Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Isolation and characterization of eight microsatellite loci in two morphotypes of the Southeast Asian army ant,Aenictus laeviceps

Eight polymorphic microsatellite markers were developed from morphotype L1 of the Southeast Asian army ant, Aenictus laeviceps in Borneo, and characterized for morphotypes L1 and L2. The number of alleles per locus ranged from nine to 24 (average 15) in morphotype L1 and two to 16 (average 9.8) in L2. Observed heterozygosity ranged from 0.75 to 0.96 in morphotype L1 and from 0.08 to 0.96 in L2. All loci were in Hardy–Weinberg equilibrium. One pair of loci showed linkage disequilibrium in morphotype L1. All markers successfully amplified in morphotype S, and some markers amplified in other Aenictus species.     

Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus

The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.     

青藏高原早熟甘蓝型春油菜遗传资源研究

利用SSR和SRAP 2种分子标记研究了69份试验材料的遗传差异及其亲缘关系.29对SSR标记共扩增出118条多态性带,多态性位点占总扩增位点的97.5%,27对SRAP引物扩增出123条多态性带,多态性比率为70.3%.两种标记聚类结果表明.在相似系数0.566处所有材料可以分为A、B 2个大类群;B类在相似系数0.620处又可分为7个亚类,10个天然双低早熟甘蓝型品系、2个甘蓝型亲本和4个新型品系聚在第1亚类中,其余的51个新型甘蓝型油菜品系分别聚在其他6个亚类中.对55份新型品系进行遗传成分分析,结果表明,每个品系都合有4种带型,各品系所舍不同带所占比率不同.对各品系中含有白菜型亲本带所占比率分别与其对应的两亲本之间的遗传距离进行相关分析,结果表明新型甘蓝型油菜品系中白菜型亲本带所占比率与白菜型素本间的遗传距离为负相关(-0.52),且达到极显著水平;与甘蓝型亲本间的遗传距离为正相关(0.31),且达到显著水平.对试验材料之间的遗传距离及其来源进行分析(除与2个白菜型亲本间),遗传距离排名前20位的都来自新型品系之间或天然品系与新型之间,最大为0.544.     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichlo? amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Development and characterization of microsatellite loci for the tropical tree pathogen Botryosphaeria rhodina

Nineteen simple sequence repeat (SSR) markers were developed for the pathogen and blue stain fungus Botryosphaeria rhodina. Eight pairs were found to be polymorphic among isolates collected from Pinus spp., whereas a further five pairs were polymorphic when isolates from Pinus spp. were compared with those from Eucalyptus spp. Nine isolates of B. rhodina collected from pines and eucalypts in South Africa, Mexico and Indonesia were used to demonstrate the range of the markers. Testing the markers yielded preliminary evidence that relationships among isolates are more closely linked to host than to geographical origin.     

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.     

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

Distribution of Simple Sequence Repeat and AFLP Markers in Molecular Linkage Map of Rice

SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) are PCR-based molecular markers developed in recent years. In this study, the authors analyzed the polymorphisms, inheritance and distribution of SSR and AFLP markers using an F2 population from a cross between cultivar "Aijiao Nante" ( Oryza sativa L. ssp. indica) and an accession of the common wild rice ( O. rufipogon Griff). A total of 200 new markers were obtained including 28 SSR and 172 AFLP markers. Six of the 28 SSR markers were developed by National Key Laboratory of Crop Genetic Improvement (NKLCGI) using DNA sequences from GenBank and the other 22 were from data published previously. The 172 AFLP markers were from a total 228 polymorphic bands amplified using 25 selected primer combinations. Mapping of the 200 new markers using NKLCGI' S previously developed RFLP map based on the same F2 population resolved these markers to all 12 rice chromosomes. Integration of the SSR and AFLP markers into the RFLP map resulted in a high density molecular linkage map containing 612 polymorphic loci.     

Signatures of speciation? Distribution and diversity of Hypoplectrus (Teleostei: Serranidae) colour morphotypes

Aim To test historical and current influences on the distributions of sympatric colour morphotypes in the coral reef fish genus Hypoplectrus. Location The Caribbean and surrounding tropical waters. These areas cover the entire distribution of the genus. Methods A large and extensive database of Hypoplectrus sightings was used to establish the distribution of colour morphotypes and test a long‐standing hypothesis regarding their origin. First, we considered the evidence for the previously proposed ‘population centre’ hypothesis, which suggests that current morphotype distributions reflect past conditions where these colour forms evolved in allopatry. Using morphotype sighting data, the existence of clusters in occurrence and density was tested. Second, we examined whether the observed patterns of morphotype co‐occurrence deviate from random expectations using null model simulations, within subregions of the distribution of the genus, to infer ecological influences on distribution. Results There is considerable variation in morphotype distribution, with even widespread morphotypes showing geographical clustering. There is also little evidence to suggest past or current geographical isolation, with only one of the 11 morphotypes (Hypoplectrus chlorurus) showing a density distribution that is consistent with the population centre hypothesis. Null model analyses show that variation in local morphotype co‐occurrence is typically significantly lower than expected under random dispersal conditions. Main conclusions Our results strongly suggest that morphotype co‐occurrence is not random, but there is no evidence to suggest a past allopatric radiation in Hypoplectrus colour. Current distributions are likely to be driven by competitive interactions and/or habitat preferences. Our study highlights the value of the Hypoplectrus species complex as a system for the study of speciation in the marine environment, and implies that these closely related morphotypes have ecological relevance rather than being simple colour variants of a single polymorphic species.     

Trypsin-like enzymes from two morphotypes of the 'living fossil' Triops (Crustacea: Branchiopoda: Notostraca)

Trypsin-like enzymes from two morphotypes (here called short and long) of the 'living fossil' Triops of Baja California Sur, Mexico were studied. Adults of both morphotypes were obtained from outdoor static cultures using dry soil from the natural habitats as a source of cysts and culture substrate. Individual and pooled extracts were made from dissected digestive tubes. The effect of pH and temperature on the trypsin activity was studied using N-alpha-benzoyl-DL-Arg-p-nitroanilide (BAPNA) as substrate. The highest proteolytic activity was found at the same pH with extracts of both morphotypes. At this pH, there was greater proteolytic activity at a lower temperature with the short morphotype extract than with the long morphotype extract. Substrate-SDS-PAGE zymograms showed bands of activity. Short morphotype extracts produced six bands; five of them were serine proteases of which three were trypsin-like enzymes. Long morphotype extracts revealed eight bands; six of them were serine proteases of which three were trypsin-like enzymes.     

Transferability of SSR markers among wheat,rye, and triticale

Simple sequence repeat (SSR) markers are a valuable tool for many purposes, such as mapping, fingerprinting, and breeding. However, they are only available in some economically important crops because of the high cost and labor intensity involved in their development. Comparative mapping reveals a high degree of colinearity between closely related species, which allows the exchange of markers between them. Our objective was to examine the transferability of SSR markers among wheat (Triticum aestivum L.), rye (Secale cereale L.), and triticale (X Triticosecale Wittmack). One hundred forty-eight wheat and 28 rye SSR markers were used to amplify genomic DNA extracted from five lines each of wheat, rye, and triticale. Transferability of wheat SSR markers to rye was 17%, whereas 25% of rye markers were amplifiable in wheat. In triticale, 58% and 39% transferability was achieved for wheat and rye markers, respectively. Wheat markers gave an average of 2.6, 2.7, and 2.4 polymorphic bands in wheat, rye, and triticale, respectively, while rye markers gave an average of 2.0 in rye and none in wheat and triticale. These transferable markers can now be exploited for further genetic and breeding studies in these species.Nebraska Agricultural Research Division, Journal Series No. 14243Communicated by B. Friebe     

Molecular variability in Brycon cf. pesu Müller and Troschel, 1845 (Characiformes: Characidae) from the Araguaia-Tocantins Basin

Brycon pesu is a small-sized fish distributed throughout the Amazon and Orinoco Basins and other coastal basins of northeastern South America. Brycon cf. pesu specimens from the Araguaia-Tocantins Basin are currently separated into two morphotypes, Brycon sp1 and Brycon sp2, owing to different coloration of their anal fin. Brycon sp2 has a reddish margin stripe on the anal fin which morphologically distinguishes it from Brycon sp1. In the present research, nuclear and mitochondrial markers were used to test the hypothesis that the Brycon sp1 and Brycon sp2 morphotypes are distinct species. Specimens from the two morphotypes were collected from the Lajeado Hydroelectric Plant and the Palmas River in the Araguaia-Tocantins Basin. Thirty-five loci obtained by the amplification of five inter-simple sequence repeat primers were analyzed but no species-specific bands were detected. Electrophoretic profiles obtained from 5S rDNA non-transcribed spacer amplification failed to show any differentiation in morphotypes. These results were corroborated by nucleotide sequence analysis of the mtDNA control region, in which 24 polymorphic nucleotide sites, representing a polymorphism rate of only 5%, were detected. The low rates of polymorphism detected by inter-simple sequence repeat, non-transcribed spacer and mtDNA D-loop markers strongly reject the hypothesis that the two morphotypes Brycon sp1 and Brycon sp2 represent distinct species within Brycon cf. pesu. Further studies are needed to obtain conclusive data on the notion that the coloration of the anal fin is an intraspecific polymorphism, possibly related to environmental factors.     

利用SSR标记构建萝卜种质资源分子身份证

为了保护我国特有的优异萝卜资源,促进资源的有效区分和合理利用,保障我国萝卜产业发展,目前需积极开展萝卜种质资源的特异性鉴定和种质识别技术研究。本研究基于SSR分子标记、信息处理和图像处理技术,筛选出22对SSR引物对75份来源和特征不同的代表性萝卜种质进行鉴定,共扩增出153条带,其中多态性条带为87条,平均多态性位点百分率为55.49%,平均每对引物可扩增出6.95条带和3.95条多态性带,有效地显示出每份萝卜种质的特异性。基于最少引物鉴定最多种质的原则,利用MATLAB程序筛选出8对SSR引物,依据8对引物的扩增数据,经过多态性谱带的有序编码转换,构建出75份萝卜种质分子身份证。结果显示利用SSR标记构建萝卜种质分子身份证进行种质资源的鉴定和保护是可行的。     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

Genetic diversity analysis of 60 ginger germplasm core accessions using ISSR and SSR markers

Molecular techniques play a critical role in studies of phylogeny and, thus, have been applied to understand the distribution and extent of genetic variation within and between species. In the present study, a genetic analysis was undertaken using molecular markers (9 ISSR and 13 SSR) on 60 ginger cultivars from different regions of the eastern coast of India (Odisha). The data obtained with 22 polymorphic markers revealed moderate to high diversity in the collection. Both ISSR and SSR markers were efficient in distinguishing all the 60 ginger cultivars. A total of 42 and 160 polymorphic bands were observed with ISSR and SSR markers, respectively. However, SSR markers were observed to be better at displaying average polymorphism (63.29%) than ISSR markers (55%). Analysis of molecular variance results showed that 52 and 66% of the variation occurred among different ginger populations, whereas 48 and 34% of the variation was found within populations, respectively, using ISSR and SSR markers, indicating that ginger cultivars display significant genetic diversity at the population level. Principal coordinates analysis and the dendrogram constructed out of combined data of both markers showed grouping of ginger accessions to their respective area of collection, indicating geographical closeness due to genetic similarity irrespective of the relationship that exists at the morphological level.     

萼花臂尾轮虫多态性及形态型转化

在实验室条件下研究了萼花臂尾轮虫3种形态型(双短侧棘刺、单短侧棘刺和无侧棘刺)个体之间的关系。从轮虫的培养水体中随机挑出若干3种形态型雌体的幼轮虫(龄长<24h),分别培养在正常培养液和晶囊轮虫培养液过滤液中。将实验轮虫的后代(F1代)置于200倍显微镜下检查其个体的形态型。实验结果表明,在正常培养液中,每种形态型轮虫个体都可以产生3种形态型的后代。当培养在晶囊轮虫培养液过滤液中时,这3种形态型轮虫的后代都具有2个显著增长的侧棘刺。此外,这3种形态型个体的轮虫能够共存于同一培养环境中,而且每种形态型个体的轮虫都具有3种生殖类型的雌体。由此推测萼花臂尾轮虫是一个由3种基础形态型个体组成的形态型轮虫复合体。     

SSR标记对不同黄籽甘蓝型油菜亲本材料的遗传分析

杂交育种中,亲本选配是育种成败的关键。本研究以重庆市油菜工程技术研究中心提供的180份甘蓝型黄子油菜亲本种质为材料,应用分布于不同连锁群的60对SSR标记进行了分析,共检测出308个标记位点,每对引物在不同亲本材料之间的等位基因数在1～11个之间,平均位点为5.1个。其中多态性位点207个,多态率达67.2%。对SSR扩增结果进行UPGMA分析,在遗传距离0.566处,180个品种(系)分为3个类群,聚类结果与种质来源比较一致,本研究为甘蓝型油菜黄子杂交育种和优势组合的选配提供了理论依据。