Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

Hu, Menglan; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xiaojin; Wang, Wenquan

doi:10.3389/fpls.2016.00680

Cited by 30 publications

(19 citation statements)

References 45 publications

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“…For virus quantification in the varieties Albert and Namikonga, a SYBR Green qRT-PCR assay was performed using published primer sets (Adams et al, 2013) and cassava 4.1_010236 (acyl co-binding A) as an internal control for relative virus quantification (Hu et al, 2016; Supplementary Table 1).…”

Section: Methodsmentioning

confidence: 99%

Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America

et al. 2019

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Cassava brown streak disease (CBSD) is a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. This necrosis can encompass large areas of the root, rendering it inedible so that the entire cassava harvest can be lost. African cassava varieties are susceptible to either of the two viruses causing the disease, cassava brown streak virus (CBSV) and Uganda cassava brown streak virus, and while there are less sensitive varieties, all cassava eventually succumb to the disease. The lack of CBSD resistance in African cassava varieties prompted this search for new sources of virus resistance in the diversity of South American cassava germplasm held in the collection at International Center for Tropical Agriculture, Columbia. Our search for CBSD resistance in South American cassava germplasm accessions revealed that most of the 238 South American cassava lines infected with CBSV established systemic virus infections with moderate to severe disease symptoms on leaves and stems. Fifteen cassava accessions did not become virus infected, remained free of symptoms, and CBSV was undetected by qRT-PCR. When tuberous roots of those lines were examined, necrotic tissue was found in eight lines and CBSV was detected. The remaining seven cassava accessions remained clear of symptoms on all tissues and organs and were virus free. A broad spectrum of virus resistance also including other virus isolates was confirmed for the breeding lines DSC167 and DSC118. While detailed infection experiments with other cassava lines selected for resistance are still ongoing, this indicates that the resistance identified may also hold against a broader diversity of CBSVs. Taken together, we present the results of a comprehensive study on CBSV resistance and susceptibility in cassava germplasm accessions from South America. The virus resistance in cassava germplasm identified provides compelling evidence for the invaluable contribution of germplasm collections to supply the genetic resources for the improvement of our crops.

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Section: Methodsmentioning

confidence: 99%

Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America

et al. 2019

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“…RevertAidTM First-Strand cDNA Synthesis Kit (Fermentas, Ontario, Canada) was used to synthesize the First-Strand cDNA. QPCR was performed according to the method of Hu et al [ 21 ], using Stratagene Mx3000P Real-Time PCR and SYBR ® Premix Ex Taq™ (TaKaRa, Tokyo, Japan). The qPCR of MePHD1 was conducted with the primers Rt PHD1 F (5′-GGTACTATCGGCCAGAGGAG-3′) and Rt PHD1 R (5′-CAGAAGTAGTCCTCAGCTCCA-3′).…”

Section: Methodsmentioning

confidence: 99%

MePHD1 as a PHD-Finger Protein Negatively Regulates ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava

Chen

Liu

et al. 2018

IJMS

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ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in the starch synthesis pathway. Its enzyme activity can determine the efficiency of starch biosynthesis. Cassava (Manihot esculenta Crantz) is the main staple crop worldwide and has a high starch content in its storage root. However, the inner regulatory mechanism of AGPase gene family is unclear. MePHD1; a plant homeodomain transcription factor; was isolated through a yeast one-hybrid screening using the promoter of ADP-glucose pyrophosphorylase small subunit1a (MeAGPS1a) as bait, and cassava storage root cDNA library as prey. This factor could bind to the MeAGPS1a promoter in vitro and in vivo, and its predicted binding region ranged from −400 bp to −201 bp, at the translation initiation site. The transcript level of MePHD1 could be induced by five plant hormones, and a temperature of 42 °C. This was down-regulated during the maturation process of the storage root. MePHD1 protein could repress the promoter activity of MeAGPS1a gene by a dual-luciferase assay; which indicated that MePHD1 is a negative regulator for the transcript level of MeAGPS1a gene.

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“…Quantitative PCR (qPCR) was performed according to Hu’s method (Hu et al, 2016) using Stratagene Mx3000P Real-Time PCR and SYBR ® Premix Ex Taq TM (TaKaRa, JPN). The primer pairs of MeSAUR1 and β -actin were list in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

MeSAUR1, Encoded by a Small Auxin-Up RNA Gene, Acts as a Transcription Regulator to Positively Regulate ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava

Chen

Liu

et al. 2017

Front. Plant Sci.

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Cassava, being one of the top three tuberous crops, features highly efficient starch accumulation in the storage root to adapt the tropical resources and environments. The molecular mechanism for the process, however, is still unclear. ADP-glucose pyrophosphorylase, the first and rate-limited enzyme in starch biosynthesis pathway, is a heterotetramer comprised of two small/catalytic and two large/modulatory subunits. To understand the regulation of MeAGPase, the promoter of a highly expressed small subunit, MeAGPs1a, was used as bait for a yeast one-hybrid assay to screen storage root cDNA library. One cDNA, coding for a small auxin-up RNA protein, named MeSAUR1, was isolated from cassava. MeSAUR1 could bind to the promoter of MeAGPS1a in yeast one-hybrid test and in vitro, and was located in cell nucleus. MeSAUR1 displayed a higher transcript level in cassava root cortex, and its expression was induced by indole-3-acetic acid, gibberellin and ethylene, but repressed by abscisic acid. A dual-luciferase interaction test further convinced that MeSAUR1 could bind to the promoter of MeAGPS1a, and positively regulate the transcription of MeAGPS1a in cassava.

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Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

Cited by 30 publications

References 45 publications

Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America

Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America

MePHD1 as a PHD-Finger Protein Negatively Regulates ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava

MeSAUR1, Encoded by a Small Auxin-Up RNA Gene, Acts as a Transcription Regulator to Positively Regulate ADP-Glucose Pyrophosphorylase Small Subunit1a Gene in Cassava

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