Fox Hunting in Wild Apples: Searching for Novel Genes in Malus Sieversii

Malus sieversii is considered the progenitor of modern apple (Malus pumila) cultivars and to represent a valuable source of genetic diversity. Despite the importance of M. sieversii as a source of disease resistance, stress tolerance, and novel fruit traits, little is known about gene function and diversity in M. sieversii. Notably, a publicly annotated genome sequence for this species is not available. In the current study, the FOX (Full-length cDNA OvereXpressing) gene hunting system was used to construct a library of transgenic lines of Arabidopsis in which each transgenic line overexpresses a full-length gene obtained from a cDNA library of the PI619283 accession of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall–early winter, a time at which many abiotic stress-adaptative genes are expressed. Over 4000 apple FOX Arabidopsis lines have been established from the pool of transgenic seeds and cDNA inserts corresponding to various Gene Ontology (GO) categories have been identified. A total of 160 inserts appear to be novel, with no or limited homology to M. pumila, Arabidopsis, or poplar. Over 1300 lines have also been screened for freezing resistance. The constructed library of transgenic lines provides a valuable genetic resource for exploring gene function and diversity in Malus sieversii. Notably, no such library of t-DNA lines currently exists for any Malus species.

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Screening for Gene Function Using the FOX (Full-Length cDNA OvereXpressor Gene) Hunting System

Methods in Molecular Biology - Plant Chemical Genomics ◽

10.1007/978-1-62703-592-7_19 ◽

2013 ◽

pp. 201-210 ◽

Cited By ~ 4

Author(s):

Mieko Higuchi-Takeuchi ◽

Minami Matsui

Keyword(s):

Gene Function ◽

Full Length ◽

Full Length Cdna ◽

Gene Hunting

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Establishment and Identification of a Normalized Full-Length cDNA Library of CCRI 36

ACTA AGRONOMICA SINICA ◽

10.3724/sp.j.1006.2009.00602 ◽

2009 ◽

Vol 35 (4) ◽

pp. 602-607 ◽

Cited By ~ 2

Author(s):

Dong WU ◽

Jun-Jie LIU ◽

Shu-Xun YU ◽

Shu-Li FAN ◽

Mei-Zhen SONG

Keyword(s):

Cdna Library ◽

Full Length ◽

Full Length Cdna

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PacBio full-length transcriptome of wild apple (Malus sieversii) provides insights into canker disease dynamic response

BMC Genomics ◽

10.1186/s12864-021-07366-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Xiaojie Liu ◽

Xiaoshuang Li ◽

Xuejing Wen ◽

Yan Zhang ◽

Yu Ding ◽

...

Keyword(s):

Dynamic Response ◽

Early Response ◽

Full Length ◽

Differentially Expressed ◽

Late Response ◽

Response Mechanism ◽

Necrotrophic Pathogen ◽

Canker Disease ◽

Malus Sieversii ◽

Highly Correlated

Abstract Background Valsa canker is a serious disease in the stem of Malus sieversii, caused by Valsa mali. However, little is known about the global response mechanism in M. sieversii to V. mali infection. Results Phytohormone jasmonic acid (JA) and salicylic acid (SA) profiles and transcriptome analysis were used to elaborate on the dynamic response mechanism. We determined that the JA was initially produced to respond to the necrotrophic pathogen V. mali infection at the early response stage, then get synergistically transduced with SA to respond at the late response stage. Furthermore, we adopted Pacific Biosciences (PacBio) full-length sequencing to identify differentially expressed transcripts (DETs) during the canker response stage. We obtained 52,538 full-length transcripts, of which 8139 were DETs. Total 1336 lncRNAs, 23,737 alternative polyadenylation (APA) sites and 3780 putative transcription factors (TFs) were identified. Additionally, functional annotation analysis of DETs indicated that the wild apple response to the infection of V. mali involves plant-pathogen interaction, plant hormone signal transduction, flavonoid biosynthesis, and phenylpropanoid biosynthesis. The co-expression network of the differentially expressed TFs revealed 264 candidate TF transcripts. Among these candidates, the WRKY family was the most abundant. The MsWRKY7 and MsWRKY33 were highly correlated at the early response stage, and MsWRKY6, MsWRKY7, MsWRKY19, MsWRKY33, MsWRKY40, MsWRKY45, MsWRKY51, MsWRKY61, MsWRKY75 were highly correlated at the late stage. Conclusions The full-length transcriptomic analysis revealed a series of immune responsive events in M. sieversii in response to V. mali infection. The phytohormone signal pathway regulatory played an important role in the response stage. Additionally, the enriched disease resistance pathways and differentially expressed TFs dynamics collectively contributed to the immune response. This study provides valuable insights into a dynamic response in M. sieversii upon the necrotrophic pathogen V. mali infection, facilitates understanding of response mechanisms to canker disease for apple, and provides supports in the identification of potential resistance genes in M. sieversii.

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Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

Agricultural Sciences in China ◽

10.1016/s1671-2927(11)60304-0 ◽

2011 ◽

Vol 10 (1) ◽

pp. 28-40 ◽

Cited By ~ 1

Author(s):

Li-na WANG ◽

Dong WU ◽

Shu-xun YU ◽

Shu-li FAN ◽

Mei-zhen SONG ◽

...

Keyword(s):

Gossypium Hirsutum ◽

Cdna Library ◽

Full Length ◽

Mads Box ◽

Mads Box Genes ◽

Gossypium Hirsutum L ◽

Full Length Cdna

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A comprehensive overview of FCGR3A gene variability by full-length gene sequencing including the identification of V158F polymorphism

Scientific Reports ◽

10.1038/s41598-018-34258-1 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 12

Author(s):

Niken M. Mahaweni ◽

Timo I. Olieslagers ◽

Ivan Olivares Rivas ◽

Stefan J. J. Molenbroeck ◽

Mathijs Groeneweg ◽

...

Keyword(s):

Gene Sequencing ◽

Full Length ◽

Comprehensive Overview ◽

Gene Variability ◽

Full Length Gene

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Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

Journal of Integrative Agriculture ◽

10.1016/s2095-3119(13)60414-1 ◽

2014 ◽

Vol 13 (2) ◽

pp. 378-386

Author(s):

Su-mei ZHAO ◽

Yong-gang LIU ◽

Hong-bing PAN ◽

Xi ZHANG ◽

Chang-rong GE ◽

...

Keyword(s):

Cdna Library ◽

Expressed Sequence Tags ◽

Full Length ◽

Full Length Cdna ◽

Expressed Sequence

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Association of the IVR Gene with Virus Localization and Resistance

10.32747/1995.7604922.bard ◽

1995 ◽

Author(s):

Gad Loebenstein ◽

William Dawson ◽

Abed Gera

Keyword(s):

Amino Acids ◽

Coat Protein ◽

Plant Viruses ◽

Full Length ◽

N Gene ◽

Complete Suppression ◽

Coat Proteins ◽

Nicotiana Glutinosa ◽

Nicotiana Debneyi ◽

Full Length Gene

We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.

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