Os11Gsk gene from a wild rice, Oryza rufipogon ... - Springer Link

Funct Integr Genomics (2012) 12:277–289 DOI 10.1007/s10142-012-0265-4

ORIGINAL PAPER

Os11Gsk gene from a wild rice, Oryza rufipogon improves yield in rice Sudhakar Thalapati & Anil K. Batchu & Sarla Neelamraju & Rajeshwari Ramanan

Received: 22 November 2011 / Revised: 27 January 2012 / Accepted: 7 February 2012 / Published online: 25 February 2012 # Springer-Verlag 2012

Abstract Chromosomal segments from wild rice species Oryza rufipogon, introgressed into an elite indica rice restorer line (KMR3) using molecular markers, resulted in significant increase in yield. Here we report the transcriptome analysis of flag leaves and fully emerged young panicles of one of the high yielding introgression lines IL50-7 in comparison to KMR3. A 66-fold upregulated gene Os11Gsk, which showed no transcript in KMR3 was highly expressed in O. rufipogon and IL50-7. A 5-kb genomic region including Os11Gsk and its flanking regions could be PCR amplified only from IL50-7, O. rufipogon, japonica varieties of rice-Nipponbare and Kitaake but not from the indica varieties, KMR3 and Taichung Native1. Three sister lines of IL50-7 yielding higher than KMR3 showed presence of Os11Gsk, whereas the gene was absent in three other ILs from the same cross having lower yield than KMR3, indicating an association of the presence of Os11Gsk

with high yield. Southern analysis showed additional bands in the genomic DNA of O. rufipogon and IL50-7 with Os11Gsk probe. Genomic sequence analysis of ten highly co-expressed differentially regulated genes revealed that two upregulated genes in IL50-7 were derived from O. rufipogon and most of the downregulated genes were either from KMR3 or common to KMR3, IL50-7, and O. rufipogon. Thus, we show that Os11Gsk is a wild rice-derived gene introduced in KMR3 background and increases yield either by regulating expression of functional genes sharing homology with it or by causing epigenetic modifications in the introgression line. Keywords Transcriptomics . O. rufipogon . Rice progenitors . Restorer line . Yield increase . Introgression . Hybrids

Introduction The microarray data have been submitted to the GEO repository and assigned GEO accession number. GSE30487. It was released on the 31st of December 2011. Electronic supplementary material The online version of this article (doi:10.1007/s10142-012-0265-4) contains supplementary material, which is available to authorized users. S. Thalapati : A. K. Batchu : S. Neelamraju Biotechnology Unit, Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India R. Ramanan (*) Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 009, Andhra Pradesh, India e-mail: [email protected] R. Ramanan e-mail: [email protected]

Microarray technique has become a useful tool for exploring genome-scale gene expression (Schena et al.1995). In plants, microarray analysis has been employed to investigate gene expression in response to hormones such as abscisic acid and gibberellin (Zhong and Burns 2003; De Paepe et al. 2004; Leonhardt et al. 2004; Yang et al. 2004), cold, high salinity and drought stress (Rabbani et al. 2003; Seki et al. 2002), pathogen infections (Marathe et al. 2004), and light/ dark conditions (Jung et al. 2008). A genome-wide transcriptome comparison of super hybrid rice and its parents has been reported in an attempt to understand molecular mechanisms in heterosis (Huang et al. 2006; Wei et al. 2009). Developing near-isogenic introgression lines by introgressing alien chromosomal segments from wild species into domesticated cultivars is an effective way of adding unutilized genetic variation in crops such as rice (Ali et

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al. 2010). The introgression of chromosomal segments from wild species results in extensive genetic and epigenetic changes in introgression lines (Wang et al. 2005, 2010). A genome-wide transcriptome analysis between introgression lines (ILs) and the recipient parent would reveal differences in expression resulting from the introgressed segments. Improved varieties of rice with increased yield are being derived by breeding and selecting from the pool of genetic variation in domesticated rice. There is a need to explore new, acceptable, and non-conventional approaches to increase rice yield. Wild relatives of rice have long been recognized as important gene sources for quantitatively inherited traits such as resistance and/or tolerance to abiotic and biotic stresses (Brar and Khush 1997; Zhang et al. 1998; Jeon et al. 2011). It has been demonstrated that wild relatives are also important sources of useful alleles for complex traits such as yield in different crop plants notably rice and tomato and yield enhancing QTLs have been mapped (Tanksley and McCouch 1997; Moncada et al. 2001; Thomson et al. 2003; He et al. 2006; Tian et al. 2006; Swamy and Sarla 2008; Swamy and Sarla 2011; Fu et al. 2010; Luo et al. 2011; Gur et al. 2011, Swamy et al. 2011). In rice, DNA segments (QTL alleles) from the wild relative Oryza rufipogon have been reported to have significant effects on yield and its component traits in the genetic background of cultivars/hybrids (Xiao et al. 1996, 1998; Ali et al. 2010). Previous studies on global gene expression generally deal with development (Wang et al. 2005; Kondou et al. 2006) and heterosis (Huang et al. 2006; Zhang et al. 2008; Wei et al. 2009) where only panicle (Peng et al. 2009) or anther (Endo et al. 2004) was used for expression analysis. Other studies deal with changes in transcriptome as related to environment such as high temperature (Yamakawa et al. 2007), drought, and high salinity (Walia et al. 2007; Pandit et al. 2010). Introgression lines derived from crosses between elite and wild species have been used in tomato to identify candidate genes for yield traits (Kamenetzky et al. 2010; Gur et al. 2011). However, there are no reports of transcriptomics of wild species-derived introgression lines in rice. This study emerged from a marker assisted backcross program to develop a near isogenic line for the major yield QTL yld2.1 identified earlier from O. rufipogon in an elite cultivar background (Marri et al. 2005). KMR3, one of the parents of mapping population is a popular restorer line used in production of high-yielding hybrid (KRH2) with wide adaptability in India. Two plants carrying flanking markers of yld2.1 were identified in the progeny of mapping population and these were backcrossed to KMR3 for 3 generations selecting for flanking markers in each generation and 500 BC3 plants were selfed (Babu et al. 2009). Several BC3F2 derived introgression lines (ILs) yielding >20% over parent KMR3 were selected and IL50 had highest grain

Funct Integr Genomics (2012) 12:277–289

yield (Sarla et al. 2009). Single plant selections were made from IL50 and advanced up to F8 generation. IL50-7, IL5010 and IL50-13 were selected for their high yield and also high yield of the hybrids derived from using these ILs as restorer lines in combination with different cms lines. These lines are stable and have most of the genome similar to KMR3 except for few chromosomal regions introgressed from O. rufipogon, which probably are responsible for increased yield phenotype. We selected one of the highest yielding ILs (IL50-7) for comparing global gene expression of flag leaf and young panicle with those of recurrent parent KMR3. Such a comparison would lead to discovery of novel yield-related genes/alleles from wild rice. Functional analysis of short listed genes to confirm their role would help in identification of specific genes/alleles contributing to increased yield. In this transcriptome study, we have identified genes from wild rice highly differentially expressed in high yielding introgression line and propose that Os11Gsk from O. rufipogon increases grain yield.

Methods Plant materials Introgression line IL50-7 and control KMR3 were selected on the basis of high seed yield in several seasons in DRR field. Thirty-day-old seedlings were transplanted in the main field in a randomized block design with three replications during Kharif 2009 at the Directorate of Rice Research (DRR) farm. Each entry was planted in ten rows of each line containing 24 plants. Single seedling was transplanted per hill at a spacing of 20×15 cm and all recommended packages of practices were followed to raise a healthy crop. Plant tissue samples, flag leaves, and young panicle (fully emerged) were collected. To test reproducibility of the microarray experiments, three biological replications were maintained separately and RNA was extracted. RNA isolation and GeneChip hybridization Flag leaves and fully emerged young panicles were collected and total RNA was extracted with TRIzol Reagent (SigmaAldrich, St Louis, MO, USA). Plant tissues were homogenized using mortar and pestle with liquid nitrogen and 1 ml of TRIzol reagent was added per 100 mg of tissue. RNA samples were processed according to Affymetrix GeneChip expression analysis technical manual. The cDNA was synthesized from 8 μg of total RNA using superscript double-stranded cDNA synthesis kit and poly (T) nucleotide primers that contain sequence recognized by T7 RNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA) as per supplier’s instructions. Biotin tagged cRNA was synthesized using Affymetrix


GeneChip IVT labeling kit and cRNA fragmentation was performed as per manufacturer’s instructions. Rice Affymetrix GeneChip (51 K oligoarray) was hybridized using 10 μg of the fragmented cRNA, washed and stained with streptavidinphycoerythrin in Affymetrix Fluidics station 450, followed by scanning in GeneChip Scanner 3000. Rice GeneChip array and probe annotation The Affymetrix rice genome array contained probe sets designed from 48,564 japonica and 10,260 indica gene sequences. Sequence information for this array was derived from the National Centre for Biotechnology Information (NCBI) UniGene build number 52 (http://www.ncbi.nlm. nih.gov/UniGene), GenBank mRNAs using the Affymetrix 3′ UTR rice genome array consisting of (57,381) gene predictions from TIGR’s osa1, version 2.0. Gene models that had any indication of transposable elements were removed from the list of TIGR genes. The array is believed to represent about 46,000 distinct rice genes. About 26,000 of these are 3′ anchored unigene ESTs and mRNA clusters, including known rice full-length cDNA clones, and 19,431 are solely from the TIGR gene predictions. Annotation of the differentially expressed probes was done using NetAffyx software of Affymetrix and further validated using BLASTX search through NCBI.

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Carlsbad, CA, USA) in a 20-μl reaction volume. For qRTPCR, each cDNA sample was diluted 1:4 in sterile ddH2O, and each reaction contained 1 μl of cDNA, 0.5 μM of each primer, and 10 μl of SYBR Green I master mix (Qiagen India Pvt. Ltd. New Delhi, India) in 20 μl aliquots. PCR was carried out in iCycler iQ5TM (BioRad Laboratories, Hercules, CA, USA), according to the manufacturer’s instruction. Thermal cycling conditions comprised of 50°C for 1 h followed by an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 55–65°C for 1 min, and extension at 72°C for 1 min. The specificity of amplification for qRT-PCR was evaluated by dissociation curves and expression levels by peak area generated from PCR runs. No-template controls were used to ensure the absence of contamination. For positive control GAPDH gene was used as a reference sample and relative quantification of genes was determined using the method of Livak and Schmittgen (2001). Gene annotation and probe set–gene association The annotation of genes and gene families used in this study were downloaded from TIGR Rice Genome Pseudomolecules Release 5. Due to the update of genome assemble and gene annotation, we revaluated definition of probe sets.

Statistical analysis of microarray data

Gene ontology analysis

The array data were analyzed using GeneChip Operating Software version GCOS 1.2 and GeneSpring software version GX 9.0.6. We used a default target intensity value setting of n0500 and scaling factor for the array in the range of 3.1–8.5. The detection calls for the probe sets were made by GCOS. Probe level normalization was performed using the robust multichip average algorithm of GeneSpring and only those probes with expression levels above the background levels with statistical significance (p66-fold), showed no detectable amplicons in KMR3 but was highly expressed in O. rufipogon and IL50-7 and genomic sequence was the same in latter two but different in KMR3. On the other hand, a highly downregulated (>90fold) gene Os12Mat, was present in KMR3 and IL50-7 but absent in O. rufipogon and its downregulation in IL50-7 could be the effect of introgression. Of the ten other DEGs that were sequenced two were O. rufipogon alleles that had introgressed into IL50-7 whereas four were KMR3 alleles and the remaining four were same in KMR3 and O. rufipogon. The two alleles derived from wild rice showed upregulation whereas KMR3 alleles showed downregulation in IL50-7. Os11Gsk and Os12Mat are two distinct examples of presence and absence variation in closely related species as reported in maize and its progenitor teosinte (SwansonWagner et al. 2010). Our results support their observation that four genes whose sequence is similar in IL50-7, KMR3 and O. rufipogon have not been selected during domestication and may not have a major role in yield. In contrast, 6 genes which were different in KMR3 and O. rufipogon and preferentially selected for up/downregulation were subjected to selection resulting in sequence changes. Most DEGs are members of paralogous families so that a few losses can be tolerated through buffering by redundant members (Peng et al. 2009). The gene Os11Gsk has a coding sequence of 444 bp and encodes for a 148 amino acid protein. BLAST search for homology of the predicted protein sequences of Os11Gsk shows ~83% similarity of 143 amino acids in the C-terminal region to the N-terminal sequences of a 406-amino acid shaggy-like GSK-3 kinase protein from Triticum aestivum. However, the Os11Gsk protein did not show any functional domains in a conserved domain search whereas the Triticum protein had a well conserved kinase domain similar to that found in glycogen synthase 3-kinases from humans. Os11Gsk gene also shows 91% homology to 108 bp region of functional GSK3 on chromosome 1 (LOC_Os01g14860) which has been shown to group phylogenetically with AtSK11 and AtSK12 genes involved in flower development (Dornelas et al. 2000). GSK-3 kinases were shown to be expressed in pollen in A. thaliana, Petunia hybrida, and Nicotania tabacum (Tichtinsky et al. 1998). The coexpression analysis from expression database showed that the expression of Os11Gsk positively correlates with the


expression of four genes including Os12Mat whereas in our study all these four genes are downregulated, showing a negative correlation with Os11Gsk, which is highly upregulated. This suggests that all these genes may be involved in a common pathway and the Os11Gsk allele derived from O. rufipogon may be regulating the other KMR3-derived genes present in IL50-7. Of the two genes that have been derived from O. rufipogon namely Os11Gsk and Os3Ein, Os11Gsk is a completely new gene introduced in KMR3 background and may have a major role to play in increasing yield of IL50-7. There are two possible roles of Os11Gsk introgression in IL50-7. One hypothesis is that Os11Gsk introgression triggers transposition of endogenous transposon resulting in epigenetic modifications in the target genes since Os11Gsk has been annotated as a transposase in the TIGR database and additional bands were observed in the genomic DNA southern analysis of introgression lines in this study. Introgression from wild rice Zizania latifolia activated endogenous cryptic transposons, Tos17 (Han et al. 2004), and DART (Wang et al. 2010) in cultivated rice resulting in structural and methylation pattern changes of the flanking genomic region. The high number of downregulated genes observed in leaves and panicles of IL50-7 and the fact that all downregulated genes are KMR3 alleles in microarray data supports this hypothesis. The other possibility is Os11Gsk from wild rice is a pseudogene and mediates trans-silencing of functional genes sharing homology with it. The large number of transcripts may trigger siRNA which act in trans to silence orthologues of Os11Gsk such as Os1Gsk coding for a functional kinase GSK3 on chromosome 1. Os1Gsk, an orthologue of A. thaliana BIN2, a GSK3 protein which is a negative regulator of the brassinosteroid pathway and known to be involved in several important agronomic traits related to yield (Koh et al. 2007; Kim et al. 2009; Divi and Krishna 2009). This hypothesis is supported by the fact that many genes in the BR signalling pathway did show differential regulation in IL50-7 line compared to KMR3 in our microarray analysis. Parental line improvement is a prerequisite for developing high yielding rice hybrids using favourable genes from distant relatives to meet target productivity. Elite parental lines can be improved for yield using wild species. Once an elite restorer line or male-sterile line is developed, it can be used to breed a series of hybrids with strong heterosis that can be applied in rice production (Deng et al. 2006). The identification of yield enhancing genes/QTLs from wild rice and introducing them into parents of hybrid rice would dramatically enhance heterosis in rice hybrids (Xiao et al. 1996; Wu 2009). There are only two reports of parental line improvement using genes from wild species of rice (Liang et al. 2004; Fu et al. 2010) and both deal with the restorer line 9311 used in China. KMR3 is a popular restorer


line used in production of Karnataka Rice Hybrid-2 (KRH2) in India. The improved restorer lines (IL50-7 and IL50-13) and other sister lines with introgressions from O. rufipogon in KMR3 background have been crossed to several CMS lines to generate hybrids that yield higher than KRH2 (unpublished data). Further work is in progress to investigate the role of candidate genes in increasing yield. Acknowledgments This work and ST were supported by a DBT grant to NS [DBT No.BT/AD/FG-2(PH-II)2009]. AKB was funded by Indian Council for Agricultural Research project 3019 (NPTC/FG/ 05/2672/33). We acknowledge the contributions of A Prasad Babu, C Surendhar Reddy, and BP Mallikarjuna Swamy in earlier work on developing and evaluating introgression lines and marker aided selection. We thank Project Director, DRR for the encouragement. We thank G. Haritha and N. Naga Deepthi for help in identifying polymorphic markers. The authors are thankful to the Sequencing laboratory CCMB, India for providing the sequencing facility used for this study.

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