TY  - CONF
AU  - Božić, Manja
AU  - Ignjatović-Micić, Dragana
AU  - Delić, Nenad
AU  - Mladenović, Marko
AU  - Vančetović, Jelena
AU  - Banović Đeri, Bojana
AU  - Nikolić, Ana
PY  - 2023
UR  - https://belbi.bg.ac.rs/
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2007
AB  - Micro RNAs (miRNAs) are known regulators of various processes in plants, including growth,
development and stress responses. They achieve this through mRNA cleavage or translational
inhibition, in a process called RNA interference. Herein, their role in chilling stress response in
young maize seedlings (Zea mays L.) is examined, using high-throughput sequencing methods.
Bringing light to all aspects of chilling stress response in maize is necessary since earlier
sowing, during colder periods, is one of the most promising strategies of avoiding maize yield
loss due to effects of climate change in these areas.
Sterilized seeds of two maize genotypes (tolerant - T and sensitive - S to low temperatures)
were germinated in the dark for five days (optimal conditions), after which the 5-d old seedlings
were exposed to chilling conditions for 6h (10° C). Samples for RNA isolation and cDNA library
preparation were taken after the treatment ended, and single-end 50 bp sequencing was
performed (Illumina® Novaseq 6000). The miRNAs were then filtered, mapped, identified
and quantified using adequate bioinformatics tools; and the differential expression analysis
was carried out using the DEGseq R package. The analysis was performed on 859 miRNAs,
after previously executed TPM normalization using the MA-plot-based method with random
sampling model (MARS). The threshold for significantly differential expression was set as the
Bayesian adjusted p-value, or q-value < 0.01 and log2 fold change > 1.
A total of 612 were expressed differentially, but only 55 miRNAs were common for both
genotypes and at the same time differentially expressed between control and treatment
conditions – 40 novel and 15 known. Half of the common miRNAs showed the same
expression patterns in both genotypes, while the other half did not. Among them, seven known
miRNAs showed opposing expression patterns between the genotypes (zma-miR167b-3p
zma-miR167e-3p, zma-miR159c-5p, zma-miR164g-3p, zma-miR166a-5p, zma-miR398a-
3p, and zma-miR528a-3p). These miRNAs were shown to have a role in various abiotic stress
responses, including drought, waterlogging, high salts – but not chilling. While the results point
to their potential role in establishing chilling tolerance in maize seedlings, further research is
necessary to confirm it and connect the miRNAs to their potential targets.
PB  - Belgrade : Institute of molecular genetics and genetic engineering
C3  - 4th Belgrade Bioinformatics Conference
T1  - Seven miRNAs potentially included in the chilling response of maize plants in early stages of development
EP  - 83
SP  - 83
VL  - 4
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2007
ER  - 

@conference{
author = "Božić, Manja and Ignjatović-Micić, Dragana and Delić, Nenad and Mladenović, Marko and Vančetović, Jelena and Banović Đeri, Bojana and Nikolić, Ana",
year = "2023",
abstract = "Micro RNAs (miRNAs) are known regulators of various processes in plants, including growth,
development and stress responses. They achieve this through mRNA cleavage or translational
inhibition, in a process called RNA interference. Herein, their role in chilling stress response in
young maize seedlings (Zea mays L.) is examined, using high-throughput sequencing methods.
Bringing light to all aspects of chilling stress response in maize is necessary since earlier
sowing, during colder periods, is one of the most promising strategies of avoiding maize yield
loss due to effects of climate change in these areas.
Sterilized seeds of two maize genotypes (tolerant - T and sensitive - S to low temperatures)
were germinated in the dark for five days (optimal conditions), after which the 5-d old seedlings
were exposed to chilling conditions for 6h (10° C). Samples for RNA isolation and cDNA library
preparation were taken after the treatment ended, and single-end 50 bp sequencing was
performed (Illumina® Novaseq 6000). The miRNAs were then filtered, mapped, identified
and quantified using adequate bioinformatics tools; and the differential expression analysis
was carried out using the DEGseq R package. The analysis was performed on 859 miRNAs,
after previously executed TPM normalization using the MA-plot-based method with random
sampling model (MARS). The threshold for significantly differential expression was set as the
Bayesian adjusted p-value, or q-value < 0.01 and log2 fold change > 1.
A total of 612 were expressed differentially, but only 55 miRNAs were common for both
genotypes and at the same time differentially expressed between control and treatment
conditions – 40 novel and 15 known. Half of the common miRNAs showed the same
expression patterns in both genotypes, while the other half did not. Among them, seven known
miRNAs showed opposing expression patterns between the genotypes (zma-miR167b-3p
zma-miR167e-3p, zma-miR159c-5p, zma-miR164g-3p, zma-miR166a-5p, zma-miR398a-
3p, and zma-miR528a-3p). These miRNAs were shown to have a role in various abiotic stress
responses, including drought, waterlogging, high salts – but not chilling. While the results point
to their potential role in establishing chilling tolerance in maize seedlings, further research is
necessary to confirm it and connect the miRNAs to their potential targets.",
publisher = "Belgrade : Institute of molecular genetics and genetic engineering",
journal = "4th Belgrade Bioinformatics Conference",
title = "Seven miRNAs potentially included in the chilling response of maize plants in early stages of development",
pages = "83-83",
volume = "4",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2007"
}

Božić, M., Ignjatović-Micić, D., Delić, N., Mladenović, M., Vančetović, J., Banović Đeri, B.,& Nikolić, A.. (2023). Seven miRNAs potentially included in the chilling response of maize plants in early stages of development. in 4th Belgrade Bioinformatics Conference
Belgrade : Institute of molecular genetics and genetic engineering., 4, 83-83.
https://hdl.handle.net/21.15107/rcub_imagine_2007

Božić M, Ignjatović-Micić D, Delić N, Mladenović M, Vančetović J, Banović Đeri B, Nikolić A. Seven miRNAs potentially included in the chilling response of maize plants in early stages of development. in 4th Belgrade Bioinformatics Conference. 2023;4:83-83.
https://hdl.handle.net/21.15107/rcub_imagine_2007 .

Božić, Manja, Ignjatović-Micić, Dragana, Delić, Nenad, Mladenović, Marko, Vančetović, Jelena, Banović Đeri, Bojana, Nikolić, Ana, "Seven miRNAs potentially included in the chilling response of maize plants in early stages of development" in 4th Belgrade Bioinformatics Conference, 4 (2023):83-83,
https://hdl.handle.net/21.15107/rcub_imagine_2007 .

TY  - CONF
AU  - Mladenović, Marko
AU  - Grčić, Nikola
AU  - Dudić, Dragana
AU  - Nikolić, Ana
AU  - Božić, Manja
AU  - Delić, Nenad
AU  - Prodanović, Slaven
AU  - Banović Đeri, Bojana
PY  - 2021
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1872
AB  - Multidisciplinary research is today commonly used in plant breeding for improving important agronomic
traits. High throughput genotyping technologies and genotype – phenotype association studies as widely
used for improving breeding programs, depend on bioinformatics analysis for extracting information from
the gathered data. In this research, among plethora of widely used bioinformatics approaches, the custom
made one was chosen, based on the current recommendations in the field.
The material includes a set of 46 maize inbred lines commonly used in maize breeding programs. Phenotyping
was done for thirteen important quantitative agronomic traits in 8 environments during two years (2018
and 2019). For the purpose of genotyping, plants of all inbred lines were grown under optimal conditions
and sampled after completing the V4 growth stage. Total RNA was isolated from the third leaf of three plants
per inbred line and used for cDNA preparation by Illumina TruSeq Stranded RNA LT kit. Pair-end RNA-Seq
based on Next Generation Sequencing methodology was performed on MiSeq Illumina sequencer using
MiSeq Reagent kit, v2 (2 x 150bp). Raw sequencing data of maize leaves’ transcriptionally active genome
regions at the moment of sampling were used for identification of single nucleotide polymorphisms (SNPs)
in each of 46 inbred lines.
Bioinformatics pipeline for data manipulation and analysis was custom made and included FastQC (for
quality control (QC) of raw data), Trimmomatic tool v0.32 (for adapter and contaminants removal, as well
as for the removal of regions with QC below 30), TopHat (insert size 130, standard deviation 50, maximum
intron size 100.000 – for mapping filtered reads onto the B73 maize reference genome v3.0), Cufflinks
v2.2.1 (for reads assembly), Cuffmerge (for the final transcriptome assembly) and an intersection output of
two independent SNPs calling tools FreeBayes and BCFtools (to minimize false positive results). With the
aim to find SNP markers which show strongly statistically supported relationship with favorable values of
investigated quantitative traits, genotype - phenotype association analysis was conducted. It was performed
using two approaches – one relying on the TASSEL software, widely used in agronomics and the other based
on machine learning software like WEKA, rarely used in agronomics. The results of two approaches were
compared and discussed.
PB  - Novi Sad : Faculty of Sciences, Department of Biology and Ecology
C3  - Biologia Serbica
T1  - Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.)
IS  - 1 (Special Edition)
SP  - 109
VL  - 43
UR  - https://hdl.handle.net/21.15107/rcub_imagine_1872
ER  - 

@conference{
author = "Mladenović, Marko and Grčić, Nikola and Dudić, Dragana and Nikolić, Ana and Božić, Manja and Delić, Nenad and Prodanović, Slaven and Banović Đeri, Bojana",
year = "2021",
abstract = "Multidisciplinary research is today commonly used in plant breeding for improving important agronomic
traits. High throughput genotyping technologies and genotype – phenotype association studies as widely
used for improving breeding programs, depend on bioinformatics analysis for extracting information from
the gathered data. In this research, among plethora of widely used bioinformatics approaches, the custom
made one was chosen, based on the current recommendations in the field.
The material includes a set of 46 maize inbred lines commonly used in maize breeding programs. Phenotyping
was done for thirteen important quantitative agronomic traits in 8 environments during two years (2018
and 2019). For the purpose of genotyping, plants of all inbred lines were grown under optimal conditions
and sampled after completing the V4 growth stage. Total RNA was isolated from the third leaf of three plants
per inbred line and used for cDNA preparation by Illumina TruSeq Stranded RNA LT kit. Pair-end RNA-Seq
based on Next Generation Sequencing methodology was performed on MiSeq Illumina sequencer using
MiSeq Reagent kit, v2 (2 x 150bp). Raw sequencing data of maize leaves’ transcriptionally active genome
regions at the moment of sampling were used for identification of single nucleotide polymorphisms (SNPs)
in each of 46 inbred lines.
Bioinformatics pipeline for data manipulation and analysis was custom made and included FastQC (for
quality control (QC) of raw data), Trimmomatic tool v0.32 (for adapter and contaminants removal, as well
as for the removal of regions with QC below 30), TopHat (insert size 130, standard deviation 50, maximum
intron size 100.000 – for mapping filtered reads onto the B73 maize reference genome v3.0), Cufflinks
v2.2.1 (for reads assembly), Cuffmerge (for the final transcriptome assembly) and an intersection output of
two independent SNPs calling tools FreeBayes and BCFtools (to minimize false positive results). With the
aim to find SNP markers which show strongly statistically supported relationship with favorable values of
investigated quantitative traits, genotype - phenotype association analysis was conducted. It was performed
using two approaches – one relying on the TASSEL software, widely used in agronomics and the other based
on machine learning software like WEKA, rarely used in agronomics. The results of two approaches were
compared and discussed.",
publisher = "Novi Sad : Faculty of Sciences, Department of Biology and Ecology",
journal = "Biologia Serbica",
title = "Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.)",
number = "1 (Special Edition)",
pages = "109",
volume = "43",
url = "https://hdl.handle.net/21.15107/rcub_imagine_1872"
}

Mladenović, M., Grčić, N., Dudić, D., Nikolić, A., Božić, M., Delić, N., Prodanović, S.,& Banović Đeri, B.. (2021). Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.). in Biologia Serbica
Novi Sad : Faculty of Sciences, Department of Biology and Ecology., 43(1 (Special Edition)), 109.
https://hdl.handle.net/21.15107/rcub_imagine_1872

Mladenović M, Grčić N, Dudić D, Nikolić A, Božić M, Delić N, Prodanović S, Banović Đeri B. Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.). in Biologia Serbica. 2021;43(1 (Special Edition)):109.
https://hdl.handle.net/21.15107/rcub_imagine_1872 .

Mladenović, Marko, Grčić, Nikola, Dudić, Dragana, Nikolić, Ana, Božić, Manja, Delić, Nenad, Prodanović, Slaven, Banović Đeri, Bojana, "Bioinformatics pipeline for genotyping and genotype - phenotype association study in maize (Zea mays L.)" in Biologia Serbica, 43, no. 1 (Special Edition) (2021):109,
https://hdl.handle.net/21.15107/rcub_imagine_1872 .

TY  - CONF
AU  - Božić, Manja
AU  - Nikolić, Ana
AU  - Dudić, Dragana
AU  - Ignjatović-Micić, Dragana
AU  - Samardžić, Jelena
AU  - Delić, Nenad
AU  - Banović Đeri, Bojana
PY  - 2021
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/1874
AB  - Finding new ways of improving crop quality, yield potential and abiotic stress tolerance are some of the most
important pursuits in crop production today. As one of the biggest causes of yield and productivity reduction
is climate change, specifically increasing temperatures and drought during the summer, a large number of
strategies is focussed on lessening their negative effects. Cropping pattern changes include earlier sowing
(early spring), when the temperatures are lower, as one of the most promising escape strategies for avoiding
high summer temperatures. Thus, development of cold tolerant maize lines became an important goal.
Comparative analysis of 46 maize inbred lines belonging to two different genetic backgrounds, one predominantly
cold tolerante (marked as Non-Lancaster) and the other predominantly cold sensitive (marked as
Lancaster) in the field, was done by whole transriptome sequencing and differential gene expression (DGE)
analysis. Plants were grown under optimal, greenhouse conditions and sampled after completing the V4
growth stage. Total RNA isolated from leaves of three plants per inbred line was used for cDNA library preparation
by Illumina TruSeq Stranded RNA LT kit. Pair-end sequencing was performed on MiSeq Illumina
sequencer using MiSeq Reagent kit, v2 (2 x 150bp). Data manipulation and analysis was performed using a
custom-made bioinformatics pipeline that included high throughput sequence data quality control (using
FastQC), removal of low quality reads (using Trimmomatic tool, version 0.32), transcriptome assembly and
mapping (using Cufflinks, version 2.2.1), expression quantification (using CuffDiff) and DGE analysis (using
BLAST2GO and GO analysis Toolkit and Database for Agricultural Community, agriGO v2).
DGE analysis revealed 77 differentially expressed genes (DEGs) between the Lancaster and the Non-Lancaster
group, 21 of which were statistically supported for differential expression between the two groups and
annotated as involved in abiotic stress responses in maize and other plant species. To test DEGs response to
cold stress expression of a subset of seven DEGs in eight inbred lines (4 belonging to Lancaster and 4 belonging
to Non-Lancaster genetic background) was analyzed under 24h long exposure to low temperatures (6/4°
C, 12h photoperiod), with sampling being done 6h and 24h after beginning of the treatment, as well as after
48h of recovery. Six DEGs showed different expression regulation dependent on cold exposure duration and
genetic background. These findings imply differently regulated processes between the analysed Lancaster
and Non-Lancaster inbred lines, contributing to their different cold response and adaptation, and will be
further used for the development of cold tolerant hybrids.
PB  - Novi Sad : Faculty of Sciences, Department of Biology and Ecology
C3  - Biologia Serbica
T1  - Differential gene expression analysis of heterotic groups’ maize inbred lines under optimal conditions led to the identification of specific gene regulation under low-temperature
IS  - 1 (Special Edition)
SP  - 106
VL  - 43
UR  - https://hdl.handle.net/21.15107/rcub_imagine_1874
ER  - 

@conference{
author = "Božić, Manja and Nikolić, Ana and Dudić, Dragana and Ignjatović-Micić, Dragana and Samardžić, Jelena and Delić, Nenad and Banović Đeri, Bojana",
year = "2021",
abstract = "Finding new ways of improving crop quality, yield potential and abiotic stress tolerance are some of the most
important pursuits in crop production today. As one of the biggest causes of yield and productivity reduction
is climate change, specifically increasing temperatures and drought during the summer, a large number of
strategies is focussed on lessening their negative effects. Cropping pattern changes include earlier sowing
(early spring), when the temperatures are lower, as one of the most promising escape strategies for avoiding
high summer temperatures. Thus, development of cold tolerant maize lines became an important goal.
Comparative analysis of 46 maize inbred lines belonging to two different genetic backgrounds, one predominantly
cold tolerante (marked as Non-Lancaster) and the other predominantly cold sensitive (marked as
Lancaster) in the field, was done by whole transriptome sequencing and differential gene expression (DGE)
analysis. Plants were grown under optimal, greenhouse conditions and sampled after completing the V4
growth stage. Total RNA isolated from leaves of three plants per inbred line was used for cDNA library preparation
by Illumina TruSeq Stranded RNA LT kit. Pair-end sequencing was performed on MiSeq Illumina
sequencer using MiSeq Reagent kit, v2 (2 x 150bp). Data manipulation and analysis was performed using a
custom-made bioinformatics pipeline that included high throughput sequence data quality control (using
FastQC), removal of low quality reads (using Trimmomatic tool, version 0.32), transcriptome assembly and
mapping (using Cufflinks, version 2.2.1), expression quantification (using CuffDiff) and DGE analysis (using
BLAST2GO and GO analysis Toolkit and Database for Agricultural Community, agriGO v2).
DGE analysis revealed 77 differentially expressed genes (DEGs) between the Lancaster and the Non-Lancaster
group, 21 of which were statistically supported for differential expression between the two groups and
annotated as involved in abiotic stress responses in maize and other plant species. To test DEGs response to
cold stress expression of a subset of seven DEGs in eight inbred lines (4 belonging to Lancaster and 4 belonging
to Non-Lancaster genetic background) was analyzed under 24h long exposure to low temperatures (6/4°
C, 12h photoperiod), with sampling being done 6h and 24h after beginning of the treatment, as well as after
48h of recovery. Six DEGs showed different expression regulation dependent on cold exposure duration and
genetic background. These findings imply differently regulated processes between the analysed Lancaster
and Non-Lancaster inbred lines, contributing to their different cold response and adaptation, and will be
further used for the development of cold tolerant hybrids.",
publisher = "Novi Sad : Faculty of Sciences, Department of Biology and Ecology",
journal = "Biologia Serbica",
title = "Differential gene expression analysis of heterotic groups’ maize inbred lines under optimal conditions led to the identification of specific gene regulation under low-temperature",
number = "1 (Special Edition)",
pages = "106",
volume = "43",
url = "https://hdl.handle.net/21.15107/rcub_imagine_1874"
}

Božić, M., Nikolić, A., Dudić, D., Ignjatović-Micić, D., Samardžić, J., Delić, N.,& Banović Đeri, B.. (2021). Differential gene expression analysis of heterotic groups’ maize inbred lines under optimal conditions led to the identification of specific gene regulation under low-temperature. in Biologia Serbica
Novi Sad : Faculty of Sciences, Department of Biology and Ecology., 43(1 (Special Edition)), 106.
https://hdl.handle.net/21.15107/rcub_imagine_1874

Božić M, Nikolić A, Dudić D, Ignjatović-Micić D, Samardžić J, Delić N, Banović Đeri B. Differential gene expression analysis of heterotic groups’ maize inbred lines under optimal conditions led to the identification of specific gene regulation under low-temperature. in Biologia Serbica. 2021;43(1 (Special Edition)):106.
https://hdl.handle.net/21.15107/rcub_imagine_1874 .

Božić, Manja, Nikolić, Ana, Dudić, Dragana, Ignjatović-Micić, Dragana, Samardžić, Jelena, Delić, Nenad, Banović Đeri, Bojana, "Differential gene expression analysis of heterotic groups’ maize inbred lines under optimal conditions led to the identification of specific gene regulation under low-temperature" in Biologia Serbica, 43, no. 1 (Special Edition) (2021):106,
https://hdl.handle.net/21.15107/rcub_imagine_1874 .