Candidate gene analysis for determinacy in pigeonpea (Cajanus spp.)
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- Mir, R.R., Kudapa, H., Srikanth, S. et al. Theor Appl Genet (2014) 127: 2663. doi:10.1007/s00122-014-2406-8
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We report a likely candidate gene,CcTFL1,for determinacy in pigeonpea through candidate gene sequencing analysis, mapping, QTL analysis together with comparative genomics and expression profiling.
Pigeonpea (Cajanus cajan) is the sixth most important legume crop grown on ~5 million hectares globally. Determinacy is an agronomically important trait selected during pigeonpea domestication. In the present study, seven genes related to determinacy/flowering pattern in pigeonpea were isolated through a comparative genomics approach. Single nucleotide polymorphism (SNP) analysis of these candidate genes on 142 pigeonpea lines found a strong association of SNPs with the determinacy trait for three of the genes. Subsequently, QTL analysis highlighted one gene, CcTFL1, as a likely candidate for determinacy in pigeonpea since it explained 45–96 % of phenotypic variation for determinacy, 45 % for flowering time and 77 % for plant height. Comparative genomics analysis of CcTFL1 with the soybean (Glycine max) and common bean (Phaseolus vulgaris) genomes at the micro-syntenic level further enhanced our confidence in CcTFL1 as a likely candidate gene. These findings have been validated by expression analysis that showed down regulation of CcTFL1 in a determinate line in comparison to an indeterminate line. Gene-based markers developed in the present study will allow faster manipulation of the determinacy trait in future breeding programs of pigeonpea and will also help in the development of markers for these traits in other related legume species.
Pigeonpea [Cajanus cajan (L.) Millsp.] is one of the most important food legume crops for arid and semi-arid regions of the world. It is grown on ~5 million hectares (ha) globally and constitutes one of the main sources of protein for >1 billion people, as well as a cash crop for millions of resource poor people living in Asia, Africa, South America, Central America and the Caribbean (Mula and Saxena 2010). The pattern and time of flowering are important adaptive traits in flowering plants controlled by physiological signals, genes, gene interactions and interactions of genes with the environment (Liu et al. 2010). Tremendous progress has been made in the area of isolation and characterization of plant genes for crop improvement due to emergence of plant genomics (Arabidopsis Genome Initiative 2000; Mouradov et al. 2002; Michael and Jackson 2013). Availability of genome sequence of a number of plant species together with comparative genomics have helped in answering some of the fundamental aspects of plant biology including identification and analysis of genes involved in adaptive traits in crop species (Cronk 2001; Foucher et al. 2003). One of the best examples of such evolutionary developmental studies in plant species is the identification and analysis of MADS box genes involved in flower development (Ma and De Pamphilis 2000). Subsequently, orthologous genes have been isolated in many species providing insights into the conservation and diversification of such genes and their functions in plant development (Hofer and Ellis 2002).
Several approaches like genetic linkage analysis, candidate gene association analysis, and heterologous transformation have been used to test for the candidacy of homologous genes from Arabidopsis into other crop species like soybean (Tian et al. 2010). These studies revealed that flowering time/flowering pattern/determinacy has been selected long ago by breeders in combination with photoperiod insensitivity to obtain varieties with shorter flowering period, earlier maturation and ease of mechanized harvest (Repinski et al. 2012). Genetic mechanism responsible for these traits has been uncovered in model plant Arabidopsis (Arabidopsisthaliana), pea (Pisum sativum), soybean (Glycine max), common bean (Phaseolus vulgaris) etc. (Foucher et al. 2003; Hecht et al. 2005; Kwak et al. 2008; Liu et al. 2010; Repinski et al. 2012). In some cases it was proved that determinacy is controlled by a single gene, whereas in other studies more than one gene was found responsible for the transition of different growth habits (Tian et al. 2010). In pea, it was shown that the determinate mutant (det) is caused by mutations in a homologue of the ArabidopsisTFL1 gene (Foucher et al. 2003). In soybean, the gene responsible for determinacy “GmTfl1” was isolated and found to complement the functions of TFL1 in Arabidopsis (Liu et al. 2010; Tian et al. 2010). Similarly, in common bean, it was proved that gene “PvTFL1y” co-segregated with the determinacy locus “fin” (Kwak et al. 2008) and later the same was validated and found as a functional homolog of ArabidopsisTFL1 gene (Repinski et al. 2012). In pigeonpea, both indeterminate (IDT) and determinate (DT) type flowering pattern exist (Mir et al. 2012b). Wild relatives and most of the cultivars have indeterminate growth habit and therefore, it is believed that determinate forms of pigeonpea were selected by farmers or breeders during pigeonpea domestication process or breeding. The availability of determinate growth habit genotypes having initial vigor and tolerance to drought and water logging have been found advantageous over indeterminate types for environments with moderate growth (5–6 t ha−1), while as IDT type lines have been found suitable for environments with high (7–8 t ha−1) growth potential (Singh and Oswalt 1992). However, only some linked markers associated with flowering pattern/determinacy have been reported recently in pigeonpea (Mir et al. 2012b). The present study reports the isolation of seven genes and identification of likely candidate gene “CcTFL1” for determinacy in pigeonpea using candidate gene sequencing, linkage mapping based association analysis, comparative genomics and differential gene expression approaches.
Materials and methods
Plant material and phenotyping
A set of 142 pigeonpea germplasm [Cajanus cajan (L.) Millsp.] accessions including 84 indeterminate (IDT) and 58 determinate (DT) accessions were selected to test associations of candidate genes/SNPs with determinacy in pigeonpea (Table S1a). For genetic mapping of candidate genes/SNPs, a bi-parental F2 mapping population derived from a cross ICPA 2039 (DT, plant height: 140 cm, days to 50 % flowering: 70 to 80 days, days to maturity: 130 to 140 days) × ICPR 2447 (IDT, plant height: 150 cm, days to 50 % flowering: 75 to 85 days, days to maturity: 125 to 135 days) comprising 188 lines was used (Table S1b). To validate the identified SNP in candidate gene “TFL1”, another F2 mapping population derived from a wide cross [C. cajan (ICPL 85010) × C. volubilis Blanco (ICP 15774)] comprising of 21 F2 lines was used (Table S1c).
Determinacy data were recorded at the Research Farm, ICRISAT, Patancheru, Hyderabad, India in the year 2009 cropping season. For both F2 mapping populations, data were recorded on single plants for plant height, flowering time and determinacy in un-replicated manner.
Total genomic DNA was extracted from DT/IDT lines, parental lines and segregating F2 progenies at an early seedling stage using a high-throughput mini DNA extraction protocol (Cuc et al. 2008). The quality and quantity of extracted DNA was checked on 0.8 % agarose gels and the DNA was normalized to 5 ng/µl for further use.
For expression profiling, two pigeonpea accessions ICPA 2039 (DT) and ICPL 87118 or Asha (IDT) were used as representatives of the two phenotypic categories. Seeds were sown in pots (three seeds per pot), and maintained in a glasshouse under controlled conditions. Plants in each pot were thinned to one healthy plant/pot at the stage, 15 days after germination (DAG). Tissues representing different developmental stages viz., root tip, roots, young leaves, mature leaves, shoot, shoot tip and flower were targeted for collection in three biological replications. Six tissue samples (excluding flower, due to limited or no flower) were harvested from individual glass-house grown pigeonpea plants at three different time points, 15DAG, 30DAG, 10 days after flowering (DAF). Seven tissue samples (including flower) were harvested at 20 DAF. Collection of tissues at different growth stages from different parts of the pigeonpea plants (vegetative vs reproductive parts) was based on the evidence that TFL1 gene shows differential expression in different parts at different stages of plant development in Arabidopsis and other related legume crops like pea, soybean and common bean (Repinski et al. 2012). Tissues were washed thoroughly with 0.1 % DEPC water, frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Total RNA was extracted from the harvested tissues using TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. RNA quality was assessed on 1.2 % formaldehyde agarose gels, while purity of RNA was assessed using a NanoVue spectrophotometer (A260/A280 ratio). First strand cDNA was synthesized from total RNA (2.5 μg) using a cDNA synthesis kit (Superscript® III, Invitrogen, CA, USA) following manufacturer’s instructions.
Selection of candidate genes
List of primer pairs used for amplification of the respective candidate genes
Kwak et al. (2008)
Kwak et al. (2008)
CcFCA (Flowering control locus A)
(EF643224, EF643225, EF643226)
Kwak et al. (2008)
Kwak et al. (2008)
CcFLD(Flowering locus D)
(EF643227, EF643228, EF643229)
Kwak et al. (2008)
Kwak et al. (2008)
(EF643231, EF643232, EF643233, EF643234)
Kwak et al. (2008)
Kwak et al. (2008)
(EF643235, EF643236, EF643237, EF643238)
Kwak et al. (2008)
Kwak et al. (2008)
CcTFL2 (Terminal Flower 2)
Kwak et al. (2008)
Kwak et al. (2008)
CcTFL1(Terminal Flower 1)
(EF643247, EF643248, EF643249, EF643250)
Kwak et al. (2008)
Allele specific primers for the gene CcTFL1
qRT-PCR primers for the gene CcTFL1
CcTFL1_e2 + 3_F
CcTFL1_e2 + 3_R
Polymerase chain reaction (PCR) and amplicon sequencing
The PCR master mix components and PCR cycle profile used were as described for candidate gene amplification/sequencing in chickpea (Gujaria et al. 2011). PCR products were separated on 1.2 % agarose gels.
PCR products were treated with exonuclease I (Exo) and shrimp alkaline phosphatase (SAP) before subjected to Sanger sequencing from both ends using respective forward and reverse primers at Macrogen Inc., Seoul, South Korea (http://www.macrogen.com/).
Sequence diversity estimation
Sequencing data were inspected manually for possible sequencing error and consensus sequences were prepared using DNA Baser v 2.9 software (http://dnabaser.com). Consensus sequences for all genotypes were aligned using Clustal W (http://www.ebi.ac.uk/Tools/clustalw2/index.html) (Thompson et al. 1994) and analyzed in BioEdit version 188.8.131.52. for SNP identification.
FASTA multiple sequence alignment files (were analyzed using the SNP DIVersity ESTimator (DIVEST) software module (http://hpc.icrisat.cgiar.org/Pise/5.a/statistics_calculation/) developed at ICRISAT (Jayashree et al. 2009) for calculating the polymorphism information content (PIC) value of individual SNPs as well as nucleotide diversity (π), number and PIC value of haplotypes for each gene.
In cleaved amplified polymorphic sequences (CAPS) assay (Konieczny and Ausube 1993) PCR amplicons were subjected to restriction enzyme digestion followed by electrophoretic separation on agarose gels (3 % agarose, 1X TBE buffer, 1 h, 120 V) and visualized by means of ethidium bromide staining (Varshney et al. 2007).
Allele-specific marker assay
Primers targeting each allele of the SNP in gene CcTFL1 and one pair of external primers were designed using the software tools Fast PCR (Kalendar et al. 2009) and Primer 3 (http://frodo.wi.mit.edu/). Primers were multiplexed into a single PCR reaction to obtain co-dominant marker. This marker assay consisted of two external common primers (external common forward primer- TFL1_PCR_CF and external common reverse primer- TFL1_PCR_CR) flanking the SNP and one internal primer targeting one SNP allele “A-allele” (TFL1_PCR_A) and the other internal primer targeting the other SNP allele “T-allele” (TFL1_PCR_T).
Genetic mapping and linkage analysis
Genotyping data generated from 188 F2 plants derived from cross ICPA 2039 × ICPR 2447 were combined with the data for 81 SSR markers already available on the same population “ICPA 2039 × ICPR 2447” (Bohra et al. 2012). Markers were tested for linkage using JoinMap® 4 program (Ooijen 2006); http://www.kyazma.nl) using LOD 3-10 and the Kosambi map function. The inter-marker distances calculated from the JoinMap® 4 program were used to construct a linkage map which was displayed using MAPCHART version 2.2 (Voorrips 2002).
Single marker regression analysis was carried out in Excel 2007 (Microsoft) using the F2 marker genotypes as independent variables and the F2 -phenotypes as dependent variables. The phenotypic data were recorded on single F2 plants. Composite interval mapping (CIM) (Zeng 1993, 1994) was conducted using WinQTL Cartographer, version 2.5 (for more details see Ravi et al. 2011; Mir et al. 2012b).
Comparative gene analysis
BLASTN analysis of CcTFL1 gene of pigeonpea was conducted against the genome sequences of common bean and soybean available at the Phytozyme database (http://www.phytozome.net/). After identification of collinear regions encompassing TFL1 orthologous in pigeonpea (chromosome 3), soybean (chromosome 19) and common bean (chromosome 1) syntenic relationships were analyzed using SyMAP 4.0 (Soderlund et al. 2011).
qRT-PCR assay for validation of CcTFL1 for determinacy
Quantitative real-time PCR (qRT-PCR) was performed using an Applied Biosystems 7500 Real-Time PCR machine and SYBR green chemistry according to the manufacturer’s instructions (Applied Biosystems, CA, USA). Gene-specific primers for qRT-PCR were designed using Primer Express software (Applied Biosystems, CA, USA). Three primer pairs were designed covering all four exonic regions of the CcTFL1 gene; one primer pair each for exons 1 and 4, one primer pair covering exons 2 and 3. Transcript levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin reference genes. PCR was carried out as described in Rawat et al. (2012) and relative expression levels were determined using the 2−ΔΔCT method and student’s t test was used to calculate significance (Livak and Schmittgen 2001).
Flowering related genes
BLASTN similarity between pigeonpea amplicons corresponding genes in soybean, common bean and Arabidopsis
Max. identity (%)
PREDICTED: floral homeotic protein
APETALA 1-like [Glycine max]
PREDICTED: Glycine max flowering time control protein FCA-like, mRNA
Phaseolus vulgaris cultivar Midas flowering locus D (FLD) gene, partial cds
Glycine max circadian clock-associated FKF1 (FKF1), mRNA > gb|DQ371902.1| Glycine max circadian clock-associated FKF1 (FKF1) mRNA, complete cds
PREDICTED: Glycine max protein GIGANTEA-like, transcript variant 2 (LOC100779044), mRNA
Arabidopsis thaliana TFL2 gene for TERMINAL FLOWER 2, complete cds
Glycine max cultivar Heimoshidou Dt1 gene
Diversity features for the candidate genes in a set of 142 Cajanus lines
No. of genotypes surveyed
Sequence data surveyed (average bp)
No. of SNPs identified
PIC of individual SNP
No. of haplotypes
PIC of haplotypes
Association between candidate genes and determinacy
Linkage analysis of candidate genes
The markers were used to score 188 lines of the F2 mapping population derived from ICPA 2039 (DT) × ICPR 2447 (IDT). The phenotypic evaluation of 188 F2 progenies for DT/IDT growth habit revealed that 152 progenies possessed IDT growth habit whereas 36 progenies possessed DT growth habit. The genotyping of this population with gene CcGI showed DT-specific fragment in 66 F2 lines and IDT-specific fragment in 15 F2 lines whereas majority of lines (106) showed heterozygous nature with only one line with missing data. Similarly, the genotyping of CcTFL1 on 188 F2 lines of bi-parental mapping population showed segregation for DT/IDT. For instance, out of 36 DT progenies, 26 showed DT fragment, 5 showed both DT and IDT fragments (heterozygous), 2 showed IDT fragment and remaining 3 showed failure in amplification (missing data). Likewise, out of 152 IDT progenies, 71 lines showed IDT fragment and 80 lines showed both DT and IDT fragment (heterozygotes) whereas one progeny showed failure in amplification (missing data).
Single marker analysis (SMA) using regression and composite interval mapping (CIM) based on our genotype and phenotype data showed association of CcTFL1 with determinacy as well as flowering time and plant height. For instance, SMA analysis of CcTFL1 with trait determinacy showed gene-trait association explaining 75 % phenotypic variation. On the other hand, CIM analysis revealed a cluster of three major QTLs one each for determinacy, flowering time and plant height present in the genomic region (24 cM) defined by CcTFL1 and CcM0126 (Fig. 6). This genomic region explains 45–96 % phenotypic variation for determinacy, 45 % for flowering time and 77 % for plant height (Fig. 6).
Further, the likely candidature of CcTFL1 for determinacy through the first approach, linkage analysis was validated through two more approaches- comparative mapping and expression profiling using qRT-PCR. The second approach (comparative mapping) was followed for the gene CcTFL1 to compare its syntenic relationship with the genomic regions harboring determinacy gene in soybean and common bean. In the third (expression analysis using qRT-PCR) approach, functional validation to confirm candidacy of CcTFL1 for determinacy in pigeonpea was conducted.
Comparative genomics analysis
Expression analysis of CcTFL1
Determinacy is one of the most important and widely studied domesticated traits in flowering plants. In order to obtain early maturing varieties with shorter flowering period, determinacy trait has been selected via domestication process together with photoperiod insensitivity (Repinski et al. 2012). Several studies have been conducted in the past in model plant Arabidopsis, pea, soybean, common bean, etc. to identify the genetic mechanism that is responsible for different forms of growth habit (Foucher et al. 2003; Hecht et al. 2005; Kwak et al. 2008; Liu et al. 2010; Tian et al. 2010; Repinski et al. 2012). In some cases it has been proved that determinacy is controlled by single gene, whereas in other studies more than one gene have been found responsible for the transition from vegetative growth to reproductive growth (Tian et al. 2010). In pea, it has been shown recently that the determinate mutant (det) is caused by mutations in a homologue of the ArabidopsisTFL1 gene. These mutations are synonymous or non-synonymous substitutions at the junction between an exon and an intron resulting in splicing failure (Foucher et al. 2003). In soybean, the gene responsible for determinacy “GmTfl1” has been isolated and found to complement the functions of TFL1 in Arabidopsis (Liu et al. 2010; Tian et al. 2010). Similarly, in common bean, it has been proved that gene “PvTFL1y” co-segregated with the determinacy locus “fin” (Kwak et al. 2008) and later the same has been validated and found as a functional homolog of ArabidopsisTFL1 gene (Repinski et al. 2012).
The same trait exists in pigeonpea also and the availability of determinate growth habit genotypes having initial vigor and tolerance to drought and water logging is advantageous over indeterminate types for environments with moderate growth (5–6 t ha−1) whereas IDT type lines are suitable for environments with high (7–8 t ha−1) growth potential (Singh and Oswalt 1992). Some inheritance studies have been conducted earlier in pigeonpea towards understanding the genetics of this important trait (Waldia and Singh 1987; Gupta and Kapoor 1991; Gumber and Singh 1997). We have tried to uncover this mechanism of transition from indeterminate growth habit to determinate growth habit in pigeonpea recently using whole-genome scanning approach using SNPs and DArT assays (Mir et al. 2012b). The present study is in continuation of our earlier efforts towards identification of definite candidates for determinacy in pigeonpea. The identification of candidate gene(s) for determinacy in pigeonpea will allow us to understand the domestication process in pigeonpea and will allow for further, and faster, manipulation of growth habit and flowering time in future breeding efforts.
Flowering-related genes and sequence diversity
The judicious selection and use of candidate genes during the present study was based on the previous information and validation of their role for determinacy and related traits in Arabidopsis, soybean and common bean (Kwak et al. 2008; Tian et al. 2010). Among all the seven genes, CcTFL1 has been reported as real candidates for the determinacy in these plant species. In pigeonpea, the occurrence of sequence variability in terms of number of SNPs, SNP frequency, nucleotide diversity and number of haplotypes among seven candidate genes strongly indicate the occurrence of different evolutionary constraints. The level of genetic diversity revealed by these gene sequences is in the range of those reported in the literature on crops like Arabidopsis, wheat, barley and sunflower (see Giordani et al. 2011). The occurrence of greater sequence diversity in the IDT group than the DT group was likely a manifestation of a domestication or breeding-driven bottleneck experienced by the DT group, which was composed entirely of the cultigen.
Furthermore, nucleotide blast and BlastX results clearly indicated that the correct TFL1 gene in pigeonpea with same internal structure as that of soybean has been isolated (Tian et al. 2010). Sequence comparison of TFL1 of all the plant species with pigeonpea TFL1 (CcTFL1) also supported these results as the CcTFL1 clustered with soybean TFL1 gene models and common bean TFL1y showing maximum similarity (Fig. 2). Similarly, TFL1 gene sequence of all the DT and IDT lines was found useful in phylogenetic classification/analysis of DT and IDT lines including wild pigeonpea accessions (Fig. 3). In summary, all these results of CcTFL1 analysis provided great support that the CcTFL1 of pigeonpea is the same as has been found in other plant species like Arabidopsis, soybean and common bean (Kwak et al. 2008; Tian et al. 2010; Repinski et al. 2012).
Candidate genes for determinacy and linkage analysis
Association analysis through single marker analysis (SMA)/single marker regression showed that this marker based on TFL1 gene contributes 75 % of phenotypic variation for determinacy in pigeonpea. Further sophisticated analysis using composite interval mapping using QTL Cartographer led to the identification of major QTL on LG09 of pigeonpea genetic linkage map of bi-parental mapping population (ICPA 2039 × ICPR 2447) segregating for determinacy, flowering time and plant height. The major QTL contributes 45–96 % phenotypic variation towards determinacy trait, 45 % towards flowering time and 77 % variation towards plant height and is defined by marker interval CcTFL1 and CcM0126. Thus these findings clearly indicated that CcTFL1 controls determinacy in pigeonpea in addition to its role in controlling flowering time and plant height. The other reason for coincidence of several QTLs for these traits could be due to linkage of genes for these traits. The likely control of TFL1 on more than one trait is also reported in earlier studies in common bean also. For instance, correlation of days to flowering, days to maturity and determinacy were reported in an earlier study in common bean (Tar’an et al. 2002). In addition, it was also found that determinacy causes an early flowering, and there is a positive correlation between earliness and plant height (PH) (Kwak et al. 2008). Mapping of candidate genes with respect to single gene or QTL for growth habit and other related traits provides a test of their possible role in those agronomic traits (Kwak et al. 2008). The isolation and mapping of candidate genes will also test the extent of conserved gene function across multiple crops.
Conversion of SNPs into marker assays revealed that only three candidate genes—CcAP, CcGI and CcTFL1 among the seven genes could be either converted into CAPS/dCAPS/PCR-based marker assays. The SNPs in gene CcAP and CcGI were converted into CAPS and dCAPS assays, respectively, while the SNP in gene CcTFL1 was converted into user friendly PCR-based marker assay. The sequencing alignment of the CcTFL1 on 142 pigeonpea germplasm lines (58 DT and 84 IDT lines) led to the discrimination of all the DT lines from the IDT lines with the exception of 4 lines using diagnostic PCR-based SNP assay. The presence of DT allele in four IDT lines could be attributed to some other genes causing variation in growth habit (Ramkumar et al. 2010). Each assay has its own advantages and disadvantages. The CAPS/dCAPS assays require additional steps of long hours with restriction digestion after PCR and sometimes followed by polyacrylamide denaturing gels for fragment separation and silver staining, thus making these markers laborious and costly for regular use in marker-assisted selection (MAS) programs. On the other hand, the PCR-based SNP markers target the functional SNPs by designing PCR primers such that a forward or reverse primer has a specific deoxynucleotide triphosphate (dNTP) at the 3′ end (Collard and Mackill 2008 ).
The developed marker assays were further directed for genotyping and genetic mapping using either only bi-parental/or bi-parental and wide cross mapping populations. However, only two genes (CcGI and CcTFL1) could be mapped on the genetic linkage map. The inability to map gene CcAP may be due to less number of markers on the map and hence no linkage with any other SSR markers in the genetic map was observed with the CAPS marker. The gene CcGI was mapped on linkage group LG02 in the vicinity of two SSR markers (CcM1235 and CcM2241) (Fig. 5). Similarly, candidate gene CcTFL1 was mapped on the terminal end of LG09 linked by the marker CcM0126 on individual genetic map of ICPA 2039 × ICPR 2447 as well as consensus map of pigeonpea (Fig. 6) developed after merging of several (5–6) genetic maps (Bohra et al. 2012). Candidate genes for determinacy/flowering time have been also mapped in some earlier studies in soybean, pea and common bean (Foucher et al. 2003; Kwak et al. 2008; Tian et al. 2010).
These findings all prove that CcTFL1 is a likely candidate for determinacy in pigeonpea and the marker based on this gene will prove useful in future marker-assisted breeding programs aiming at pigeonpea improvement by making use of both DT and IDT lines in crossing programs together.
Comparative genomics analysis and expression profiling of CcTFL1
Comparative genomics analysis has been performed to confirm and validate our results that CcTFL1 is the candidate gene for determinacy in pigeonpea. Comparison with genome sequences of soybean and common bean revealed conservation of eight genes indicating the orthologous nature of CcTFL1 gene and the high-level of conservation of gene sequence in homologous region across three Phaseoloid legumes. In fact, the same genomic region was found to contain GmTFL1 in soybean and PvTFL1 in common bean (Fig. 8) (Tian et al. 2010; Repinski et al. 2012).
Furthermore, expression profiling of CcTFL1 supported the results obtained through sequencing and linkage analysis. Overall lower levels of expression of CcTFL1 were evident in the DT line ICPA 2039 relative to those in the IDT line Asha across multiple tissues and developmental stages (Fig. 9), as it was observed in other legumes such as pea (Foucher et al. 2003) and soybean (Jung et al. 2012). Prior studies have focused on elucidating genes whose expression differs within the same individual, using the different tissue types or between individuals using same tissue (Li et al. 2009; Tian et al. 2010). In the present study gene expression analysis was performed in contrasting genotypes as well as across different developing stage tissues. Further analysis is necessary to elucidate the mechanistic basis for the observed down-regulation of the CcTFL1 in pigeonpea. In particular it remains to be determined whether the assorting SNP within intron 2 of CcTFL1 affects transcript stability as observed for regulation of the RFL gene in rice (see Prasad et al. 2003) or underlies quantitative control of expression as has been observed in soybean recently (see Ping et al. 2014). In this context, 1,060 bp immediately upstream of the start codon of CcTFL1 was sequenced for 10 DT and 4 IDT lines (data not shown). Although sequence analysis did not identify polymorphism among DT and IDT lines, the possibility of additional SNP(s) in the cis regions further upstream or in 3′ untranslated regions of CcTFL1 that may be causal to transition of IDT to DT cannot be excluded. Also of interest is whether the pattern of expression differences between IDT and DT lines may relate to the perennial plant cycle of pigeonpea, which contrasts with the annual habit of other plant species where CcTFL1 orthologs have been characterized. Nevertheless, our data strongly implicate CcTFL1 as the likely genetic basis for the evolution of the determinacy trait in cultivated pigeonpea, paving the way for marker-assisted selection for this trait in pigeonpea breeding.
RKV conceived, designed and coordinated the experiments. RRM, KHB, SS and RKS performed genotyping/experimental setup. RRM, KHB, RKS, RVP and RKV analyzed the data. RKS and KBS performed the field experimentations/selections. RKV, AS and SA contributed reagents/materials/analysis tools. RKV, RRM, KHB, RKS and RVP wrote the paper.
This study was supported by Theme Leader Discretionary Grant from CGIAR Generation Challenge Programme and United States Agency for International Development (USAID) and Department of Biotechnology (DBT), Government of India. Thanks are due to Ashish Kumar, Abhishek Bohra, Neha Gujaria, A. BhanuPrakash, Reetu Tuteja, Abdul Gafoor and Srivani Gudipati for their help in different experiments. This work has been undertaken as part of the CGIAR Research Program on Grain Legumes. ICRISAT is a member of CGIAR Consortium.
Conflict of interest
The authors declare that they have no conflict of interest.
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