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Objective: EST-SSR loci were identified and analyzed based on the transcriptome sequencing data in Polygonatum cyrtonema, in order to develop SSR markers suitable for evaluation and application of germplasm resources on P. cyrtonema. Methods: SSR loci were identified and analyzed in all of 126 544 Unigenes by using MISA tool. SSR primers were designed by using Primer 3.0 software and 50 pairs of SSR primers were randomly selected for validation test. Results: A total of 12 317 SSR loci, including the types of 2-6 nucleotide repeats with occurring frequency of 1/5.91 kb, were identified from 9 982 Unigenes in P. cyrtonema transcriptome. The distribution frequency of SSRs was 9.73%. Dinucleotide repeat was the main type, accounting for as much as 53.14% of all SSRs, followed by trinucleotide repeat (33.31%). The validation test on 50 pairs of SSR primers showed that 29 of them (58%) generated fragments with expected molecular size from P. cyrtonema. The capillary electrophoresis using fluorescence-labeled SSR markers showed that nine genotypes were identified at seven SSR loci in P. cyrtonema, which further demonstrated the validity of these SSR primers. Conclusion: There are numerous SSRs in P. cyrtonema transcriptome with high frequency, rich repeat types and relatively high polymorphic, which will provide abundant valuable candidate markers for genetic diversity analysis and genetic mapping construction in P. cyrtonema, also can be used as a technical tool for molecular identification among Polygonatum species and for molecular marker assistant breeding in superior cultivars of P. cyrtonema.
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Objective: To develop the EST-SSR molecular identification system of Lilium lancifolium, Lilium davidii var. willmottiae, Lilium regale, Lilium casa blanca and Lilium brownie var. viridulum, and analyze the development efficiency and identification ability of EST-SSR molecular marker technology for Lilium genus. Methods: The MISA.pl program was used to identify the SSR locus of the EST gene sequence published by NCBI. The EST-SSR primers were generated by Primer3 program module, and the primers were screened by PCR amplification and agarose gel electrophoresis. The primary screening primers were verified by polyacrylamide gel electrophoresis, and the characteristic bands of different germplasms were labeled and analyzed to construct an identification system. Results: A total of 199 pairs of SSR primers were designed. After screening 26 pairs of primers, two pairs of highly efficient primers were obtained. The molecular identification system constructed by primer JZ391 can effectively identify the mixed commercial materials, which had certain practical value. Conclusion: Based on the extreme genetic characteristics of the research materials, this identification marker development is very efficient. The results confirm that the genetic basis of the species is an important factor affecting the development efficiency of its EST-SSR molecular marker. At the same time, this case can be used as a reference for the development of EST-SSR markers for other Chinese medicinal herbs similar to Lilium genus.
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Objective To explore genetic diversity of and genetic relationships among 18 Picria felterrae populations to provide references for the resource assessment and utilization. Methods The genetic diversity of 18 P. felterrae populations were analyzed using the EST-SSR primer development technology and SSR molecular markers, and cluster analysis was performed based on genetic distances to determine the relationships among those populations. Results A total of 48 pairs of polymorphic primers were selected from 100 pairs of EST-SSR markers, of which 20 pairs were randomly selected and used for amplification of 18 populations. A total of 71 alleles were amplified, 3.55 alleles per primer. Among the primers, the percentage of polymorphic loci (P) varied from 0 to 40.7%, with an average of 19.9%; The polymorphism information content (PIC) varied from 0 to 0.794 1, 0.397 7 on average; The Shannon diversity information index (I) varied from 0 to 1.814 3, with an average of 0.808 4; Obs_Het varied from 0 to 0.442 3, with an average of 0.212 7; And the Exp_Het varied from 0 to 0.826 9, with an average of 0.455 8. For the 18 populations, the Inbreeding Coefficient (Fis) varied from -0.095 3 to 0.663 9, with an average of 0.159 2; The inbreeding coefficient of subgroups (Fit) varied from 0.062 6 to 0.858 7, with an average of 0.537 2; The genetic differentiation coefficient (Fst) varied from 0 to 0.686, with an average of 0.449 6; The gene flow (Nm) varied from 0.114 4 to 0.759 4, with an average of 0.306 1. For the 18 samples tested, the gene diversity index (Nei) varied from 0 to 0.401 6, the I varied from 0 to 0.620 9, Wuzhou Guangxi having the maximum value and Longtan Yunnan the minimum value. Menglong and Jingha, two towns in Yunnan, had the shortest genetic distance (0.031 9), whereas Longzhou Guangxi and Menghai Yunnan had the maximum genetic distance (0.963 8). The 18 populations could be divided into four groups at the location where genetic distance was 0.321 3. The three populations in Guangxi belonged to the same group, populations from Menglong, Menglun and Mengzhe of Yunnan belonged in the same group, populations from Mengsong Yunnan became an independent group, and the rest belonged in the fourth group. Conclusion The genetic differentiation levels of 18 populations were not consistent, and the heterogeneity difference was significant. The gene flow among populations was small, which indicated that the population gene exchange was low. A certain inbreeding rate exists among the populations. The relationship among populations was influenced by geographical isolation and environmental factors. Key words:
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Objective The genetic diversity of the natural populations of Gardenia jasminoides were investigated to provide scientific basis for its resources protection and rational utilization. Methods Fourteen pairs of EST-SSR primers were screened in 19 natural populations of 573 individuals to calculate the genetic parameters of G. jasminoides, and further cluster analysis was then carried out. Results Fourteen pairs of EST-SSR primers generated 75 loci, which showed high genetic diversity maintained in natural populations of G. jasminoides (He = 0.703). Mean population gene diversity (Nei) within populations was 0.603, the Shannon’s diversity index (I) was 1.10. Moderate genetic differentiation (Fst= 0.141) and high gene flow (Nm = 1.523) among populations have been showed too. AMOVA analysis revealed that genetic variation within populations was the main sources of total variation. The Mantle test showed there was no significant correlation between genetic distances and geographic distances. Moreover, significant bottlenecks effects in two-phased model of mutation (TPM) test in 73.7% populations were detected in recent history. Conclusion The results in this study indicated that high level genetic diversity were existed in the natural G. jasminoides populations.
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Objective To analyze the genetic diversity and variation of Psammosilene tunicoides in Yunnan-Guizhou provincial region. Methods The genetic diversity of different populations of P. tunicoides with high polymorphism was analyzed by using EST-SSR primers developed from transcriptomic sequencing technique. Results A total of 17 530 SSR-containing EST sequences were obtained by transcriptomic sequencing, 14 pairs of polymorphism EST-SSR primers were used to analyze the genetic diversity of 17 populations of P. tunicoides in Yunnan-Guizhou region, the results showed that the P. tunicoides in different populations had a high level of genetic diversity with the polymorphic information content (PIC) in the range from 0.350 0 to 0.795 0 in locus level; and had a lower value in group level with percentage of polymorphic bands (PPB) of 64.29%-100%. And the range of Nei’s genetic similarity coefficient was from 0.188 2 to 0.477 7 with mean value of 0.323 2. The gene flow in P. tunicoides in Yunnan-Guizhou region was small with Nm mean value of 0.302 0, and there is a large genetic differentiation between groups with Fst mean value of 0.452 9. Conclusion Transcriptomic sequencing enriched P. tunicoides EST database. The genetic diversity of P. tunicoides might be related to the long evolutionary history of reproductive pattern and distribution area. And there was a highly genetic diversity among the populations of P. tunicoides in Yunnan-Guizhou region, it might be that the geographic barrier cut off the genes exchange among different populations.
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Objective: To expand the exploitation and genetic diversity conservation of medicinal leech resources, the effective functional molecular markers for identification are needed to be developed by transcriptome-SSR analysis. Methods: Totally 14 samples including Poecilobdella javanica and Hirudo nipponia of Hirudinidae, Whitmania pigra Whitman of Haemopidae and Heamadipsa cavatuses sp. nov. of Haemadipsidae were used in transcriptomic analysis by RNA-seqencing, the simple sequence repeats (SSR) distribution characteristics of four species were compared, and transcriptomic comparation of hematophagia samples and hungry samples were carried out to investigate the anticoagulation involved unigene. Finally, the data in two groups were combined to mining functional EST-SSR involved in anticoagulation. Results: The SSR density analysis showed the gap lengths of SSR in transcriptome of Poecilobdella javanica, Hirudo nipponia, Whitmania pigra Whitman, and Heamadipsa cavatuses were 4 723, 6 026, 8 059, and 8 144 bp. In the four species, trinucleotide repeats occupied overwhelming majority in composition of EST-SSR; On the whole, distribution of SSR types can distinct from each other, and Heamadipsa cavatuses was particularly different from others. Moreover, we found that the anticoagulation involved unigene group containing SSR had high fragment-polymophism. Conclusion: For the four leech species, the characteristics of density, composition, and distribution of EST-SSR can reflect its classification relationship. Meanwhile, the EST-SSRs in unigenes which involved in anticoagulation and overlaped in four leech species are ideal fragment group for screening anticoagulation functional marker.
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Ligusticum chuanxiong is a well-known traditional Chinese medicine plant. The study on its molecular markers development and germplasm resources is very important. In this study, we obtained 24 422 unigenes by assembling transcriptome sequencing reads of L. chuanxiong root. EST-SSR was detected and 4 073 SSR loci were identified. EST-SSR distribution and characteristic analysis results showed that the mono-nucleotide repeats were the main repeat types, accounting for 41.0%. In addition, the sequences containing SSR were functionally annotated in Gene Ontology (GO) and KEGG pathway and were assigned to 49 GO categories, 242 KEGG pathways, among them 2 201 sequences were annotated against Nr database. By validating 235 EST-SSRs,74 primer pairs were ultimately proved to have high quality amplification. Subsequently, genetic diversity analysis, UPGMA cluster analysis, PCoA analysis and population structure analysis of 34 L. chuanxiong germplasm resources were carried out with 74 primer pairs. In both UPGMA tree and PCoA results, L. chuanxiong resources were clustered into two groups, which are believed to be partial related to their geographical distribution. In this study, EST-SSRs in L. chuanxiong was firstly identified, and newly developed molecular markers would contribute significantly to further genetic diversity study, the purity detection, gene mapping, and molecular breeding.
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In order to saturate a sunflower genetic map and facilitate marker-assisted selection (MAS) breeding for stress response, it is necessary to enhance map saturation with molecular markers localized in linkage groups associated to genomic regions involved in these traits. This work describes the identification and characterization of 1,134 simple sequence repeat (SSR) containing expressed sequence tags (EST) from unigenes available databases. Twelve of these functional markers as well as 41 public SSR markers were successfully localized in linkage groups, thus contributing to the saturation of specific regions on a reference genetic-linkage-map derived from recombinant inbred lines (RIL) mapping population from the cross between PAC2 x RHA266 lines. The enriched map includes 547 markers (231 SSR, 9 EST-SSR, 3 insertions/deletions (InDel) and 304 amplified fragment length polymorphisms (AFLP) distributed in 17 linkage groups (LG), spanning genetic size to 1,942.3 cM and improving its mean density to 3.6 cM per locus. As consequence, no gaps longer than 13.2 cM remain uncovered throughout the entire map, which increases the feasibility of detecting genes or traits of agronomic importance in sunflower.
Subject(s)
Chromosome Mapping , Helianthus/genetics , DNA, Plant/genetics , Agriculture , Amplified Fragment Length Polymorphism Analysis , Breeding , Genetic Linkage , Genetic Markers , INDEL Mutation , Microsatellite Repeats , Polymerase Chain Reaction , Polymorphism, GeneticABSTRACT
Making use of the gene resources of wild type peanuts is a way to increase the genetic diversity of the cultivars. Marker assisted selection (MAS) could shorten the process of inter-specific hybridization and provide a possible way to remove the undesirable traits. However, the limited number of molecular markers available in peanut retarded its MAS process. We started a peanut ESTs (Expressed Sequence Tags) project aiming at cloning genes with agronomic importance and developing molecular markers. In this study we found 610 ESTs that contained one or more SSRs from 12,000 peanut ESTs. The most abundant SSRs in peanut are trinucleotides (66.3 percent) SSRs and followed by dinucleotide (28.8 percent) SSRs. AG/TC (10.7 percent) repeat was the most abundant and followed by CT/GA (9.0 percent), CTT/GAA (7.4 percent), and AAG/TTC (7.3 percent) repeats. Ninety-four SSR containing ESTs were randomly selected for primer design and synthesis, of which 33 pairs could generate good amplification and were used for polymorphism assessment. Results showed that polymorphism was very low in cultivars, while high level of polymorphism was revealed in wild type peanuts.
Subject(s)
Arachis/genetics , Cloning, Molecular , Expressed Sequence Tags , Microsatellite Repeats , DNA, Plant/genetics , Crop Production , Arachis/growth & development , Base Sequence , Genetic Markers , Polymerase Chain Reaction , Polymorphism, Genetic , Selection, GeneticABSTRACT
Objective To analyze the simple sequence repeat(SSR)information in expressed sequence tag(EST)resource of Saruma henryi and lay a solid foundation for the development of EST-SSR markers in this species.Methods ESTs of S.henryi were downloaded from GenBank and used to perform the contig assembly using Sequencher 4.8.Uni-ESTs were obtained and screened for SSR-containing unigenes using SciRoKo 3.4.The distributing frequency of the EST-SSRs and the basic characteristics of motifs were analyzed.Results A total of 10 274 ESTs of S.henryi were retrieved and were assembled into 6 643 non-redundant Uni-ESTs with a total length of 5.11?106 bp.In all,the data mining yielded 1 408 SSR loci,which corresponded to 1 232 Uni-ESTs(18.55%).On average,EST-SSRs spanned 22.30 bp,and occurred every 3.63 kb in length.In S.henryi,mononucleotide repeats predominated with an occurrence frequency of 12.24%.Dinucleotide repeats followed with a frequency of 5.01%.The most frequent one was A/T among all the repeat motifs,then followed by AG/CT.Conclusion SSRs in ESTs of S.henryi display a relatively high level of occurrence frequency and show abundance of types.