Towards sex identification of Asian Palmyra palm (Borassus flabelliferL.) by DNA fingerprinting, suppression subtractive hybridization andde novotranscriptome sequencing

BackgroundAsian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing.MethodsDNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries andde novotranscriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers.ResultsThrough extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Althoughde novotranscriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species.

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Peer Review #1 of "Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing (v0.1)"

10.7287/peerj.7268v0.1/reviews/1 ◽

2019 ◽

Keyword(s):

Peer Review ◽

Dna Fingerprinting ◽

Suppression Subtractive Hybridization ◽

Transcriptome Sequencing ◽

De Novo ◽

Subtractive Hybridization ◽

Sex Identification ◽

De Novo Transcriptome ◽

De Novo Transcriptome Sequencing

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Peer Review #2 of "Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing (v0.2)"

10.7287/peerj.7268v0.2/reviews/2 ◽

2019 ◽

Keyword(s):

Peer Review ◽

Dna Fingerprinting ◽

Suppression Subtractive Hybridization ◽

Transcriptome Sequencing ◽

De Novo ◽

Subtractive Hybridization ◽

Sex Identification ◽

De Novo Transcriptome ◽

De Novo Transcriptome Sequencing

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Peer Review #2 of "Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing (v0.1)"

10.7287/peerj.7268v0.1/reviews/2 ◽

2019 ◽

Keyword(s):

Peer Review ◽

Dna Fingerprinting ◽

Suppression Subtractive Hybridization ◽

Transcriptome Sequencing ◽

De Novo ◽

Subtractive Hybridization ◽

Sex Identification ◽

De Novo Transcriptome ◽

De Novo Transcriptome Sequencing

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Large-scale gene gains and losses molded the NLR defense arsenal during the Cucurbita evolution

Planta ◽

10.1007/s00425-021-03717-x ◽

2021 ◽

Vol 254 (4) ◽

Author(s):

Giuseppe Andolfo ◽

Cristina S. Sánchez ◽

Joaquìn Cañizares ◽

Maria B. Pico ◽

Maria R. Ercolano

Keyword(s):

Large Scale ◽

Ad Hoc ◽

De Novo ◽

Gene Annotation ◽

Death Process ◽

Pcr Analysis ◽

Gene Gain ◽

R Genes ◽

Protein Classes ◽

Species Specific

Abstract Main conclusion Genome-wide annotation reveals that the gene birth–death process of the Cucurbita R family is associated with a species-specific diversification of TNL and CNL protein classes. Abstract The Cucurbitaceae family includes nearly 1000 plant species known universally as cucurbits. Cucurbita genus includes many economically important worldwide crops vulnerable to more than 200 pathogens. Therefore, the identification of pathogen-recognition genes is of utmost importance for this genus. The major class of plant-resistance (R) genes encodes nucleotide-binding site and leucine-rich repeat (NLR) proteins, and is divided into three sub-classes namely, TIR-NB-LRR (TNL), CC-NB-LRR (CNL) and RPW8-NB-LRR (RNL). Although the characterization of the NLR gene family has been carried out in important Cucurbita species, this information is still linked to the availability of sequenced genomes. In this study, we analyzed 40 de novo transcriptomes and 5 genome assemblies, which were explored to investigate the Cucurbita expressed-NLR (eNLR) and NLR repertoires using an ad hoc gene annotation approach. Over 1850 NLR-encoding genes were identified, finely characterized and compared to 96 well-characterized plant R-genes. The maximum likelihood analyses revealed an unusual diversification of CNL/TNL genes and a strong RNL conservation. Indeed, several gene gain and loss events have shaped the Cucurbita NLR family. Finally, to provide a first validation step Cucurbita, eNLRs were explored by real-time PCR analysis. The NLR repertories of the 12 Cucurbita species presented in this paper will be useful to discover novel R-genes.

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Identification of New Potential Regulators of the Medicago truncatula—Sinorhizobium meliloti Symbiosis Using a Large-Scale Suppression Subtractive Hybridization Approach

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-3-0321 ◽

2007 ◽

Vol 20 (3) ◽

pp. 321-332 ◽

Cited By ~ 28

Author(s):

Laurence Godiard ◽

Andreas Niebel ◽

Fabienne Micheli ◽

Jérôme Gouzy ◽

Thomas Ott ◽

...

Keyword(s):

Signal Transduction ◽

Suppression Subtractive Hybridization ◽

Medicago Truncatula ◽

Large Scale ◽

Sinorhizobium Meliloti ◽

Zinc Finger Protein ◽

Gene Clusters ◽

Subtractive Hybridization ◽

Symbiotic Interaction ◽

Genes Encoding

We set up a large-scale suppression subtractive hybridization (SSH) approach to identify Medicago truncatula genes differentially expressed at different stages of the symbiotic interaction with Sinorhizobium meliloti, with a particular interest for regulatory genes. We constructed 7 SSH libraries covering successive stages from Nod factor signal transduction to S. meliloti infection, nodule organogenesis, and functioning. Over 26,000 clones were differentially screened by two rounds of macroarray hybridizations. In all, 3,340 clones, corresponding to genes whose expression was potentially affected, were selected, sequenced, and ordered into 2,107 tentative gene clusters, including 767 MtS clusters corresponding to new M. truncatula genes. In total, 52 genes encoding potential regulatory proteins, including transcription factors (TFs) and other elements of signal transduction cascades, were identified. The expression pattern of some of them was analyzed by quantitative reverse-transcription polymerase chain reaction in wild-type and in Nod¯ M. truncatula mutants blocked before or after S. meliloti infection. Three genes, coding for TFs of the bHLH and WRKY families and a C2H2 zinc-finger protein, respectively, were found to be upregulated, following S. meliloti inoculation, in the infection-defective mutant lin, whereas the bHLH gene also was expressed in the root-hair-curling mutant hcl. The potential role of these genes in early symbiotic steps is discussed.

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Identification of Defense-Related Rice Genes by Suppression Subtractive Hybridization and Differential Screening

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.5.685 ◽

2001 ◽

Vol 14 (5) ◽

pp. 685-692 ◽

Cited By ~ 93

Author(s):

Lizhong Xiong ◽

Min-Woo Lee ◽

Min Qi ◽

Yinong Yang

Keyword(s):

Suppression Subtractive Hybridization ◽

Zinc Finger Protein ◽

De Novo ◽

Subtractive Hybridization ◽

Defense Responses ◽

Rice Cultivar ◽

Differential Screening ◽

Mitogen Activated Protein Kinase ◽

Japonica Rice ◽

Bacterial Colony

Identification of host genes involved in defense responses is one of most critical steps leading to the elucidation of disease resistance mechanisms in plants. In this study, two different cloning strategies were employed to identify defense-related genes from a tropical japonica rice cultivar (Oryza sativa cv. Drew). With the use of bacterial colony arrays, differential screening of a blast fungus (Pyricularia grisea)-induced rice cDNA library led to the isolation of 22 distinct rice genes that are expressed differentially in response to blast infection. Sequence analysis indicates that most of them are full-length cDNAs encoding pathogenesis-related proteins or other relatively abundant proteins. In combination with treatments of cycloheximide plus jasmonic acid (JA) or benzothiadiazole (BTH) in rice seedlings, the polymerase chain reaction-based suppression subtractive hybridization also was conducted to search for immediate early (IE) defense-related genes whose transcription is independent of de novo protein synthesis. The initial screening of only 768 subtracted clones resulted in the identification of 34 distinct IE genes that are induced by JA, BTH, and/or blast infection. Database searches revealed that these IE genes encode putative mitogen-activated protein kinase, diacylglycerol kinase, zinc finger protein, RelA-SpoT protein, ankyrin-containing protein, ABC transporter, β-ketoacyl-CoA synthase, and other potential defense-signaling components. Further characterization of these novel IE genes will likely facilitate the elucidation of defense signal transduction in rice plants.

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