Development Of genic-SSR Markers Research Articles

Development of Genic-SSR Markers in Myrtle by RNA-seq

Development of Genic-SSR Markers in Myrtle by RNA-seq

Comparative de novo transcriptome analysis identifies salinity stress responsive genes and metabolic pathways in sugarcane and its wild relative Erianthus arundinaceus [Retzius] Jeswiet

Erianthus arundinaceus [Retzius] Jeswiet, a wild relative of sugarcane has a high biomass production potential and a reservoir of many genes for superior agronomic traits and tolerance to biotic and abiotic stresses. A comparative physiological, anatomical and root transcriptome analysis were carried out to identify the salt-responsive genes and metabolic pathways associated with salt-tolerant E. arundinaceus genotype IND99-907 and salinity-sensitive sugarcane genotype Co 97010. IND99-907 recorded growth of young leaves, higher proline content, higher relative water content, intact root anatomical structures and lower Na+/K+, Ca2+/K+ and Mg2+/K+ ratio as compared to the sugarcane genotype Co 97010. We have generated four de novo transcriptome assemblies between stressed and control root samples of IND99-907 and Co 97010. A total of 649 and 501 differentially expressed genes (FDR<0.01) were identified from the stressed and control libraries of IND99-907 and Co 97010 respectively. Genes and pathways related to early stress-responsive signal transduction, hormone signalling, cytoskeleton organization, cellular membrane stabilization, plasma membrane-bound calcium and proton transport, sodium extrusion, secondary metabolite biosynthesis, cellular transporters related to plasma membrane-bound trafficking, nucleobase transporter, clathrin-mediated endocytosis were highly enriched in IND99-907. Whereas in Co 97010, genes related to late stress-responsive signal transduction, electron transport system, senescence, protein degradation and programmed cell death, transport-related genes associated with cellular respiration and mitochondrial respiratory chain, vesicular trafficking, nitrate transporter and fewer secondary metabolite biosynthetic genes were highly enriched. A total of 27 pathways, 24 biological processes, three molecular functions and one cellular component were significantly enriched (FDR≤ 0.05) in IND99-907 as compared to 20 pathways, two biological processes without any significant molecular function and cellular components in Co 97010, indicates the unique and distinct expression pattern of genes and metabolic pathways in both genotypes. The genomic resources developed from this study is useful for sugarcane crop improvement through development of genic SSR markers and genetic engineering approaches.

Development of genic-SSR markers and their application in revealing genetic diversity and population structure in an Eastern and North-Eastern Indian collection of Jack (Artocarpus heterophyllus Lam.)

Jack (Artocarpus heterophyllus) is a multi-purpose out-breeding tree species of the family Moraceae. We generated 42,928,887 high-quality expressed sequence reads, assembled them into 89,356 unigenes, and discovered 16,853 unigene-based perfect SSRs in A. heterophyllus. Thirty-eight polymorphic SSRs were used to analyze the genetic diversity and population structure of 224 germplasm accessions of A. heterophyllus constituting three populations from three agro-climatic zones, namely Eastern Plateau and Hills, Middle Gangetic Plain Region, and Eastern Himalayan Region, encompassing five Eastern and North-Eastern states of India. At the 38 SSR loci, we detected 142 alleles with a mean of 3.74 alleles per locus. The PIC values for the loci ranged from 0.25 to 0.69. The maximum genetic diversity was recorded in Eastern Plateau and Hills (I = 0.98, He = 0.52). The ANOVA analysis indicated significantly higher within-population variation (90%) than between populations (10%). The indirect estimation of gene flow (Nm) from PhiPT indicated significant gene flow among all three populations. The population structure analysis showed at least four distinct groups among the three populations with different introgression degrees. The NJ-based clustering grouped the 224 germplasm accessions into three main clusters, each with three sub-clusters. However, we did not observe distinct geographical structure among populations except some clustering among the germplasm accessions of the populations of geographically close locations. The transcriptome dataset and the SSR markers developed in the study would boost the species' molecular characterization, conservation, and specific need-based improvement.

Discovery of novel genic-SSR markers from transcriptome dataset of an important non-human primate, Macaca fascicularis

Macaca fascicularis, also known as the cynomolgus macaque, is an important non-human primate animal model used in biomedical research. It is an Old-World primate widely distributed in Southeast Asia and is one of the most abundant macaque species in Malaysia. However, the genetic structure of wild cynomolgus macaque populations in Malaysia has not been thoroughly elucidated. In this study, we developed genic-simple sequence repeat (genic-SSR) markers from an in-house transcriptome dataset generated from the Malaysian cynomolgus macaque via RNA sequencing, and applied these markers on 26 cynomolgus macaque individuals. A collection of 14,751 genic-SSRs were identified, where 13,709 were perfect SSRs. Dinucleotide repeats were the most common repeat motifs with a frequency of 65.05%, followed by trinucleotide repeats (20.55%). Subsequently, we designed 300 pairs of primers based on perfect di- and trinucleotide SSRs, in which 105 SSRs were associated with functional genes. A subset of 30 SSR markers were randomly selected and validated, yielding 19 polymorphic markers with an average polymorphism information content value of 0.431. The development of genic-SSR markers in this study is indeed timely to provide useful markers for functional and population genetic studies of the cynomolgus macaque and other related non-human primate species.

Development of genic-SSR markers and genetic diversity of Indian lettuce (Lactuca indica L.) in South Korea.

Indian lettuce (Lactuca indica L.) is an undomesticated wild plant with high economic potential. We know little about the plant's genome, such as its DNA markers, making genetic research using this plant difficult. In this study, 100 genic simple sequence repeat (SSR) primers with a 99-250bp target amplicon were synthesized from L. indica transcriptomic sequences. These primers were examined in 8 diverse L. indica accessions, and 90 polymorphic SSRs were obtained. Twenty-three of the 90 polymorphic SSRs were used to investigate transferability to another two Lactuca species, Lactuca serriola and Lactuca sativa. Genetic diversity was investigated in 77 Lactuca accessions, including 73 L. indica collected from across South Korea, 2 L. serriola, and 2 L. sativa. Our genic-SSR markers were highly polymorphic with a mean polymorphic information content of 0.61 and, on average, 10.83 alleles per locus. The average expected heterozygosity (0.76) was higher than the observed heterozygosity. An analysis of molecular variance revealed that most of the total variance in our population is attributable to genetic variation among accessions, rather than among provinces. STRUCTURE, unweighted neighbor-joining phylogenetic trees, and principal coordinate analyses resulted in three clusters, where northern and central-southern L. indica accessions were grouped into two clusters with some admixture. The L. serriola and L. sativa accessions did not produce a separate cluster due to a small sample size. These results show our SSR markers will be useful in germplasm assessment and genetic studies of L. indica and other Lactuca species.

De novo Assembly, Characterization of Immature Seed Transcriptome and Development of Genic-SSR Markers in Black Gram [Vigna mungo (L.) Hepper

Black gram [V. mungo (L.) Hepper] is an important legume crop extensively grown in south and south-east Asia, where it is a major source of dietary protein for its predominantly vegetarian population. However, lack of genomic information and markers has become a limitation for genetic improvement of this crop. Here, we report the transcriptome sequencing of the immature seeds of black gram cv. TU94-2, by Illumina paired end sequencing technology to generate transcriptome sequences for gene discovery and genic-SSR marker development. A total of 17.2 million paired-end reads were generated and 48,291 transcript contigs (TCS) were assembled with an average length of 443 bp. Based on sequence similarity search, 33,766 TCS showed significant similarity to known proteins. Among these, only 29,564 TCS were annotated with gene ontology (GO) functional categories. A total number of 138 unique KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified, of which majority of TCS are grouped into purine metabolism (678) followed by pyrimidine metabolism (263). A total of 48,291 TCS were searched for SSRs and 1,840 SSRs were identified in 1,572 TCS with an average frequency of one SSR per 11.9 kb. The tri-nucleotide repeats were most abundant (35%) followed by di-nucleotide repeats (32%). PCR primer pairs were successfully designed for 933 SSR loci. Sequences analyses indicate that about 64.4% and 35.6% of the SSR motifs were present in the coding sequences (CDS) and untranslated regions (UTRs) respectively. Tri-nucleotide repeats (57.3%) were preferentially present in the CDS. The rate of successful amplification and polymorphism were investigated using selected primers among 18 black gram accessions. Genic-SSR markers developed from the Illumina paired end sequencing of black gram immature seed transcriptome will provide a valuable resource for genetic diversity, evolution, linkage mapping, comparative genomics and marker-assisted selection in black gram.

Development and characterization of genic SSR markers for mungbean (Vigna radiata (L.) Wilczek)

Mungbean (Vigna radiata (L.) Wilczek) is an important food legume, but has limited genomic resources. In this study, the development of genic SSR markers from mungbean transcriptome sequences and their transferability to related Vigna species is presented. A total of 12,596 EST sequences from mungbean genotype ‘Jangan’ were searched for the presence of SSRs and 2,299 SSR motifs from 1,848 SSR containing unique ESTs were identified with an average of one SSR per 3.4 kb. Trinucleotide motifs (48 %) were the most abundant repeats followed by di- (25 %), tetra- (15 %), hexa- (7 %) and penta- (5 %) nucleotide repeats. Based on these SSR containing sequences, PCR primer pairs were successfully designed for 1,742 SSR loci and randomly selected 100 primer pairs were further used for characterization. Sequences analyses indicate that about 45 % of the SSR motifs were present in the coding sequences (CDS) and 55 % were located in the untranslated regions (UTRs). Trinucleotide repeats (62 %) were preferentially present in the CDS. Randomly selected 27 SSR markers were used to study the genetic relationship among 20 mungbean genotypes. Twenty one (78 %) genic SSR markers showed polymorphism and the number of alleles ranged from two to six with an average PIC value of 0.34. Cluster analysis based on similarity coefficient clearly separated the cultivated and the wild genotypes into separate groups. Transferability study showed that 97 % SSR markers were transferable to eight other Vigna species, hereby expanding their utility. This study gives an insight into frequency, type and distribution of SSRs in the genic region of mungbean and report the development of a large set of SSR markers for mungbean. These genic SSR markers will provide an important genomic resource for qualitative and quantitative traits mapping, marker assisted selection and genetic diversity studies in mungbean and related Vigna species.