DE NOVO TRANSCRIPTOME SEQUENCING AND METABOLOMIC PROFILING TO DISCOVER PUTATIVE GENES OF LITTLE MILLET (Panicum sumatrense L.) ASSOCIATED TO DROUGHT TOLERANCE 3624
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Date
2022-10
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JAU JUNAGADH
Abstract
Little millet (Panicum sumatrense), having relatively diploid genome
(2n=4x=36) with unknown genome size. It is one among the minor millets grown to a
limited extent all over India up to altitudes of 2100 m. This crop is resistant to adverse
agro-climatic conditions. The complex carbohydrates, phenolic compounds,
antioxidants help to prevent metabolic disorders like diabetes, cancer, obesity etc.,
Although minor millets were superior to other cereals with many nutritional benefits
due to its proximate composition of protein, fat, carbohydrates, ash, moisture, and
energy, their utilization was limited because of low palatability, the coarseness of
grain, and lack of diversified food preparations. Hence evaluation of the nutritional
quality of millets would help to understand, diversifying the usage of millets in
ensuring food, nutritional security in the ever-changing modern world. Keeping theses
benefits in the mind present study was undertaken to find out putative genes and
metabolites responsible for drought tolerance. The little millet genotype OLM 203/Tarini tolerant to drought was given in control and stressed condition in pots. 23
days old regularly watered seedling (control) and 32 days old water stressed seedlings
were utilized for present experiment.
In the current study, transcriptome analysis identified key genes regulating
drought tolerance viz, in all 04-sample, number of reads varied 582,326 to 1,302,251
and average read length 497.13 to 589.32 bp. After trimming, there was decrease in
number of reads, average read length and trimmed percentage which is varied
between 569,157 to 1,265,909, 501.4 to 588.85, 90.74 to 97.74 % respectively. As
well as, in all four samples number of total raw reads and average read length were
3,188,195 and 531.64 respectively and after trimming by CLC-GWB v20.0 number of
raw reads decreased to 3,048,100 trimmed reads, trimmed read length 532.60, and
trimmed percentage 95.24 %. Clean reads were obtained after quality control and they
were subsequently used for De novo assembly and expression analysis. The generated
draft assembly was 214498057 bp was approximately percentage of the total genome
size of little millet is unknown. The maximum and minimum contig size was 40533
and 400bp in leaf and 398 base pairs in root respectively. However, the average size
of contigs was 1138.04 bp and N50 was 1642 bp. Total de nono assembly were
generated assembled reads of 270248626 to 214498057 with GC (%) was 48.39 from
four samples with a hit length of 13112251 to 12759055 with GC (%) was 46.99.
Differential expression analysis, total number of genes in comparison of 23 days leaf
vs 32 days leaf (treated) were observed 7264, and in comparison of 23 days root vs 32
days root (treated) were extracted 7606.
In expression profiling of 134 important DEGs, up or down regulated 20
important drought tolerance or resistance responsive genes in control and treated root
of OLM-203 genotype was observed such as, in leaf XB34, and RS193 (up-regulated
genes), WLIM1 (down regulated genes). In case of roots, SKI35 (up-regulated genes)
while, MPK6 and TCMOp1 (down regulated genes). However, in leaf, up-regulated
top 10 important drought tolerance or resistance responsive genes (LHW, CML25,
PLP2, OBE1, SKI35p1, ATESY, SKI35p2, C3H7, PSAH1, PAP14) and in root
MPK6, SPT41, THIC, LRX6, LRX5, COQ4, DMP5, LRX5, IAA6 and SERL2 genes
were found to be switched on. Among these top 10 genes excluding SPT41, THIC
(taking part in transcription process) genes takes part in drought tolerance in the
genotype.
There are top twenty commonly expressed drought tolerant or resistance genes
in leaf and root of OLM-203 genotype viz., AX22A, BOB2, C3H32, C71B8, CAHC,
CAL, CCS1, DGDG1, FLA2, GL52, GMPP2, HAK18, IAA6, IDL2, M3K1, NAC81,
OHP1, PRX2C, SCP44 and TPR3. These all genes are responsive for positive
regulator of stress signaling and drought tolerance mechanism.
There are 36 up-regulated and 21 down-regulated serine transcripts were
expressed in root and leaf of OLM-203 genotype of little millet respectively. ‘SKI35’
(Log₂ FC=7.66) up-regulated and ‘ALA2’ (Log₂ FC= -5.82) down-regulated genes
are serine regulated differential expression genes in root of OLM-203 genotype.
‘NPRT1’ (Log₂ FC= 2.89) up-regulated and ‘KN7F’ (Log₂ FC= -4.11) down regulated genes are serine regulated differential expression genes in leaf of OLM-203
genotype. A perusal of dendrogram was clearly indicated that 134 drought tolerance
genes were considered and three genes IAA6, ALA2, and HP301 among 134 genes
played a key role during the drought or stress tolerance of the genotype.
Most GO gene annotations of leaf samples were categorized as having
‘biological process’ (35%), followed by ‘cellular component (19%) and ‘molecular
function’ (46%). Within the top represented category of ‘cellular process (125
contigs)’ were enriched, as was ‘metabolic process’ (117 contigs) and the next most
enriched ortholog-biosynthesis of ‘cellular component’ (150) found in this category.
Most GO gene annotations of root samples were categorized as having ‘biological
process’ (573 contigs), followed by ‘molecular function (401 contigs) and ‘cellular
components (166 contigs)’. In root samples, 1140 genes were found which are most
enriched with biological process, molecular function and cellular component, whereas
in leaf samples 670 genes most enriched ortholog-biological process, molecular
function and cellular component.
Enzyme commission (EC) numbers were assigned for control leaf vs treated
leaf with 105 enzyme codes for 692 unique sequences, whereas in case of control root
vs treated root with 105 enzyme codes for 699 unique sequences.
Among all the leaf samples, the GO level distribution graph showed 1270 total
annotations with mean level 6.036%, and having standard deviation 2.509, whereas in
root samples containing 1617 total annotations with mean level 5.947%, and having
standard deviation 2.516. Several putative functions were identified functional
domains such as zinc finger, drought tolerance, serine related proteins.
The confirmation results of repeats obtained by STRING 10.0 shown in MCL
clustering of repeats depicted the 11 gene clustered with the specific repeats obtained
from STRING 10.0. Six protein sequences (genes) viz., IPR019734 Tetratricopeptide,
IPR013105 Tetratricopeptide, IPR018108 Mitochondrial subs, IPR019734
Tetratricopeptide string MCL clusters, IPR013105 Tetratricopeptide string MCL
clusters, IPR002885 Pentatricopeptide string MCL clusters deciphered the role of its
genes were regulated by their 11 genes cluster jointly. While, Venn diagram showing
a total of 134 genes were found commonly shared and unique DEGs in leaf and root
and 27 up-regulated genes were found commonly in leaf and root.
There are 18 enzymes were analyzed and showed 44 KEGG pathways were
found to be regulated. There were unique and common sequences regulate the 53
pathways. There are 35 unique and common KEGG pathways regulated by 58
enzymes of OLM-203 genotype. Total 147 KEGG pathways were commonly
operated among control and treated samples of root and leaf. There are 15 and 35
number of unique and common KEGG pathways regulated by specific enzyme and
sequences in root and leaf of genotype respectively up to 32 days of the genotype.
Using the NIST library for the metabolomics study, metabolites were
identified as sugars, sugar alcohols, sugar acids, fatty acids, and others such as
dicarboxylic acid, diterpene alcohol, organic acid, and sugar amine. The detailed of
total number of sixty (60) metabolites produced in both samples. In the mean decrease
accuracy, metabolites were linolenic acid, mannonic acid, hexadecanoic acid,
pentonic acid, glucopyranosid, glucohexodialdose, and malic acid were present in
higher concentrations in the OLM-203 genotype. In treated samples, glucose,
threonine, acetamide, serine, oleic acid, fructose glucose oxime, and hydroquinone
were intensively observed, while in control sample, linolenic acid, mannonic acid,
hexadecanoic acid, pentonic acid, glucopyranosid, glucohexodialdose, and malic acid
were observed in OLM-203. Sugar also acts as a signaling molecule and helps to
modulate the plant’s growth, development, and response to multiple stresses.
Mannose was found to be accumulated in the leaf of OLM-203 (Tarini) in stress
conditions; similar findings were reported earlier in the drought-tolerant little millet.
Genomic and metabolomics changes were observed during drought treatment
in tolerant genotype ‘OLM-203/Tarini’. It was observed that the typical increase of
stress-related genes and metabolites. Therefore, it can sum-up that ‘OLM-203/Tarini’
genotype was well tolerated genotype and functions was normal even under water
stress, although the exact complex mechanism of tolerance remains to be investigated.
There were 128 EST-SSR identified which serves as a resource of high quality
transcripts for gene discovery and development of functional markers associated with
water stress tolerant. In the present study, sequence collection represents a major
transcriptomics resource for little millet genotype and the large number of genetic
markers predicted should contribute to future research thrust.