with dramatic DPM, the American redstart (Setophaga ruticilla). Repeat song structure of territorial yearling and adult males differed significantly, based on discriminant analysis of nine non-collinear song features. Combined with previous evidence of delayed maturation in territorial (serial) song, we provide the first evidence for DSM in different song types used in different behavioral contexts during the breeding season of a migratory songbird. Within adults, variation in repeat song was associated with pairing, earlier onset of breeding, and number of offspring sired, suggesting a potential benefit for expressing more adult-like song. We found no relationship between the expression of adult-like repeat song and plumage in either age class. These results indicate that delayed maturation of repeat song in yearling redstarts is not due to a generalized delay in signal development, and suggest further work is needed to determine whether mate attraction (for both yearling and adult males) provides greater pressure to sound more “adult-like” than to look more “adult-like”.     

入侵植物加拿大一枝黄花和本地一枝黄花的遗传多样性比较

为了阐明加拿大一枝黄花成功入侵的机制,利用简单序列重复区间标记(ISSR)方法对加拿大一枝黄花和本地一枝黄花的遗传多样性进行比较研究。从100条引物中筛选出12条引物用于PCR扩增,利用POPGEN32软件对2种一枝黄花进行遗传多样性分析。结果表明,加拿大一枝黄花在物种水平上的多态位点百分率为95.19%,Nei’s基因多样性指数为0.308 5,Shannon’s信息指数为0.415 8;本地一枝黄花在物种水平上的多态位点百分率(89.80%)、Nei’s基因多样性指数(0.249 1)和Shannon’s信息指数(0.383 4)都比加拿大一枝黄花小。加拿大一枝黄花和本地一枝黄花居群间遗传分化系数分别为0.118 2和0.131 3,居群内变异分别为0.881 8和0.868 7,表明2个物种居群间的遗传分化不明显,遗传一致度高,且主要的遗传变异存在于居群内。入侵植物加拿大一枝黄花具有较高遗传多样性,且高于本地一枝黄花,这可能是加拿大一枝黄花成功入侵的原因之一。     

Genotypic variation in element concentrations in brown rice from Yunnan landraces in China

The mineral elements present in brown rice play an important physiological role in global human health. We investigated genotypic variation of eight of these elements (P, K, Ca, Mg, Fe, Zn, Cu, and Mn) in 11 different grades of brown rice on the basis of the number and distance coefficients of 282 alleles for 20 simple sequence repeat (SSR) markers. Six-hundred and twenty-eight landraces from the same field in Yunnan Province, one of the largest centers of genetic diversity of rice (Oryza sativa L.) in the world, formed our core collection. The mean concentrations (mg kg⁻¹) of the eight elements in brown rice for these landraces were P (3,480) > K (2,540) > Mg (1,480) > Ca (157) > Zn (32.8) > Fe (32.0) > Cu (13.6) > Mn (13.2). Mean P concentrations in brown rice were 6.56 times total soil P, so the grains are important in tissue storage of P, but total soil K is 7.82 times mean K concentrations in brown rice. The concentrations of the eight elements in some grades of brown rice, on the basis of the number and distance coefficients of alleles for 20 SSR markers for the landraces, were significantly different (P < 0.05), and further understanding of the relationship between mineral elements and gene diversity is needed. There was large variation in element concentrations in brown rice, ranging from 2,160 to 5,500 mg P kg⁻¹, from 1,130 to 3,830 mg K kg⁻¹, from 61.8 to 488 mg Ca kg⁻¹, from 864 to 2,020 mg Mg kg⁻¹, from 0.40 to 147 mg Fe kg⁻¹, from 15.1 to 124 mg Zn kg⁻¹, from 0.10 to 59.1 mg Cu kg⁻¹, and from 6.7 to 26.6 mg Mn kg⁻¹. Therefore, germplasm evaluations for Ca, Fe, and Zn concentrations in rice grains have detected up to sevenfold genotypic differences, suggesting that selection for high levels of Ca, Fe, and Zn in breeding for mass production is a feasible approach. Increasing the concentrations of Ca, Fe, and Zn in rice grains will help alleviate chronic Ca, Zn, and Fe deficiencies in many areas of the world.     

不同冬夏寄主棉蚜种群重复序列引物DNA多态性分析

应用重复序列引物ＰＣＲ技术研究了中国不同冬夏寄主上棉蚜种群的ＤＮＡ多态性,通过筛选出的３种适用于棉蚜的重复序列引物,用它们的扩增结果进行相似性指数Ｉｓ和Ｎｅｉ’ｓ遗传距离Ｄ的计算,并根据遗传距离对所研究的棉蚜种群做聚类分析,结果表明,用遗传距离指标可以将冬夏寄主上的棉蚜分开,并且在不同的冬寄主之间,较适宜越冬的不同寄主上的棉蚜呈现出更大的相似性。在实验数据的分析过程中,还发现遗传距离指标比相似性指     

分子生物学技术在菌根研究中的应用及其进展

近几年来,菌根分子生物学得到了较快发展;尤其是一些用于首根真菌ITS区域的特异引物先后设计并合成成功,进一步推动了菌根学在分子水平上的研究。分子生物学技术在留根真菌分类鉴定及亲缘关系研究上应用较多。在菌根菌竟争性和持续性研究上．DNA分析技术已成为最重要的研究手段之一。在这些研究中,专为菌根真菌ITS区域分析而设计的ITS1-F和ITS4-B两种特异引物应用最为普遍。本文对菌根DNA分析技术、PCR－RFLP技术做了简单介绍;综述了PCR、ITh－RFLP等分析技术在菌根菌分类、亲缘关系及菌种持续性等方面的研究和应用情况。