An Ac/Ds-mediated Insertional Mutagenesis System for Functional Genomics in Sorghum

Transgenic sorghum (cv M 35-1) plants, carrying insertional inactivation tagging Ds, were crossed with iAc plants as the source of transposase to produce mutagenic F1 plants carrying both Ds and iAc. Screening of F2 plants for the presence of Ds (Ds ), transposed (TDs) and absence of iAc (iAc) could identify eight Ds tagged insertional inactivation tagged mutants. Ds transpositions unlinked to the original site of launch pad (T-DNA+Ds) integration were more frequent (57%) than linked transpositions. Analysis of flanking sequence tags of the Ds among the four mutants showed gene tagging in all. Preliminary phenotyping of the Ds tagged mutants (without ascertaining their zygosity) under green house conditions showed no major difference for morphological traits when compared to M 35-1. With the demonstration of both transiently expressed transposase (TET) system and hybridization of iAc and Ds plants to generate Ds tagged mutants, now it should be possible to develop more mutants for sorghum functional genomics.


Introduction
Sorghum [Sorghum bicolor (L.) Moench] is an important staple food and fodder crop grown worldwide. It is known for high water use efficiency, tolerance to low soil fertility and drought. As a model C 4 grass, sorghum was considered for genome sequencing (Paterson et al., 2009). Approximately 98% of the total predicted genes (34 496) have been placed in their chromosomal context (Paterson et al., 2009). Comparative genomics (Jaiswal et al., 2006), TILLING (Xin et al., 2008), mapping (Billot et al., 2013) and trancsriptomics (Dugas et al., 2011) have been widely attempted to identify and study the genes.
Gene disruption with DNA tags provides a direct way of studying gene function (Springer, 2000). T-DNA and transposable element (TE) are the DNA tags used for gene tagging with the latter offering the advantage of developing multiple independent tagged mutants from a few individual starter lines. The ability of TEs to re-transpose from the tagged sites allowing the recovery of revertants offers a unique method of gene function validation. Two-element system of transposon tagging entails a modified autonomous element that expresses a functional transposase but does not undergo transposition. This immobile autonomous element is used along with a non-autonomous element, which can be transposed by the transposase. Two element system with immobile Ac (iAc) and Ds of maize is most widely used for transposon mutagenesis.
Insertional inactivation tagging is the major method of developing mutants through Ds mutagenesis (Springer, 2000;Waki et al., 2013). Insertion of the DNA tag results in gene disruption (inactivation) leading to loss-of-function in insertional inactivation tagging.
Previous study with transiently expressed transposase (TET) system in sorghum has demonstrated that insertional inactivated Ds-tagged mutants could be generated as early as in the first tissue culture generation (T 0 ) (Verma et al., 2011). In addition, mutagenic double transformants were also produced, which could generate mutants in the subsequent generation. But a more general method of developing Ds tagged mutants involves generating independent transgenics with Ds and iAc, and effecting crosses between them to obtain mutagenic F 1 plants. The F 2 plants showing excision and re-insertion of Ds without containing iAc are recognized as Ds tagged mutants. Alternatively, mutagenic T 0 plants are obtained by double transformation through co-transformation or super-transformation, and Ds-tagged mutants are obtained in subsequent generations. This study describes the development of insertional inactivation tagged mutants in sorghum and checking for Ds transpositions linked and unlinked to original site of Ds integration.

Confirmation of Ds and iAc starter plants
For insertional inactivation, six events of pUR224NA and five events of pNU435 were checked for the T-DNA+Ds copy number. Of the six events of pUR224NA, four events showed the presence of single copy T-DNA+Ds. Among the five events of pNU435, four events carried single copy of T-DNA+Ds as tested by  2 analysis.

Developing mutagenic F 1 plants
For insertional inactivation tagging, seven plants from four different events carrying single copy Ds of pUR224NA were used as female parents. Similarly, eight plants from four different events carrying single copy Ds of pNU435 were used as female parents. Fifteen plants from four different events of pKU352NA were used as male parents (source of transposase). Crossing was done by using simple hand emasculation and pollination.

Confirmation of F 1 plants
From the seven crosses involving four events of pUR224NA parent, a total of 18 true F 1 s were obtained. Out of 135 F 1 plants obtained from 8 crosses involving four events of pNU435, 18 were found to be true F 1 s. F 1 plants were bagged to ensure selfing. Panicles were harvested at physiological maturity and the F 2 seeds were collected.
Identifying Ds tagged mutants Screening of 80 F 2 plants involving four events of pNU435 and pUR224NA showed the absence of iAc (iAc -), presence of transposed Ds (TDs + ) in eight plants, indicating that they were insertional inactivated mutants.

Checking linked and unlinked transpositions of Ds
Of the four Ds tagged mutants obtained from pUR224NA, IIM1 and IIM4 gave an amplification of 450 bp with bar PCR, and three Ds tagged mutants obtained from pNU435, IIM6 gave an amplification of 270 bp with barnase PCR Determining tagged gene among mutants Site of insertion of Ds was checked in four mutants (IIM2, IIM3, IIM6, and IIM7) that showed unlinked transposition. Blast results showed that flanking sequence obtained from IIM2 had homology on chromosome number 6 and Ds insertion was between 767 bp and 768 bp of gene coding rice cyclin family protein (cyclin-T1-4). Similarly IIM3 showed insertion of Ds in Sb07g017390 on chromosome number 7.
Ds tagged mutant IIM6 obtained from the launch pad of pNU435 had showing homology with region on chromosome number 8. The Ds insertion was between 1,287 bp and 1,288 bp of Sb08g021000 gene. A FST of 29 bp was recovered from IIM7, and it showed homology to region on chromosome number 1. The insertion inactivation Ds tag was inserted between 3,033 bp and 3,034 bp of Sb01g008710 gene.

Phenotypic evaluation of the mutants
The Ds tagged mutants were observed in for the traits like days to 50 percent flowering, plant height at maturity, number of leaves per plant, number of internodes per plant, panicle length, panicle breadth, number of primaries per panicle, test weight, number of grains per panicle and grain yield per plant as defined by Sorghum Descriptor (IPGRI, 1993)

Discussion
From the large number of Ds launch pad and iAc plants produced in our previous studies in the background of M 35-1, a popular variety of sorghum, those receiving the Ds from pUR224NA and pNU435 were used as the launch pad lines for insertion inactivation tagging, .Plants receiving the iAc from pKU352NA were used as the source of transposase for both the types of tagging.
In general, starter lines with low copy launch pads are used (Upadhyaya et al., 2002) for transposon mutagenesis. Such Ds starter lines not only simplify the mutant analysis, but also ensure higher activity of Ds (Kolesnik et al., 2004). In this regard, improvements in the Ds constructs have played a key role in generating starter lines with low copy launch pad as it has been demonstrated with pNU435 (Upadhyaya et al., 2006;Bhat et al., 2011). In this study, Ds launch pad lines carrying single copy T-DNA+Ds as identified by Southern hybridization (Verma et al., 2011) and segregation analysis were selected for initiating transposon tagging. Further, a few plants with 2~3 copies of the launch pad as checked by Southern hybridization were also selected ( Figure 1). Ds starter plants showing normal phenotype were used for generating Ds-tagged mutants. Figure 1 Sothern hybridization for Ds launch pad lines Note: 1: pUR224NA-T 0 (40)-T 1 (1)-T 2 (5)-T 3 (8)-T 4 ); 2: pUR2 24NA-T 0 (50)-T 1 (7)-T 2 In total, four events each for pUR224NA, pNU435 and pKU352NA were employed for crossing since the use of multiple events of Ds starter lines provides an opportunity for genome saturated mutagenesis (Upadhyaya et al., 2006). Of the twenty two crosses effected, 15 involved the parents with insertional inactivation tagging Ds. (Table 1). F 1 s carrying both iAc and Ds (iAc + and Ds + ) were regarded as mutagenic as they can give rise to Ds tagged mutants. Since the parents used for crossing were not confirmed to be homozygous, the F 1 s were tested for the presence of Ds and iAc. From the seven crosses involving pUR224NA Ds parents, 18 F 1 plants showed the presence of both iAc and Ds. Amplification of 497 bp amplicon with iAc (3333:3829) PCR confirmed the presence of iAc. Similarly, the presence of Ds was confirmed with the amplification of a 436 bp product with uidA PCR. Another 18 F 1 s originating from eight crosses involving pNU435 parental lines were also mutagenic for insertion inactivation tagging. The F 1 plants showed amplification with uidA PCR and iAc (3333:3829) PCR. Overall, 36 mutagenic F 1 s were obtained for insertion inactivation tagging. F 1 plants were bagged to ensure selfing. Seeds were harvested at physiological maturity and the F 2 plants were raised.
F 2 plants that were devoid of iAc but carried the transposed Ds (TDs + ) as confirmed by Ds excision and Ds re-insertion were selected as Ds tagged stable mutants. Of the 80 F 2 plants involving pUR224NA Ds parent, only 27 plants (against expected 60) carried the Ds as checked by uidA PCR (Figure 2A). Origin of such a low frequency of Ds carrying plants could be due to excision without re-integration of the Ds in the genome as reported earlier (Lazarow et al., 2012).
Among the 27 plants carrying Ds, 11 were devoid of iAc, indicating that the Ds in such plants is stable. Of the 11 plants, four plants showed Ds excision as they could amplify 460 bp amplicon with 35S:bar Ds excision PCR. Remaining seven plants failed to amplify 460 bp product, indicating that the Ds was intact within the T-DNA. As expected, 35S:bar Ds excision PCR with the plasmid DNA of pUR224NA failed to amplify the 460 bp product ( Figure 2B). Therefore, those four plants that did not carry iAc (iAc -), but carried transposed Ds (TDs + ) were considered as stable insertional inactivation tagged mutants (IIM1, IIM2, IIM3 and IIM4) ( Table 2).
pUR224NA has bar gene as the Ds excision reporter. Upon spraying Basta to 10 day old 20 F 2 seedlings from the cross involving pUR224NA_T 0 (50) event of Ds launch pad, only four plants survived, indicating the Ds excision in these plants. Subsequently, the Basta surviving plants were checked with 35S:bar Ds  Screening of 80 F 2 plants involving four events of pNU435 showed the absence of iAc (iAc -), presence of transposed Ds (TDs + ) in three plants, indicating that they were also IIMs (IIM6, IIM7 and IIM8). Thus eight insertional inactivated mutants were obtained in this study.  (Figure 3). The other two mutants, IIM2 and IIM3 had unlinked transposition as they failed to amplify 450 bp product In previous studies with a large number of rice mutants, linked transpositions varied from 36%~67% with the majority being within 1 cM of the Ds launch pad (Upadhyaya et al., 2002;Kim et al., 2004). This observation was consistent with the reports both from maize, the original source of Ds (Dooner et al., 1991) and other heterologous systems such as Arabidopsis (Bancroft et al., 1993;Keller et al., 1993;Raina et al., 2002), tobacco (Jones et al., 1990) and barley (Koprek et al., 2000). In this regard, the use of a new transposon system, Spm/dSpm (Kumar et al., 2005) might be more efficient for genome-wide coverage as it could result in 85% unlinked transpositions (Kumar et al., 2005). However, the behaviour of Ds to transpose to a linked site has been exploited for localized mutagenesis in Arabidopsis (Muskett et al., 2003) and rice (Upadhyaya et al., 2006).
Once the Ds tagged mutants are identified, generally they are phenotyped for gross morphological mutations, and analyzed for the tagged gene. Otherwise, the mutants are checked for the site of Ds transposition, and subsequently phenotyped under normal and/or special conditions. In this study, four mutants with unlinked transposition (IIM2, IIM3, IIM6, and IIM7) were analyzed for the site of Ds insertion. It was found that Ds inserted in genic region in all the mutants ( Table 2). The high frequency of gene tagging could be due to preferential transposition of Ds into gene coding regions as shown in Arabidopsis (Parinov et al., 1999;Raina et al., 2002) and rice (Enoki et al., 1999;Greco et al., 2001;Kolesnik et al., 2004).
The phenotypic evaluation of the mutants (without ascertaining their zygosity) in comparison with M 35-1 for morphological traits like days to 50% flowering, plant height at maturity, number of leaves per plant, number of internodes per plant, panicle length, panicle breadth, number of primaries per panicle, test weight, number of grains per panicle and grain yield per plant showed no major differences. It is too early to speculate on the utility of these mutants for functional genomics. For instance, phenotypic evaluation of insertional inactivation mutants with homozygous mutants under normal or special conditions might give the clear idea about the use of such Ds tagged mutants. However, having demonstrated that both transiently expressed transposase (TET) system and crossing of iAc and Ds lines could yield Ds tagged mutants, now it should be possible to generate more mutants and to analyze them for gene discovery in sorghum.

Ds and iAc starter lines
The features of pNU435 and pKU352NA have been previously described (Verma et al., 2011). pNU435 (GenBank Acc. No. DQ225750) has bidirectional gene trap cassettes (eyfp as Ds 5' trap tracer, uidA as Ds 3' trap reporter), bar as a transformation/Ds re-insertion marker, hph as an excision marker, and a plasmid rescue cassette. In addition, barnase serves as a counter selector for T-DNA integrations with vector backbone (VB), direct repeat and gene trap. pUR224NA (GenBank Acc. No. DQ225746) has hph as selectable marker, uidA as Ds trap reporter, bar as excision marker and a plasmid rescue cassette. pKU352NA (GenBank Acc. No. DQ225751) has hph as transformation marker, sgfpS65T as an iAc tracer and intron interrupted ubiquitin driven iAc element (Figure 4). M 35-1, a rabi genotype of sorghum was used to develop several Ds and iAc starter lines in the previous studies (Verma et al., 2011; at the Department of Biotechnology, UAS, Dharwad, India. Plants transformed with pNU435 and pUR224NA constructs were used as the Ds starter lines for insertional inactivation tagging. iAc starter plants as the source of transposase for insertional inactivation tagging were developed by transforming with pKU352NA. These plants were confirmed for the presence of Ds element by uidA PCR (pUR224NA and pNU435). iAc element was confirmed by iAc (3333:3829) PCR. The primers used for the various PCRs and the expected product size are listed in Table 3. The copy number of the launch pad (T-DNA+Ds)  (Dellaporta et al., 1983) and checking with PCR. Panicles from the PCR confirmed plants were bagged to ensure selfing. Panicles were harvested at physiological maturity and the F 2 seeds were collected.

Identifying Ds tagged mutants
The F 2 plants were checked for the presence of Ds and iAc using PCR. Ds excision from the launch pad of pNU435 was checked by Ds3':hph PCR where the forward primer anneals Ds 3' end and the reverse primer anneals to hph. The amplification of the PCR product would indicate the intact launch pad without Ds excision. Since pUR224NA carried bar gene as Ds excision reporter, the corresponding 10 day old F 2 seedlings were sprayed with herbicide Basta (Bayer Crop Sciences Ltd., Mumbai, India) containing glufosinate-ammonium (13.5% w/w) at 12 ppm concentration as standardized previously (Verma, 2010). Plants surviving the herbicide spray were confirmed for Ds excision by 35S:bar PCR, where the forward and reverse primers anneal to CaMV35S and bar flanking the Ds. Amplification of PCR product with 35S:bar PCR indicates the Ds excision.

Checking linked and unlinked transpositions of Ds
Ds tagged mutants were checked for the linked or unlinked Ds transpositions depending on the presence or absence of empty launch pad (T-DNA without Ds), respectively. The presence of empty launch pad of pUR224NA was checked using bar PCR while barnase PCR and uidA PCR were used to check for the empty launch pad of pNU435.

Identifying the tagged gene among the mutants
The gene tagged by Ds among the mutants was identified by recovering the sorghum endogenous sequences flanking the 5' end of Ds by TAIL-PCR. Ds 5' specific nested primers; RB94:3-1 (primary TAIL-PCR), RB96:3-2 (secondary TAIL-PCR) and RB98:3-3 (tertiary TAIL-PCR) in combination with arbitrary degenerate (AD) primers were used in TAIL-PCR.

Phenotypic evaluation of the mutants
The Ds tagged mutants were observed in comparison to the control plant for the traits like days to 50 percent flowering, plant height at maturity, number of leaves per plant, number of internodes per plant, panicle length, panicle breadth, number of primaries per panicle, test weight, number of grains per panicle and grain yield per plant as defined by Sorghum Descriptor (IPGRI 1993).