Genome-wide identification and allele mining of LEA3 family genes in Sorghum bicolor (L).

Moench

Karthikeyan Mayandi1, G. Sandeep Kumar1, P. Purvi Kalyan1 and Monika Dalal1, 2 *

1 Directorate of
Sorghum Research, Rajendra Nagar Hyderabad
2 National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, 110012, India

* Email of Corresponding author: monika@nrcpb.org

Introduction

Adaptability to drought and high temperature, high genetic diversity and availability of genome sequence makes sorghum an
attractive model for functional genomics of traits important for abiotic stress tolerance. Moreover, Global climate change
necessitates breeding for crops that are more adaptive to several abiotic stresses. Drought tolerance of sorghum has been
partly attributed to duplications and expansion of certain gene families. Hence, it would be desirable to study if superior
alleles of already known and validated genes that confer drought tolerance are available in sorghum genome. LEA (Late
Abundant Embryogenesis) protein are one such class of genes which have been shown to confer abiotic stress tolerance in
different crops. Though there are several studies on LEA from different plant species. Sorghum LEA proteins have not been
studied so far. Hence, to gain insight in to LEA3 family in sorghum, a genome wide analysis of LEA3 genes and allele mining
of LEA3 genes was carried out in sorghum.
Phylogenetic analysis of LEA genes of rice and sorghum
Translated sequences of both SbLEA3 and OsLEA3 were used for phylogenetic analysis. The phylogenetic tree revealed
three clusters supported with bootstrap value of 100 each (Fig. 1A). The sorghum and rice LEA3 sequences appeared highly
conserved with two sequences each from the OsLEA3 group clustering with that of sorghum LEA3A and 3B subgroup
respectively. The gene structure of the sorghum and rice LEA3 genes revealed differences in gene organization (Fig.1B).
a) b)

Objectives
5 3
0bp 200bp 400bp 600bp 800bp 1000bp 1200bp 1400bp 1600bp

➢ To identify LEA3 genes in sorghum genome

➢ To study the expression of LEA3 genes during seed development and abiotic stresses Fig. 1 Phylogenetic relationship among the Group 3 LEA proteins of sorghum and
➢ To identify allelic variations in diverse sorghum genotypes rice and comparison of their intron –exon organization

Expression analysis of SbLEA3 genes

Methods The expression of these four SbLEA3 genes were studied in 15 day old developing seed and mature seed of BTx623 genotype.
Two of the genes belonging to SbLEA3A (Sb09g027110 and Sb03g032380) were found to express at low level in developing
Identification of LEA3 genes in sorghum genome
seed and their expression increased significantly at maturity (Fig. 2A). The genes from subgroupSbLEA3B expressed only at
The LEA3 sequences of barley (P14928, Q43478), rice (Q94JF2, Q6KA29, Q8S7U3), and maize (Q42376) were used as
maturity. The response of these genes to different abiotic stresses and ABA treatment was also studied in 14 day old seedlings
query for BLAST search in sorghum genome (www.phytozome.net.in). Putative LEA3 sequences thus obtained were further
by semi-quantitative RT-PCR. Three of the genes (Sb09g027110, Sb03g032380 and Sb01g036790) were regulated by all three
searched for LEA3 protein specific domain in Pfam database (http://pfam.sanger.ac.uk/) with E-value cutoff of 0.01 and
kinds of stresses that is osmotic, salt and ABA (Fig 2B). . None of the genes was responsive to cold stress. The gene
default setting of SMART database (http://smart.embl-heidelberg.de/). The amino acid sequence alignments were performed
Sb01g046000 showed only ABA inducible expression (Fig 2B).
using the ClustalW algorithm and subsequently dendrogram was drawn using Neighborhood Joining method in MEGA 5.05
software (Tamura et al. 2011; http://megasoftware.net/ ). Control PEG Salt Cold ABA

Sb09g027110
Plant materials and growth conditions Sb09g027110

Sb03g032380
Seedlings of sorghum [Sorghum bicolor (L.) Moench] genotype BT×623 were raised in pots filled with soil under natural Sb03g032380

environmental conditions. For studying expression of SbLEA3 genes in seeds, developing seeds were sampled at 15 days Sb01g036790
Sb01g036790

after anthesis and at maturity. For stress treatments, the 14-days old sorghum seedlings were carefully removed from pots Sb01g046000
Sb01g046000

and the soil was washed away from roots. The whole seedlings were used for abiotic stress treatments by immersing the
roots in water (control), 10% PEG6000 (-1.48MPa) or 150mM NaCl solution. For imposing cold stress, seedling roots were
immersed in ice-cold water and placed at 4C. ABA treatment was given by applying 100M ABA on the leaves. From the
control and treated seedlings leaf samples were collected at 3 h and then frozen in liquid nitrogen for total RNA isolation.
LEA3 expression analysis
Total RNA was isolated from seeds, control and stressed leaf tissues using RNeasy plant Mini Kit (QIAGEN). For expression
analysis, two step Semi-quantitative RT-PCR was carried out. PCR products were fractionated in a 3% agarose gel.
Allele mining of LEA3 genes in sorghum
LEA3 gene (Sb09g027110) was cloned and sequenced from five contrasting genotypes. SNPs and InDels were identified
and validated by CAPS/dCAPS / deletion specific primers. The 226 genotypes from sorghum minicore collection were
screened for deletions in promoter region of the gene.
Actin
Actin
Fig. 2 Expression analysis of LEA3 genes from Sorghum. A) expression of SbLea3 genes during seed development; B)
response of SbLEA3 genes to different abiotic stresses. Seedlings were subjected to 10% PEG6000, 150 mM NaCl, Cold
(4°C) and exogenous ABA (100M) treatments. Untreated plants were referred as control.
Allele mining of LEA3 genes in sorghum
The overlapping fragments of SbLEA3 genes encompassing promoter to 3’ UTR (2.464 kb) were cloned and sequenced from 5
diverse sorghum genotypes .These sequences were analysed for allelic polymorphism. Total 14 SNPs and 9 Indels were identified
(Table 3, Fig. 3). Sequence variation was observed across the length of the gene however the number of NSPs /Indels were more
in promoter region.
Table 3 Summary of SNPs/Indels in LEA3 gene from sorghum genotypes

Results and Discussion SNPs and Indels in LEA3 gene Validation of SNP/Indels SNP/Indel based change in coding sequence
(I)

Genome wide identification of LEA3 genes in sorghum (I)

Four putative sorghum LEA3 genes were identified in sorghum genome. Based on frequency of 11-amino acid motifs (II)

these were further classified into SbLEA3A (Sb09g027110 and Sb03g032380) and SbLEA3B (Sb01g036790 and
Sb01g046000) subgroups (Table 1). The SbLEA3 proteins are highly hydrophilic in nature as evident from their individual (II)

GRAVY scores Among these proteins, Sb01g036790 had mitochondria as potential target while Sb01g046000.1 was
predicted to be secretary in nature (Table 3). Sb01g046000 has a trans-membrane region between amino acid residues 13-
33 at N terminal end of the protein.
Unrestricted Restricted

Table 1 The sorghum LEA3 sequences with

motifs and number of 11mer repeats Fig. 3 Identification and validation of SNPs and Indels in LEA3 gene from five contrasting
genotypes of sorghum
Motifs
S. No. Sequences Sub Group Motif
1 2 3 4 5 repeats A subset of 224 genotypes from minicore collection of sorghum were screened for allelic polymorphism using

1 Sb09g027110 5(8x) two deletion specific primers in the promoter region (Fig. 4). From the analysis, a total of 7 variants or allelic
2 Sb03g032380 3B 5(2x) forms were detected.
3 Sb01g036790 4(6x)
4 Sb01g046000 3A 2(2x) No of genotypes Indel 1 (-1043) Indel 2 (-371) Representatives
160bp
140bp
76 Deletion Insertion M35-1 , CSV216R

34 Insertion Deletion BTx623, B35

85 Insertion Insertion IS19153, IS20697

160bp
140bp Insertion&
15 Insertion IS30092, IS28313
Deletion

Insertion&
10 Insertion IS26749 , IS7957
Table 2 General characteristics of LEA 3 Deletion
Insertion& Insertion&
proteins from sorghum 3
Deletion Deletion
IS29519 , IS22720

S. Sequence Id Protein GRAVY Subcellular 3 NA Insertion IS29950 , IS28141

No. length location

1 Sb09g027110.1 203 -1.245 other Fig. 4 Allelic variation at LEA3 locus in sorghum minicore genotypes
2 Sb09g027110.2 201 -1.221 other
3
4
Sb03g032380.1
Sb03g032380.2
216
201
-0.875
-0.810
other
other
Conclusion
5 Sb01g036790 352 -1.047 Mitochondria
6 Sb01g046000.1 351 -0.906 secretary
➢ Sorghum genome encodes four LEA3 genes.
➢ These genes are developmentally regulated and are induced by different abiotic stresses
➢ The coding region of LEA3 gene appears to be highly conserved while promoter region showed significant allelic variation
Acknowledgement ➢ Eleven SNP/Indel based markers were developed
➢ The markers developed in this study can be useful for studying their association with phenotypic changes and subsequently can be exploited for marker
This work was funded by the Indian Council of Agricultural Research (ICAR)-sponsored National assisted breeding programme.
Agricultural Innovation Project (NAIP). The authors acknowledge ICRISAT for providing the minicore
collection of sorghum