Development of EST‐SSR markers for diversity and breeding studies in opium poppy

All publicly available opium poppy expressed sequence tag (EST) sequences, totalling 20 885, were assembled into unigenes and examined for simple sequence repeats (SSRs). Nearly 19% of the 14 957 unigenes contained SSRs with 4% harbouring more than one SSR. Average density of the SSRs was 1 SSR per 3.6 kb of non‐redundant EST sequence. Trinucleotide SSRs were most frequently identified (39%), and many of the most prevalent motifs were AT‐rich. Flanking primers were designed for 86% of the SSRs and 67 primer pairs were tested on 37 opium poppy accessions and seven related species. All markers were transferable to the related species. Polymorphism information content (PIC) values for the markers were intermediate for comparisons within opium poppy (average of 0.27) and slightly higher for comparisons across species (average of 0.29). The markers were found to be useful for diversity analysis as they successfully distinguished among Turkish opium poppy accessions and land races.     

枳壳EST-SSR标记的开发

GenBank上已公布的枳壳EST序列为开发新的SSR标记提供了宝贵的数据资源。本研究利用在线SSR鉴定软件SSRIT分析来自枳壳EST数据库的11029条Unigene序列。分析结果共发现327条EST序列含有348个SSR位点,占总数的2.96%。其中,三核苷酸重复的SSR类型最多,共有161个,占检索总数的46.26%。Primer 3.0设计合成58对EST-SSR引物,其中36对能扩增出产物,6对引物产生多态性分离,分别占所设计引物总数的62.07%和10.34%。本文研究成果为今后枳壳遗传多样性分析、遗传图谱构建及比较基因组等研究方面奠定了基础。     

Genetic diversity in groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes and detection of marker trait associations for plant habit and seed size using genomic and genic SSRs

Groundnut (Arachis hypogaea L.) an important oilseed crop in India is known to have narrow genetic base. Therefore, the assessment of genetic diversity and detection of marker-trait association are important objectives for the genetic improvement of groundnut. The present study involved the development of 192 SSR markers from Arachis genomic survey sequences. From these, seven polymorphic SSRs along with 15 other genomic SSRs, 19 genic SSRs, and three STS markers were used to detect genetic diversity among 44 groundnut genotypes. These polymorphic SSR markers amplified 155 bands (76 genomic and 79 genic), of these 128 bands (67 genomic and 61 genic) were polymorphic. The genomic SSR exhibited 88.1% and genic SSRs displayed 77.2% allelic polymorphism. The polymorphic information content (PIC) of the markers ranged from 0.04 to 0.95. The pair-wise genetic similarity ranged from 24.2 to 90.7% for genomic SSR and 32.9 to 97.9% for genic SSR markers. Cluster analysis based on the pooled data from both genomic and genic SSRs revealed a dendrogram which could distinguish all the genotypes. Further, the AMOVA analysis detected 16.7% genetic variation due to differences in seed size and 13.0% due to plant habit. Based on locus-by-locus AMOVA and Kruskal-Wallis ANOVA and further confirmation by discriminant analysis and general linear model, six markers were found to be associated with plant habit and four markers with seed size.     

Simple sequence repeats as useful resources to study transcribed genes of cotton

Microsatellites or Simple Sequence Repeats(SSRs) are informative molecular genetic markers in many crop species. SSRs are PCR-based, highly polymorphic, abundant, widely distributed throughout the genome and inherited in a co-dominant manner in most cases. Here we describe the presence of SSRs in cDNAs of cotton. Thirty one SSR primer pairs of 220 (∼14%) tested led to PCR amplification of discrete fragments using cotton leaf cDNA as template. Sequence analysis showed 25% of 24randomly selected cDNA clones amplified with different SSR primer pairs contained repeat motifs. We further showed that sequences from the SSR-containing cDNAs were conserved across G. barbadense and G. hirsutum, revealing the importance of the SSR markers for comparative mapping of transcribed genes. Data mining for plant SSR-ESTs from the publicly available databases identified SSRs motifs in many plant species,including cotton, in a range of 1.1 to4.8% of the submitted ESTs for a given species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Developing and validating microsatellite markers in elephant grass (<Emphasis Type="Italic">Pennisetum purpureum</Emphasis> S.)

Elephant grass [Pennisetum purpureum S.; syn. Cenchrus purpureus (Schumach.) Morrone] is an important global forage crop and is recognized for high yields of herbage with good nutritive value. It also has high biomass potential to be utilized as a biofuel feedstock. Whereas several previous genetic studies adapted simple sequence repeat (SSR) markers from pearl millet [Pennisetum glaucum (L.) R.Br.] for investigations in elephant grass, the present study developed SSR markers from 3536 DNA sequences derived from 16 elephant grass entries. A total of 3866 SSRs were identified including 1028 monomeric, 2019 dimeric, 735 trimeric, 49 tetrameric, 20 pentameric and 15 hexameric repeat motifs. Three hundred and seven sequences contained more than one repeated motif, and 154 SSRs were present in compound formation. Susequenctly,  four elephant grass and two pearl millet genotypes were chosen to validate 727 SSR markers. Of these, 628 markers produced visually detectable amplification products, including 73 (11.6%) polymorphic ones across all six genotypes. Polymorphism between the four elephant grass genotypes was revealed by 316 (50.6%) markers with diversity index values ranging from 0.75 to 0.38. Dimeric SSRs had the highest polymorphic rate (48.7%). These validated SSR markers had 58.6% (368 of 628) transferability rate to pearl millet. The availability of these polymorphic SSR markers will support advanced genetic studies in P. purpureum and its relatives.     

Identification and characterization of EST-SSRs in finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

In recent years, microsatellites have become the most used markers for studying population genetic diversity. The increased availability of the DNA sequences has given the possibility to develop EST-derived SSR markers. A total of 1,927 ESTs of Eleusine coracana available in the NCBI database were mined for SSRs. Di-nucleotides are the most occurring motifs accounting for more than 50% of the repeats, of which GA was the most abundant motif and tetra-nucleotides are the least occurring motifs. Of the 132 markers identified, 30 primer pairs based were synthesized. SSR markers were used for variety discrimination and genetic assessment in 15 finger millet accessions; 20 primers showed polymorphism and 13 primers were identified as having a PIC value above 0.5. On the basis of the distribution of these polymorphic alleles, the 15 accessions were classified into two groups. This study has demonstrated the potential of EST-derived SSR primer pairs in finger millet. These primers will serve as valuable source for further breeding programs.     

香蕉根系转录组SSR位点信息分析

分析香蕉根系转录组中的SSR位点信息,并设计SSR引物,为开发新的分子标记奠定基础。利用MISA工具对香蕉根系转录组unigene序列进行SSR检索。从25158条unigene中共发现4663个SSR,分布在3820条unigene序列中,出现频率为18.53%,平均每7.77 kb含有1个SSR位点,共有58种重复基元。香蕉根系转录组中,二、三核苷酸重复基元所占比例最大,分别为40.50%和40.63%;二者分别以AG/CT和AGG/CCT重复基元为主,分别占该重复基元的88.03%和30.83%。SSR重复次数以5~9次为主,基序长度主要分布于12~20 bp,平均长度为18.80 bp。香蕉根系转录组SSR位点频率、密度较高,类型多样,在香蕉遗传多样性研究及分子标记辅助育种中有较大应用潜能。     

Characterization of microsatellite markers,their transferability to orphan legumes and use in determination of genetic diversity among chickpea (Cicer arietinum L.) cultivars

The microsatellite or simple sequence repeat (SSR) marker is the most preferred marker because of its many desirable properties. It is important to increase the genic and genomic resources particularly in legumes because the SSR markers currently available in chickpea, pigeonpea, horsegram, blackgram, and cowpea are very limited. In the present study, 201 pairs of SSR markers comprising of 172 genic and 29 genomic SSRs were screened against 11 chickpea genotypes, among which 153 produced monomorphic and 48 produced polymorphic bands. The polymorphic information content ranged from 0.152 to 0.373 for both genic and genomic SSRs. Among the polymorphic markers, two-three alleles were detected for genic and two-four alleles for genomic SSRs. A unique banding pattern could be found for all the genotypes within 48 polymorphic SSR markers and cultivar specific markers could be identified for seed purity test. We have also studied the ability of chickpea genic and genomic SSRs to amplify distantly related but important legumes viz., horsegram, blackgram, cowpea, pigeonpea, and soybean. Out of 201 chickpea SSR primer pairs, 66.7% in blackgram, 62.2% in horsegram, 61.7% in redgram, 54.7% in cowpea, and 62.7% in soybean produced amplification. The transferability of about 60.0% of the chickpea SSRs to distantly related legumes could be considered successful. In the present study, 134, 133, 126, 124, and 110 new SSR primers for blackgram, horsegram, soybean, redgram, and cowpea pulse crops, respectively, were identified. It is an important addition to the already available genomic resources in these crops. In addition, among genic primer pairs, 12 in horsegram, three in soybean, 13 in redgram, and eight in cowpea, and among genomic primer pairs, two in horsegram and four in redgram were polymorphic even in the two-three genotypes tested indicating their potentially for application in genetic studies and mapping.     

鹅掌楸EST-SSR引物开发及通用性分析

通过对6520条鹅掌楸EST序列进行检索,在364条ESTs中发现394个SSRs,鹅掌楸EST-SSRs平均密度为每8.5kb含有1个SSR;在检索出的SSRs中,二核苷酸重复单元的SSRs类型最多,占总数的61.9%。利用SSR-ESTs序列共设计176对EST-SSR引物,其中132对在鹅掌楸上有扩增产物,66对扩增出多态,多态性引物占所设计引物的36.9%。这批EST-SSR引物在物种之间具有较高的通用性。研究表明在鹅掌楸中表现多态的66对EST-SSR引物,85%在中国马褂木中有扩增,54%在白玉兰中有扩增。     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.     

濒危珍稀植物半枫荷转录组中SSR位点分析

分析半枫荷转录组中的SSR位点信息,并设计简单重复序列(SSR)引物,以期为半枫荷EST-SSR分子标记提供有力工具。利用MISA工具筛选了半枫荷转录组测序获得的77629条Unigenes,对其SSR位点信息进行了分析;在此基础上利用Primer 3.0设计SSR引物,并随机选择50对SSR引物对4株不同来源的半枫荷进行多态性扩增分析。在半枫荷的转录组中,共找到15041个SSRs,分布于10669条Unigenes,SSR位点发生频率为13.74%,含多个位点的序列数为3114,占SSRs位点总数的29.19%,以复合形式出现的位点数2044个,占SSRs位点总数的19.16%,SSRs的平均距离是3.2 kb。SSRs位点中二碱基重复是主要类型,占总SSRs的42.17%;其次是单碱基重复基序(38.25%)SSRs。所包含的重复基元中,单碱基重复基元A/T(5572),二碱基重复基元AG/CT(4845)是优势重复基元类型,分别占总SSRs的37.05%、32.21%。利用Primer 3共设计出28590对SSR引物。随机选择50对引物进行PCR扩增,其中44对(88.0%)扩增出清晰、可重复的条带,15对(34.1%)扩增条带表现出多态性。半枫荷转录组SSR位点出现频率高,类型丰富;大量的SSR为其遗传多样性分析、分子标记辅助育种和遗传图谱构建提供了丰富的候选分子标记。     

Transferability of rice SSR markers to Miscanthus sinensis, a potential biofuel crop

Miscanthus sinensis (M. sinensis) is a perennial C₄ photosynthesis grass, with high yield, high efficiency of water usage, low fertilizer requirement, tolerance to extreme environments, and is one of the plant species with good biofuel potential. Simple sequence repeat (SSR) markers are highly informative and widely used in plant genetic studies. In this study, 88 SSR primer pairs derived from the rice genome, including 47 EST-SSRs (eSSRs) and 41 genomic SSRs (gSSRs), were evaluated for cross-species transferability to M. sinensis. Forty-one SSR primer pairs in total could successfully amplify DNA fragments in M. sinensis, showing an overall transferability rate of 46.6 % between rice and M. sinensis. The transferability rate of eSSR (51.1 %) was higher than that of gSSR (41.5 %). A total of 140 SSR loci and 340 alleles in the set of rice and M. sinensis germplasm collections were detected. Nei’s gene diversity varied from 0 to 0.72 and averaged 0.35. Shannon’s information index varied from 0 to 1.49 and averaged 0.56. The observed heterozygosity ranged from 0 to 0.95 and averaged 0.08. Thirty-nine loci (27.86 %) were shown heterozygosity out of 140 SSR loci. A dendrogram based on genetic distance showed a significant geographic differentiation. Our results indicated that 46.6% of the rice SSR markers are transferable to M. sinensis, and are useful for germplasm evaluation and genetic analysis in M. sinensis.     

茶树花转录组微卫星分布特征

利用Perl语言,对茶树花转录组序列进行大通量SSR位点的发掘,发现含SSR的序列10 290条,共12 582个SSR,平均2.41 kb出现一个SSR。在茶树花的转录组中共发现340种碱基重复模式,所占比例最高的是(AG/CT)n(44.99%)。在49 586条注释成功的茶树花Unigene中,共发现10 490个SSR位点,其中位于编码区的1917个,其出现频率仅为0.102 SSR/1000 bp,而非编码区为3.072 SSR/1000 bp。在基因编码区中出现频率最高的是三碱基微卫星(1140,59.5%),其次是六碱基微卫星(524,27.3%)。茶树花转录组所含微卫星以重复长度小于20 bp的序列最多,大于20 bp的仅为25.2%。茶树花转录组中,含微卫星基因的平均表达水平显著低于不含微卫星基因,其中含复杂微卫星基因的平均基因表达水平最低。     

苦荞全基因组SSR位点特征分析与分子标记开发

目前苦荞SSR多态性标记数量较少,根据已发表的苦荞基因组测序数据,利用MISA软件对1~6核苷酸重复的SSR位点进行了查找和序列特征分析,批量设计引物并对引物进行了有效性和多态性检测。结果表明,苦荞基因组中共检测到1 640个SSR位点,其中三核苷酸重复型SSR最多,占比63.29%,五核苷酸重复型最少,仅占0.12%。AT/TA、AAG/CTT、ACC/GGT和ATC/GAT为出现频率较高的重复基序。苦荞基因组SSR序列长度变化范围为12~476bp,平均长度23.14bp,长度12~19bp的占比71.71%,长度≥20bp的占比28.29%。根据不同类型SSR位点设计并合成引物479对,选择200对引物对5份苦荞资源和3份甜荞资源进行多态性检测,有56对扩增出多态性条带,17对在苦荞种质中产生多态性条带,48对在甜荞种质中产生多态性条带,9对同时在两种种质中产生多态性条带。利用苦荞全基因组序列可实现SSR标记的批量开发,可鉴定出适用于苦荞和甜荞遗传多样性分析、遗传图谱构建和品种鉴定等研究的SSR引物。     

Microsatellite polymorphism in Pisum sativum

Pisum sativum sequences were retrieved from Genbank/EMBL databases and searched for all possible dinucleotide and trinucleotide tandem repeats. One‐hundred and seventy‐one simple sequence repeats (SSRs) were found among 663 sequences. The different dinucleotide or trinucleotide motifs occurred at varying frequencies. CT/AG was the most frequent dinucleotide, and TCT/AGA the most frequent trinucleotide. Forty‐three microsatellite markers were generated from these sequences and used to assess the genetic variability among 12 pea genotypes. Thirty‐one were polymorphic among the genotypes and the average number of variants per marker was 3.6 when considering only polymorphic markers. Overall, the number of variants for a given SSR marker was correlated with the length of the SSR but some 12‐bp long SSRs showed the same degree of polymorphism as longer ones. The groupings resulting from the SSR genotyping among the 12 genotypes gave an interesting insight into the possible origin of one recent cultivar. Database‐derived SSR markers are highly variable. They can provide useful information on the genetic diversity among P. sativum cultivated types.