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De novo assembly and characterization of transcriptomes of early-stage fruit from two genotypes of Annona squamosa L. with contrast in seed number.

Gupta Y, Pathak AK, Singh K, Mantri SS, Singh SP, Tuli R - BMC Genomics (2015)

Bottom Line: The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb.A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database.This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

View Article: PubMed Central - PubMed

Affiliation: National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology (DBT), C-127, Industrial Area, Phase-8, -160071, Mohali, India. yogesh@nabi.res.in.

ABSTRACT

Background: Annona squamosa L., a popular fruit tree, is the most widely cultivated species of the genus Annona. The lack of transcriptomic and genomic information limits the scope of genome investigations in this important shrub. It bears aggregate fruits with numerous seeds. A few rare accessions with very few seeds have been reported for Annona. A massive pyrosequencing (Roche, 454 GS FLX+) of transcriptome from early stages of fruit development (0, 4, 8 and 12 days after pollination) was performed to produce expression datasets in two genotypes, Sitaphal and NMK-1, that show a contrast in the number of seeds set in fruits. The data reported here is the first source of genome-wide differential transcriptome sequence in two genotypes of A. squamosa, and identifies several candidate genes related to seed development.

Results: Approximately 1.9 million high-quality clean reads were obtained in the cDNA library from the developing fruits of both the genotypes, with an average length of about 568 bp. Quality-reads were assembled de novo into 2074 to 11004 contigs in the developing fruit samples at different stages of development. The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb. Assembled unigenes were functionally annotated by querying against the protein sequences of five different public databases (NCBI non redundant, Prunus persica, Vitis vinifera, Fragaria vesca, and Amborella trichopoda), with an E-value cut-off of 10(-5). A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database. These sequences could be genes specific to Annona sp. or belong to untranslated regions. Several of the unigenes representing pathways related to primary and secondary metabolism, and seed and fruit development expressed at a higher level in Sitaphal, the densely seeded cultivar in comparison to the poorly seeded NMK-1. A total of 2629 (Sitaphal) and 3445 (NMK-1) Simple Sequence Repeat (SSR) motifs were identified respectively in the two genotypes. These could be potential candidates for transcript based microsatellite analysis in A. squamosa.

Conclusion: The present work provides early-stage fruit specific transcriptome sequence resource for A. squamosa. This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

Show MeSH
Early-stage developing fruits (0, 4, 8, and 12 DAP) in Sitaphal and NMK-1.
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Fig2: Early-stage developing fruits (0, 4, 8, and 12 DAP) in Sitaphal and NMK-1.

Mentions: Pollens were collected from flowers, in male stage, as described by Jalikop and Kumar [4]. The flowers, in female stage, were hand self-pollinated, using freshly collected pollens, in the morning (06.00 and 10.00 h). All the flowers were pollinated at the same time to avoid confounding effect of environment on fruit development. In each pollinated flower, the floral tube was plugged with cotton to prevent contamination of outside pollen. Flowers in similar stages were tagged and left as un-pollinated controls to examine seed numbers in fruits, developed from hand self-pollination and natural open-pollination (Figure 1c). The experiment was performed on three plants (three biological replicates) each of both the genotypes, during July, 2012. Developing fruits were harvested at 4, 8, and 12 days after pollination (DAP) (Figure 2). The gynoecium comprising of unfertilized ovules (0 DAP) was harvested. All the stamens were removed surrounding the gynoecium before harvesting. The 0, 4, 8 and 12 DAP samples were surface sterilized by using absolute ethanol before harvesting. The samples were frozen in liquid nitrogen immediately after harvest, and stored at −80°C until use.Figure 2


De novo assembly and characterization of transcriptomes of early-stage fruit from two genotypes of Annona squamosa L. with contrast in seed number.

Gupta Y, Pathak AK, Singh K, Mantri SS, Singh SP, Tuli R - BMC Genomics (2015)

Early-stage developing fruits (0, 4, 8, and 12 DAP) in Sitaphal and NMK-1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4336476&req=5

Fig2: Early-stage developing fruits (0, 4, 8, and 12 DAP) in Sitaphal and NMK-1.
Mentions: Pollens were collected from flowers, in male stage, as described by Jalikop and Kumar [4]. The flowers, in female stage, were hand self-pollinated, using freshly collected pollens, in the morning (06.00 and 10.00 h). All the flowers were pollinated at the same time to avoid confounding effect of environment on fruit development. In each pollinated flower, the floral tube was plugged with cotton to prevent contamination of outside pollen. Flowers in similar stages were tagged and left as un-pollinated controls to examine seed numbers in fruits, developed from hand self-pollination and natural open-pollination (Figure 1c). The experiment was performed on three plants (three biological replicates) each of both the genotypes, during July, 2012. Developing fruits were harvested at 4, 8, and 12 days after pollination (DAP) (Figure 2). The gynoecium comprising of unfertilized ovules (0 DAP) was harvested. All the stamens were removed surrounding the gynoecium before harvesting. The 0, 4, 8 and 12 DAP samples were surface sterilized by using absolute ethanol before harvesting. The samples were frozen in liquid nitrogen immediately after harvest, and stored at −80°C until use.Figure 2

Bottom Line: The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb.A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database.This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

View Article: PubMed Central - PubMed

Affiliation: National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology (DBT), C-127, Industrial Area, Phase-8, -160071, Mohali, India. yogesh@nabi.res.in.

ABSTRACT

Background: Annona squamosa L., a popular fruit tree, is the most widely cultivated species of the genus Annona. The lack of transcriptomic and genomic information limits the scope of genome investigations in this important shrub. It bears aggregate fruits with numerous seeds. A few rare accessions with very few seeds have been reported for Annona. A massive pyrosequencing (Roche, 454 GS FLX+) of transcriptome from early stages of fruit development (0, 4, 8 and 12 days after pollination) was performed to produce expression datasets in two genotypes, Sitaphal and NMK-1, that show a contrast in the number of seeds set in fruits. The data reported here is the first source of genome-wide differential transcriptome sequence in two genotypes of A. squamosa, and identifies several candidate genes related to seed development.

Results: Approximately 1.9 million high-quality clean reads were obtained in the cDNA library from the developing fruits of both the genotypes, with an average length of about 568 bp. Quality-reads were assembled de novo into 2074 to 11004 contigs in the developing fruit samples at different stages of development. The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb. Assembled unigenes were functionally annotated by querying against the protein sequences of five different public databases (NCBI non redundant, Prunus persica, Vitis vinifera, Fragaria vesca, and Amborella trichopoda), with an E-value cut-off of 10(-5). A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database. These sequences could be genes specific to Annona sp. or belong to untranslated regions. Several of the unigenes representing pathways related to primary and secondary metabolism, and seed and fruit development expressed at a higher level in Sitaphal, the densely seeded cultivar in comparison to the poorly seeded NMK-1. A total of 2629 (Sitaphal) and 3445 (NMK-1) Simple Sequence Repeat (SSR) motifs were identified respectively in the two genotypes. These could be potential candidates for transcript based microsatellite analysis in A. squamosa.

Conclusion: The present work provides early-stage fruit specific transcriptome sequence resource for A. squamosa. This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

Show MeSH