scholarly journals Defining the ‘HoneySweet’ insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica)

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Ann M. Callahan ◽  
Tetyana N. Zhebentyayeva ◽  
Jodi L. Humann ◽  
Christopher A. Saski ◽  
Kelsey D. Galimba ◽  
...  

Abstract‘HoneySweet’ plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, ‘HoneySweet’ DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum ‘Improved French’. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the ‘HoneySweet’ transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

1997 ◽  
Vol 48 (4) ◽  
pp. 503 ◽  
Author(s):  
K. W. Jayasena ◽  
B. J. Ingham ◽  
M. R. Hajimorad ◽  
J. W. Randles

The coat protein gene of a South Australian strain of alfalfa mosaic virus (AMV-N20 [NcS]) has been cloned, sequenced, and transferred into Nicotiana tabacum L. cv. Xanthi via Agrobacterium tumefaciens under the control of the CaMV 35S promoter. A number of lines (T0 generation) were selected with the coat protein gene either in sense orientation (CP+) or in antisense orientation (CP–). The T0 plants were tested for their gene expression and susceptibility to the homologous AMV strain. A significant delay in the onset of symptoms and a reduction in virus accumulation was observed in CP+ plants mechanically inoculated with AMV. CP– plants were also significantly protected but less so than the CP+ plants. Plants transformed with the expression vector only (CP0) showed a minor resistance to local infection on inoculated leaves compared with untransformed plants. The strategy of coat protein mediated protection (CPMP) using the CP gene in either messenger sense or antisense would therefore be appropriate for testing on economically important pasture legumes.


HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1130d-1130
Author(s):  
Guowei Fang ◽  
Rebecca Grumet

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.


1994 ◽  
Vol 14 (1) ◽  
Author(s):  
Ralph Scorza ◽  
Michel Ravelonandro ◽  
AnnM. Callahan ◽  
JohnM. Cordts ◽  
Marc Fuchs ◽  
...  

1989 ◽  
Vol 17 (4) ◽  
pp. 1768-1768 ◽  
Author(s):  
B. Prill ◽  
E. Maiss ◽  
U. Timpe ◽  
R. Casper

GigaScience ◽  
2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Xuewei Li ◽  
Ling Kui ◽  
Jing Zhang ◽  
Yinpeng Xie ◽  
Liping Wang ◽  
...  

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