Annotation and validation of genes involved in photosynthesis and starch synthesis from a <i>Manihot</i> full-length cDNA library

doi:10.15302/J-FASE-2016113

Front. Agr. Sci. Eng.

2016, Vol. 3

Issue (4) : 308-320 https://doi.org/10.15302/J-FASE-2016113

RESEARCH ARTICLE

Annotation and validation of genes involved in photosynthesis and starch synthesis from a Manihot full-length cDNA library

Yang ZHANG¹,Xin CHEN¹,Haiyan WANG¹,Zhiqiang XIA¹,Peng LING²,Wenquan WANG¹(

)

¹. Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
². Department of Horticultural Sciences, Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA

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Abstract

A full-length cDNA library from leaf and root tissues of cassava (Manihot esculenta) Arg7 and one accession of its wild ancestor W14 (M. esculenta ssp. flabellifolia) has been constructed. The library is comprised of four sub-libraries, containing 32640 recombinant clones, 6028 cDNA clones from their 5′ ends, and 128 clones from the 3′ ends were sequenced. In total, 5013 high-quality expressed sequence tags (ESTs) and 1259 unigenes were obtained. Of these, 746 unigenes were identified by their sequence homologies to ESTs from model plants, and 323 unigenes were mapped onto 114 different KEGG pathways. From these, 24 differentially expressed genes involved in starch metabolism and photosynthesis were identified and five of them were selected to compare their expression level between Arg7 and W14. Notably, Arg7 has a higher net photosynthesis rate in leaves, higher ribulose-1,5-bisphosphate carboxy-lase oxygenase activities in leaves, and higher AGPase activity in roots. This resource is the first EST collection from wild cassava and should be of value for gene discovery, genome annotation and studies of Manihot evolution.

Keywords Manihot esculenta expressed sequence tag unigene pathway expression pattern

Corresponding Author(s): Wenquan WANG

Just Accepted Date: 25 October 2016 Online First Date: 09 November 2016 Issue Date: 22 January 2017

Cite this article:

Yang ZHANG,Xin CHEN,Haiyan WANG, et al. Annotation and validation of genes involved in photosynthesis and starch synthesis from a Manihot full-length cDNA library[J]. Front. Agr. Sci. Eng. , 2016, 3(4): 308-320.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2016113
https://academic.hep.com.cn/fase/EN/Y2016/V3/I4/308

Tab.1 Genes of interest, primers and size of amplification products

Tab.2 Summary of library properties and assembly results after sequencing the clones and their distributions among cDNA sub-libraries

Fig.1 Analysis of cassava ESTs assembled in terms of the number of ESTs per unigene. A total of 783 unigenes were represented by a single EST.

Tab.3 The 323 unigenes mapped to KEGG pathways

Fig.2 The differential distribution of annotated unigenes in different pathways related to carbohydrate and energy metabolism among the four sub-libraries.

Fig.3 Net photosynthetic rates in the leaves of Arg7 and W14 during the growing period 120, 160, and 200 d after planting. All measurements were made at 10:00 am. Each data point represents the mean±SD of three replicates.

Fig.4 Diurnal fluctuations in the expression of selected genes in cassava leaves measured using real-time PCR, with variations in transcript levels associated with changes in light intensity between 8:00 h and 18:00 h. (a) MerbcS; (b) MeFNR; (c) Mendh; (d) Meppc; (e) MeLSU; (f) MeInv. Each data point represents the mean±SD of nine replicates, with values indicating fold-changes in relative gene expression following normalization relative to the actin cassava gene. Asterisks above each bar indicate the degree of significance differences in the expression levels between the Arg7 cultivar of Manihot esculenta and the W14 variety of Manihot flabellifolia (*, P<0.05; **, P<0.01).

Fig.5 Expression levels of selected genes implicated in photosynthesis and carbohydrate metabolism in cassava, using real-time PCR. (a) MeFNR; (b) Mendh; (c) Meppc; (d) MeLSU; (e) MeUGP; (f) MeInv. Each data point represents the mean±SD of nine replicates, with values indicating fold-changes in relative gene expression following normalization relative to the actin cassava gene. AR 120 d, AR 160 d and AR 200 d are levels in Arg7 storage roots 120, 160, and 200 d after planting, respectively.

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