molecular evolution of 5s rdna region in vigna subgenus ceratotropis and its phylogenetic...

ORIGINAL ARTICLE

Molecular evolution of 5S rDNA region in Vigna subgenusCeratotropis and its phylogenetic implications

Ajay Saini Æ Narendra Jawali

Received: 27 October 2008 / Accepted: 9 March 2009 / Published online: 20 May 2009

� Springer-Verlag 2009

Abstract The evolution of 5S rRNA gene unit (5S gene

unit) was studied among the ten species belonging to Vigna

subgenus Ceratotropis by sequencing and analyzing the

intra- and inter-specific sequence heterogeneity. The 5S unit

from these species ranged from 214 to 342 bp in length as a

result of several indels in the intergenic spacer (IGS) region.

A large deletion ([100 bp) was found specifically in the

IGS of V. radiata accessions. IGS showed high sequence

variation with more than 50% polymorphic and 35.4%

parsimony informative sites. However, the coding region

(5S gene) was highly conserved, both in length and in

sequence. Intra-genomic and intra-specific divergence was

observed among some species, which indicated that the 5S

unit is evolving at different rates among the Vigna species.

Most Vigna species harbored one type of 5S unit indicating

complete homogenization among them. Vigna glabrescens,

a tetraploid species, also showed single type of 5S rDNA

from only one of the diploid progenitor indicating loss or

homogenization of the other type. However, V. nakashimae

and V. riukiuensis harbored multiple, diverse, ‘intra-geno-

mic 5S types’ indicating that 5S rDNA is not completely

homogenized by concerted evolution and is still evolving.

In general, the phylogeny based on IGS sequences was in

agreement with many of the earlier reports except some

surprising observations such as, V. glabrescens clustered

with V. mungo in section Ceratotropis and unlike most of

the species, wild and cultivated types of V. umbellata were

present in different subclusters. Presence of divergent 5S

sequences in V. nakashimae and V. riukiuensis caused

errors in phylogeny reconstruction at species level and

suggested a horizontal ‘gene transfer’ as a result of inter-

species hybridization. The comparative analysis showed

that 5S IGS sequences have better phylogenetic utility than

chloroplast DNA sequences, such as atpB-rbcL and is

comparable to ITS1 and ITS2 in this respect.

Keywords Vigna � Ceratotropis � 5S rRNA gene unit �Intergenic spacer � Intra-genomic 5S type �Recombinant 5S type � Phylogenetic analysis

Introduction

The genus Vigna Savi is divided into seven subgenera

among which subgenera Ceratotropis, Plectotropis and

Vigna include several cultivated species (Marechal et al.

1978). Subgenus Ceratotropis is a homogeneous and spe-

cialized group of species of Asiatic origin (Baudoin and

Marechal 1988) of which several are domesticated and are

of agricultural importance such as V. radiata, V. mungo,

V. angularis, V. aconitifolia and V. umbellata. In general,

these species are diploid (2n = 22) except V. glabrescens,

which is a natural amphidiploid (2n = 44). Earlier classi-

fication of species within this subgenus was based on

morphological, biochemical, cytological and palynological

characters (Verdcourt 1970; Marechal et al. 1978, 1981).

Egawa et al. (1988) investigated relationships among some

Ceratotropis species on the basis of pollen stainability and

pattern of meiotic chromosome pairing in the F1 hybrids.

Earlier classifications are widely accepted but there are still

several taxonomic riddles needed to be resolved (Dana and

Karmakar 1990).

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00606-009-0178-4) contains supplementarymaterial, which is available to authorized users.

A. Saini � N. Jawali (&)

Molecular Biology Division, Bhabha Atomic Research Centre,

Trombay, Mumbai 400 085, India

e-mail: [email protected]

123

Plant Syst Evol (2009) 280:187–206

DOI 10.1007/s00606-009-0178-4

http://dx.doi.org/10.1007/s00606-009-0178-4

Relationships among the species belonging to subgenus

Ceratotropis were also analyzed by RFLP (Fatokun et al.

1993) and RAPD (Kaga et al. 1996). Both studies con-

firmed the phylogeny based on the morphological charac-

ters (Baudoin and Marechal 1988). RAPD analysis also

indicated a higher intra-specific variation in V. radiata

compared to other Ceratotropis species. Other molecular

methods like biochemical markers (Jaaska and Jaaska

1990) and AFLP (Yoon et al. 2000) have been used to

analyze genetic diversity and relationships among Vigna

species. Recently, relationships among the Vigna species

have been also analyzed using internal transcribed spacer

(ITS) and chloroplast spacer region (Doi et al. 2002; Goel

et al. 2002). Hemleben and Werts (1988) characterized the

5S rRNA gene unit of V. radiata. Organization of this

region has not been analyzed in the other related Vigna

species and hence the phylogenetic potential of 5S inter-

genic spacer (IGS) region remains unutilized in inferring

relationships.

The 5S ribosomal RNA gene unit (or 5S gene unit)

belongs to the family of functional, tandem repetitive

sequences, present at one or several chromosomal locations

(Long and David 1980; Sastri et al. 1992). The 5S rRNA

gene unit includes a coding region and an IGS, present

between two successive coding regions (Fig. 1). In

eukaryotes, 5S gene units are present at chromosomal

location different from 18S-5.8S-26S rDNA and depending

on the number of repeat units, the loci are referred to

as major and minor (Sastri et al. 1992; Dubcovsky and

Dvorak 1995). The copy number of 5S rRNA genes is often

higher than that of other rRNA genes (18S, 5.8S and 26S)

and it varies from 1000 to 100000 (Schneeberger et al.

1989). Among the species belonging to Vigna, limited

information is available on the 5S rDNA. In V. radiata, the

copy number of 5S gene is more than 4000 (Hemleben and

Werts 1988) and in V. unguiculata (subgenus Vigna), 5S is

present at two loci (Galasso et al. 1995).

The 5S gene is highly conserved across diverse taxa

(Danna et al. 1996) and this is attributed to structural

constraints associated with its function. Hence, the 5S gene

itself has limited phylogenetic utility as the region provides

few characters due to its small size (Halanych 1991; Steele

et al. 1991). However, the conserved nature of the 5S gene

allows design of universal primers for amplification of IGS

region (Baker et al. 2000). IGS is highly variable in length

(80–900 bp) and sequence (Danna et al. 1996) and hence

can yield large number of informative characters making it

a region useful for inferring relationships at lower taxo-

nomic levels such as genus and species (Appels et al. 1989,

1992; Reddy and Appels 1989; Baum and Appels 1992;

McIntyre et al. 1992; Moran et al. 1992; Playford et al.

1992; Udovicic et al. 1995; Persson 2000; Becerra 2003).

Multi-copy genes including ribosomal RNA genes gener-

ally do not show variation within and between individuals

of a species. This has been attributed to homogenization of

these multi-copy sequences through a ‘molecular drive’

process (Dover 1986). However, variation in the 5S gene

units within a species have been reported among several

species and this is attributed to weak homogenizing

mechanisms in those species (Scoles et al. 1988; Baum and

Appels 1992; Appels et al. 1992; Kellogg and Appels 1995;

Cronn et al. 1996 Baum and Johnson 1998 and Sastri et al.

1992).

This is the first report of use of 5S IGS region for

inferring relationships among species belonging to genus

Vigna. The objectives of the present study were as fol-

lows: (1) to understand the organization and evolution of

the 5S rDNA among the Vigna species belonging to

subgenus Ceratotropis, (2) to assess the intra- and inter-

species heterogeneity in the 5S rDNA gene unit and (3) to

evaluate the phylogenetic utility of 5S IGS and use it

to infer relationships among the Vigna species in the

subgenus.

Materials and methods

Plant material

A total of 25 taxa belonging to ten Vigna species of sub-

genus Ceratotropis were obtained from National Botanic

Garden of Belgium (Table 1).

Fig. 1 Schematic representation of a 5S rRNA gene unit, showing the coding region (5S gene), intergenic spacer (IGS) and the binding sites of

two outward primers (VR5SL and VR5SR). The position of single base overlap at the 50 ends of both the primers is also indicated by arrowhead

188 A. Saini, N. Jawali

123

DNA extraction, PCR amplification and Agarose gel

electrophoresis

Genomic DNA was isolated from leaves of 4 to 5-week-old

individual plants by the method of Krishna and Jawali

(1997). The DNA was treated with RNAse, further purified

and quantitated according to Prasad et al. (1999).

The complete 5S gene unit (gene ? IGS) was PCR

amplified using primers VR5SL (50 CCATCAGAACTC

CGCAGTTA 30) and VR5SR (50 GGATCCGGTGCAT

TAGTGCT 30). The primers were designed in the con-

served regions of 5S gene (identified by multiple align-

ments of several 5S sequences of plants available in the

GenBank database). Two primers were designed from the

same position, position 35 of the 5S gene, VR5SR from

the region 35–16 and VR5SL from the region 35–54

(Fig. 1; Supp. Fig. 3).

Proofreading thermostable enzyme, Vent DNA poly-

merase (Exo?, New England BioLabs Inc., USA.) was

used for PCR amplification to reduce the errors in the

sequence. The reaction mixture (25 ll) contained 20 mM

Tris–HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4,

2 mM MgSO4, 0.1% TritonX-100, 0.2 lM primer, 0.2 mM

of each dNTP, 1.0 Unit of Vent DNA polymerase (exo?)

and 25 ng of genomic DNA. The PCR amplification was

carried out in an Eppendorf Mastercycler Gradient using

the following temperature cycles: 5 min at 94�C for initial

denaturation; 35 cycles of 94�C for 40 s, 55�C for 40 s and

72�C for 40 s; followed by 10 min at 72�C. Negative

controls (where genomic DNA was not added in the

Table 1 List of the Vigna species analyzed along with the length, %G ? C content and GenBank accession numbers of the complete 5S unit

Accessions of Vigna species Accession

numberaCountry

of origin

5S rRNA gene unit GenBank

accession No.Length

(bp)

% G ? C

1. Vigna radiata (L.) R. Wilczek NI 1012 India 215 46.5 AY884267

2. Vigna radiata (L.) R. Wilczek var. setulosa NI 1135 India 214 44.8 AY884268

3. Vigna radiata (L.) R. Wilczek var. radiata NI 127 Guyana 215 46.5 AY884269

4. Vigna radiata (L.) R. Wilczek var. sublobata NI 634 India 215 46.0 AY884271

5. Vigna radiata (L.) R. Wilczek var. sublobata NI 1607 Cameroon 215 45.1 AY884270

6. Vigna mungo (L.) Hepper NI 1397 Thailand 331 47.4 AY884262

7. Vigna mungo (L.) Hepper var. silvestris NI 1490 Thailand 330 45.5 AY884263

8. Vigna mungo (L.) Hepper var. mungo NI 515 Australia 331 47.4 AY884265

9. Vigna mungo (L.) Hepper var. silvestris NI 635 India 331 47.7 AY884266

10. Vigna mungo (L.) Hepper var. mungo NI 208 Zaire 331 47.7 AY884264

11. Vigna umbellata (Thunb.) Ohwi & H.Ohashi var. umbellata NI 137 – 329 49.6 AY884275

12. Vigna umbellata (Thunb.) Ohwi & H.Ohashi var. umbellata NI 300 India 329 49.6 AY896865

13. Vigna umbellata (Thunb.) Ohwi & H.Ohashi var. gracilis(Prain) Marechal, Mascherpa & Stainier

NI 571 Lao 328 48.4 AY896866

14. Vigna umbellata (Thunb.) Ohwi & H.Ohashi var. gracilis(Prain) Marechal, Mascherpa & Stainier

NI 1398 Thailand 342 48.9 AY896867

15. Vigna trilobata (L.) Verdc. NI 451 Sri lanka 331 47.4 AY884274

16. Vigna trilobata (L.) Verdc. NI 1439 Indonesia 331 47.4 AY884272

17. Vigna trilobata (L.) Verdc. NI 251 India 331 47.4 AY884273

18. Vigna angularis (Willd.) Ohwi & H.Ohashi var. nipponensis(Ohwi) Ohwi & H.Ohashi;

NI 1634 Japan 339 49.3 AY884257

19. Vigna angularis (Willd.) Ohwi & H.Ohashi var. angularis; NI 307 – 331 51.4 AY884258

20. Vigna cf. minima (Roxb.) NI 1377 Thailand 338 46.4 AY884260

21. Vigna glabrescens Marechal, Mascherpa & Stainier NI 532 Philippines 331 47.7 AY884259

22. Vigna aconitifolia (Jacq.) Marechal NI 51 India 332 45.5 AY884256

23. Vigna nakashimae (Ohwi) Ohwi & H.Ohashi; NI 1703 Japan 329–339 48.4–49.6 AY896868–71

24. Vigna riukiuensis (Ohwi) Ohwi & H.Ohashi; NI 1635 Japan 327–339 47.4–50.1 AY896872–75

25. Vigna cf. minima (Roxb.) NI 970 India 335 47.2 AY884261

26. Vigna radiata (L.) R. Wilczekb – – 215 45.1 M18861

27. Glycine max (L.) Merrb – – 330 45.2 X15199

a Accession number of the National Botanic garden of Belgiumb Published sequences of V. radiata (Hemleben and Werts 1988) and Glycine max (Gottlob-McHugh et al. 1990) used for analysis

Analysis of 5S rDNA in Vigna subgenus Ceratotropis 189

123

PCR mix) were also included for each set of samples

analyzed.

The PCR amplified products were analyzed by electro-

phoresis at 8–10 V/cm in 19 TBE and 2.5% agarose gel

(Sigma-Aldrich Corporation, USA). The gels were stained

by ethidium bromide and viewed under UV. The images

were grabbed by gel documentation system (Syngene

Corporation, UK) using GeneSnap software. The PCR

product size was estimated by GeneTools software.

Cloning and sequencing of 5S gene unit

The major 5S fragment as well as the additional frag-

ment(s), if observed, were selectively amplified by band-

stab PCR method (Bjourson and Cooper 1992) using the

same PCR components and thermal cycling conditions as

mentioned above.

The PCR products were purified by ethanol precipitation

and ligated into the vector plasmid Bluescript (Stratagene)

at the EcoRV site using the Rapid DNA ligation kit (Roche

Molecular Biochemicals, Germany) according to the pro-

tocols provided by the manufacturer. Ligation product was

transformed into E. coli strain DH5a (Sambrook et al.

1989) and the cells were plated on LB plates containing

ampicillin (100 lg/ml), 5-bromo-4-chloro-3-indolyl-b-D-

galactoside (X-Gal) and isopropylthio-b-D-galactoside

(IPTG) and incubated at 37�C overnight. The colonies

carrying recombinant plasmids (white colonies) were

stabbed with a fine sterile needle tip and immersed briefly

into a PCR tube containing 25 ll PCR reaction mixture as

described above. The insert was PCR amplified from 10

randomly chosen clones of each accession, by using plas-

mid primers, P1 (50 CGACGTTGTAAAACGACGGCC

AGT 30) and P2 (50 CACACAGGAAACAGCTATGACC

ATG 30) in an Eppendorf Mastercycler Gradient PCR

machine. The thermal cycling conditions used were, how-

ever, slightly different: 5 min at 94�C for initial denatur-

ation; 35 cycles of 94�C for 40 s, 60�C for 40 s and 72�C

for 1 min; followed by 10 min at 72�C. The PCR products

were purified by ethanol precipitation and subsequently

used for sequencing.

Both strands of the insert were sequenced using primers P1

and P2, respectively. Cycle-sequencing was done using ABI

PRISM Big Dye Terminator Ready Reaction kit (Applied

Biosystems) according to the protocol provided by the

manufacturer. At least three clones belonging to a PCR

product were sequenced, whereas, for some species such as

V. glabrescens, V. nakashimae and V. riukiuensis, ten clones

were sequenced (see ‘‘Results’’ and ‘‘Discussion’’). PCR

products were purified and applied to an ABI 377 automated

DNA sequencer (Applied Biosystems). Sequence informa-

tion was extracted and edited using Sequence Analysis

Software (Applied Biosystems) and used for further analysis.

The sequences were submitted to GenBank database and the

accession numbers are listed in Table 1 (complete 5S units)

and Table 3 (truncated 5S units).

Sequence analysis

Since the primers were designed in the coding region and

start from the same position, the 5S unit sequence obtained

has IGS in the middle, flanked by parts of 5S gene (Fig. 1).

The sequence also contained a duplicated site at both ends

of PCR product. Hence, prior to analysis, the duplicated

site was removed from one end and the sequences were

rearranged to obtain coding region followed by the spacer.

Multiple sequence alignment of the complete 5S unit

sequences obtained in this study along with the sequences of

V. radiata and Glycine max was performed using ClustalX

(Thompson et al. 1997) software using default values for gap

opening and gap extension penalties of 15.0 and 6.66,

respectively. The 5S gene unit sequences of the additional

smaller fragments obtained in some species were aligned

with the complete 5S gene unit sequence from the same

species to detect variation. As reported earlier by others, 5S

from Vigna species was found to be highly conserved, hence

only IGS sequences were used for analyzing the intra- and

inter-species heterogeneity. The multiple sequence alignment

was assessed manually, edited using GeneDoc software

(Nicholas et al. 1997) and analyzed using MEGA version 2.1

(Kumar et al. 2001) and DAMBE software (Xia and Xie

2001). Kimura two-parameter model (Kimura 1980) in

MEGA was used along with pairwise deletion option for

estimating genetic distances.

Phylogenetic analysis

Phylogenetic analysis was done by neighbor-joining (Saitou

and Nei 1987) using Kimura two-parameter model (Kimura

1980) and maximum-parsimony (Fitch 1971) using Close-

Neighbor-Interchange method (CNI, with search level ‘3’;

Nei and Kumar 2000) methods in MEGA software (version

2.1, Kumar et al. 2001). Different measures of homoplasy,

such as consistency index (CI, Kluge and Farris 1969),

retention index (RI) and rescaled CI (Farris 1989a, b) were

also calculated using MEGA to estimate the amount of

phylogenetic information and homoplasy in the dataset.

Glycine max was used as outgroup species for phylogenetic

reconstruction. The truncated 5S gene unit sequences were

not included in phylogenetic analysis. Since V. radiata

sequences showed a long deletion (of [100 bp) in the IGS

region compared to rest of the sequences, phylogenetic

analysis was carried out by both including and excluding

V. radiata. Statistical analysis of both the neighbor-joining

and the maximum-parsimony tree was carried out by boot-

strap method (Felsenstein 1985).


123

Results

PCR amplification: length variation of 5S rRNA

gene unit

The 5S gene unit was PCR-amplified from all the 25 Vigna

accessions (Table 1) using the primers VR5SL and VR5SR

designed in this study, which showed the utility of these

primers in amplifying the 5S gene unit from diverse taxa.

Considerable length variation (*220 to *340 bp) of 5S

gene unit was observed among Vigna accessions (Fig. 2).

No detectable variation in length of 5S gene unit was

observed among taxa belonging to a species, except in case

of cultivated (var. umbellata) and wild (var. gracilis) types

of V. umbellata (Lanes 11–14, Fig. 2). The 5S gene unit

from V. radiata accessions was the smallest (*220 bp,

Lanes 1–5, Fig. 2) and was comparable to the size reported

by Hemleben and Werts (1988). Interestingly, the sizes of

the 5S gene unit from nine other Vigna species (including

V. mungo, a species closely related to V. radiata) in the

subgenus Ceratotropis, were significantly larger (*310–

340 bp).

Apart from a major PCR product (5S gene unit),

substantially smaller, additional product(s) were also

obtained in most of the taxa analyzed (Fig. 2). However,

a slightly longer product in addition to the major

PCR product was detected only in V. nakashimae and

V. riukiuensis (Lanes 23, 24; Fig. 2). The additional

fragments (small and long), could not be eliminated by

optimizing PCR conditions suggesting that they are 5S

length variants and not non-specific bands (Supp. Fig. 1).

The small fragments from V. radiata var. setulosa

(*150 and *90 bp fragments), V. mungo (*150 bp

fragment from NI 515 and NI 208), V. umbellata

(*200 bp fragment from NI 137 and NI 300; *150 bp

fragment from NI 571) and V. glabrescens (*230 and

*200 bp fragments) and the long fragment from both

V. nakashimae and V. riukiuensis were isolated and

characterized as detailed in materials and methods.

Variation in the 5S rRNA gene unit within and between

Vigna species

The complete 5S gene units and the additional fragments

from all the 25 Vigna accessions were sequenced and the

length and sequence characteristics are described in

Tables 1, 2 and 3.

The length of the 5S gene unit (gene ? spacer) among the

Vigna species ranged from 214 to 342 bp (Table 1). The 5S

gene unit from V. radiata var. setulosa (wild type) was the

smallest (214 bp) and the one from V. umbellata var. gracilis

(NI 1398) was the largest (342 bp). Vigna angularis and

V. umbellata showed high intraspecific length variation (8

and 14 bp, respectively) in 5S gene unit, whereas remaining

species either showed low (1–2 bp) or no intraspecific length

variation (Table 1). The 5S rRNA genes are generally

homogenized as a result of concerted evolution, however, in

two related species, V. nakashimae and V. riukiuensis mul-

tiple ‘intragenomic 5S rDNA length variants’ were detected.

Both V. nakashimae and V. riukiuensis harbored four ‘in-

tragenomic 5S types’ each with a length variation of 2–10 bp

2–12 bp, respectively (Table 1; Supp. Table 1A).

The G ? C content of the 5S gene unit showed a wide

variation, ranging from 44.8% (in V. radiata var. setulosa)

to 51.4% (in V. angularis var. angularis) (Table 1).

Presence of ‘5S pseudogene units’ in Vigna species

The small fragments present in addition to the major

fragment (Fig. 2) were characterized from a few Vigna

species and compared with the complete 5S gene unit

of the corresponding species (Fig. 3; Supp. Fig. 2A–D).

These small fragments contained indels and substitutions in

5S gene as well as in the IGS region (30 downstream and

50 upstream regions) (Table 2; Fig. 3) that is essential for

the expression of 5S gene (Scoles et al. 1988). Hence, these

sequences were termed as putative 5S pseudogene units.

The 5S pseudogenes (140 and 93 bp) from V. radiata var.

setulosa showed both substitutions and small deletions in

Fig. 2 Variation in the length of 5S rRNA gene unit among species

belonging to subgenus Ceratotropis analyzed. Numbers on top of the

lanes indicate the Vigna species listed in Table 1 (in the same order).

Lane ‘M’ indicates the marker, 100 bp ladder. Arrows indicate

additional fragments observed, where smaller fragments were seen in

several accessions whereas longer ones were observed only in

V. nakashimae, lane 23 and V. riukiuensis, lane 24


123

the coding region, while large deletions (71 and 94 bp)

were observed in the IGS (Table 2; Fig. 3a). In V. mungo,

the 5S pseudogenes contained only deletions in both cod-

ing region (at 30 end) and IGS (151 bp deletion that

included 30 downstream and mid-spacer region) (Table 2;

Fig. 3b). Similarly, the V. umbellata 5S pseudogenes

contained only deletions in the coding (28 bp at the 30 end)

and spacer region (169 bp deletion in the 30 downstream

and mid-spacer region) (Table 2; Fig. 3c). The 5S pseu-

dogenes from V. glabrescens showed substitutions as well

as deletion. The 230 bp fragment showed deletions in the

coding region (at 50 end), while IGS showed a few inser-

tions (1–2 bp) and long deletion (85 bp in the 50 upstream

spacer region). However, the 200 bp truncated 5S unit

showed a complete 5S gene and only some part of 30

downstream spacer (important for transcription termina-

tion), while a long deletion (134 bp) removed the

remaining part of the spacer region (Table 2; Fig. 3d).

The 5S pseudogene fragments were of low intensity

compared to major 5S fragments (Fig. 2), which indicated

that these might be present in small copy number and might

be present at some minor 5S loci in the genome. It is

possible that some pseudogene sequences might not have

been amplified due to variations at primer binding sites.

The putative 5S pseudogene sequences were not included

for subsequent phylogenetic analysis.

Variation in 5S rRNA gene within and between Vigna

species

Length, G ? C content and sequence variation

Among the Vigna species analyzed, the length of the 5S

rRNA gene was invariant (118 bp), however, the G ? C

content showed a variation of 3.4% (range 52.5–55.9%;

Table 3). The ‘intragenomic 5S types’ from V. riukiuensis

and V. nakashimae 5S gene showed more variation in the

G ? C content than intraspecific variation observed in

certain Vigna species analyzed (Table 3, Supp. Table 1A).

5S gene showed only few (16) polymorphic sites that

included both transitions (10, CT = AG) and transversions

(5, TG [ TA) (Fig. 4 and Supp. Fig. 3). Although the 5S

gene showed low sequence divergence, the order of

nucleotide abundance was not conserved among the Vigna

species (Table 3, Supp. Table 1B).

Variation in IGS region within and between Vigna

species

Length and G ? C content

Unlike the 5S gene, IGS showed extensive length variation,

ranging from 96 bp (V. radiata var. setulosa) to 224 bp

Table 2 Length and sequence variations in the 5S pseudogenes characterized in four Vigna species belonging to the subgenus Ceratotropisalong with their GenBank accession numbers

Species Total Length

(in bp) and

(GenBank

Acc. No.)a

5S coding region Intergenic spacer (IGS) region

Substitutions Insertion/deletionb Substitutions Insertion/deletionc

Length

(in bp)

Position Length

(in bp)

Position

V. radiata var. setulosa (NI 1135) 140 (AY896880) 11: Tn, 6; Tv, 5 01 (D) 13 0 73 (D) 141–214

93 (AY896881) 05: Tn, 3; Tv, 2 01 (I)

05 (D)

07 (D)

14 (D)

66

87–91

98–104

106–119

0 94 (D) 120–214

V. mungo var. mungo (NI 515) 163 (AY896879) 0 15 (D) 103–118 0 151 (D) 119–270

V. mungo var. mungo (NI 208) 163 (AY896878) 0 15 (D) 103–118 0 151 (D) 119–270

V. umbellata var. gracilis (NI 571) 145 (AY896882) 0 28 (D) 90–118 0 169 (D) 119–288

V. glabrescens (NI 532) 230 (AY896876) 03: Tn, 2; Tv, 1 01 (D)

01 (D)

14 (D)

17

19

1–14

13: Tn, 8; Tv, 5 01 (I)

02 (I)

02 (I)

85 (D)

126

144–145

163–164

246–331

200 (AY896877) 01: Tn, 0; Tv, 1 0 – 01: Tn, 0; Tv, 1 134 (D) 133–267

Tn Transitions, Tv Transversionsa Refers to the total length of the 5S rRNA pseudogene unit (including coding and spacer region) along with GenBank Accession Nob Refers to the insertion (I)/deletion (D) in coding region: length and the respective positionc Refers to the indels in IGS region. The position of indels mentioned is according to alignment of pseudogene sequences and the complete 5S

unit of each species done separately


123

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)G

?C

(in

%)

TC

AG

Len

gth

(bp

)G

?C

(in

%)

TC

AG

V.

rad

iata

NI1

01

21

18

55

.12

2.0

26

.32

2.9

28

.89

73

6.0

45

.42

4.7

18

.61

1.3

V.

r.se

tulo

saN

I11

35

11

85

5.1

22

.02

6.3

22

.92

8.8

96

32

.34

6.9

24

.02

0.8

8.3

V.

r.ra

dia

taN

I12

71

18

55

.12

2.0

26

.32

2.9

28

.89

73

6.0

45

.42

4.7

18

.61

1.3

V.

r.su

blo

ba

taN

I63

41

18

55

.12

2.0

26

.32

2.9

28

.89

73

5.0

47

.42

3.7

17

.51

1.3

V.

r.su

blo

ba

taN

I16

07

11

85

5.1

22

.02

6.3

22

.92

8.8

97

33

.04

7.4

23

.71

9.6

9.3

V.

mu

ng

oN

I13

97

11

85

5.1

22

.02

6.3

22

.92

8.8

21

34

2.8

34

.32

8.2

23

.01

4.6

V.

m.

silv

estr

isN

I14

90

11

85

5.1

22

.02

6.3

22

.92

8.8

21

24

0.1

36

.32

5.9

23

.61

4.2

V.

m.

mu

ng

oN

I51

51

18

55

.12

2.0

26

.32

2.9

28

.82

13

43

.63

3.8

30

.02

3.0

13

.1

V.

m.

silv

estr

isN

I63

51

18

55

.12

2.0

26

.32

2.9

28

.82

13

43

.63

3.8

30

.52

2.5

13

.1

V.

m.

mu

ng

oN

I20

81

18

55

.12

2.0

26

.32

2.9

28

.82

13

43

.63

3.8

30

.52

2.5

13

.1

V.

u.

um

bel

lata

NI1

37

11

85

5.1

22

.02

6.3

22

.92

8.8

21

14

6.5

29

.92

9.9

23

.71

6.6

V.

u.

um

bel

lata

NI3

00

11

85

5.1

22

.02

6.3

22

.92

8.8

21

14

6.0

30

.32

9.4

23

.71

6.6

V.

u.

gra

cili

sN

I57

11

18

54

.32

2.0

26

.32

3.7

28

.02

10

45

.33

3.8

28

.62

1.0

16

.7

V.

u.

gra

cili

sN

I13

98

11

85

5.1

22

.02

6.3

22

.92

8.8

22

44

5.5

33

.52

9.0

21

.01

6.5

V.

tril

ob

ata

NI4

51

11

85

5.1

22

.02

6.3

22

.92

8.8

21

34

3.2

32

.92

9.6

23

.91

3.6

V.

tril

ob

ata

NI1

43

91

18

55

.12

2.0

26

.32

2.9

28

.82

13

43

.23

2.4

31

.02

4.4

12

.2

V.

tril

ob

ata

NI2

51

11

85

5.1

22

.02

6.3

22

.92

8.8

21

34

3.2

32

.43

1.0

24

.41

2.2

V.

a.

nip

po

nen

sis

NI1

63

41

18

55

.12

2.0

26

.32

2.9

28

.82

21

46

.23

3.5

30

.82

0.4

15

.4

V.

a.

an

gu

lari

sN

I30

71

18

55

.92

1.2

27

.12

2.9

28

.82

13

48

.43

1.0

31

.02

0.7

17

.4

V.

min

ima

NI1

37

71

18

55

.12

2.0

26

.32

2.9

28

.82

20

41

.83

5.5

25

.92

2.7

15

.9

V.

min

ima

NI9

70

11

85

4.2

22

.92

5.4

22

.92

8.8

21

74

3.4

34

.62

6.3

22

.11

7.1

V.

gla

bre

scen

sN

I53

21

18

54

.22

2.9

25

.42

2.9

28

.82

13

44

.23

3.8

29

.62

2.1

14

.6

V.

aco

nit

ifo

lia

NI5

11

18

53

.42

3.5

26

.12

2.7

27

.72

14

41

.13

3.6

30

.42

5.2

10

.7

V.

na

kash

ima

eaN

I17

03

11

85

5.1

–5

3.4

22

.0–

23

.72

5.4

–2

6.3

22

.9–

23

.72

7.1

–2

8.8

21

1–

22

14

4.6

–4

7.5

32

.4–

35

.12

8.0

–2

9.2

20

.1–

21

.31

6.5

–1

8.3

V.

riu

kiu

ensi

saN

I16

35

11

85

2.5

–5

4.2

22

.0–

22

.92

5.4

–2

6.3

22

.9–

25

.42

7.1

–2

8.8

20

9–

22

14

3.5

–4

7.5

31

.2–

34

.42

8.7

–3

0.3

20

.5–

22

.01

4.8

–1

7.8

IGS

reg

ion

of

V.

rad

iata

acce

ssio

ns

was

con

sid

erab

lysm

all

inle

ng

than

dm

ore

‘Tri

ch’

com

par

edto

rest

of

the

Vig

na

spec

ies

aS

pec

ies

that

sho

wed

mu

ltip

le‘i

ntr

agen

om

ic5

Su

nit

s’


123

(V. umbellata var. gracilis, NI 1398) (Table 3). Substantial

length variation was observed within certain species (V.

angularis, V. umbellata) and among the ‘intra-genomic 5S

types’ from V. nakashimae and V. riukiuensis (Supp.

Table 1A).

Wide variation (16.1%) in the G ? C content was also

observed in the IGS region, among the species analyzed

(Table 3). Variation observed in the G ? C content

among ‘intra-genomic 5S types’ from V. nakashimae and

V. riukiuensis was more than the intraspecies variation

observed in certain species such as V. umbellata and

V. mungo (Table 3; Supp. Table 1A).

Variation at specific indel regions in the IGS

A total of nine indels localized in specific regions were

identified in the 5S IGS that contributed to the length

variation within and among the Vigna species. Of the nine

indels, five (#1–#5) were more than 2 bp in length (Fig 4;

Supp. Fig 3).

Indel region #5 (125 bp) was completely absent among

wild (var. setulosa and var. sublobata) and cultivated (var.

radiata) types of V. radiata and this deletion was respon-

sible for the small size (214–215 bp) of the 5S gene unit in

V. radiata as compared to the rest of the Vigna species

(Fig. 4). The indel region #5 was not completely deleted in

remaining species but showed four small indels regions

(#5a to #5d, Fig 4; Supp. Fig 3), which showed variability

(presence/absence) among different Vigna species includ-

ing the 5S types from V. nakashimae (‘A’ and ‘B’) and

V. riukiuensis (type ‘A’). Regions #5c and #5d showed

insertion in a few IGS sequences as result of duplication of

the adjacent sequence motif’s ‘TTACC’ (#5c) and ‘CC’

(#5d) (Fig. 4; Supp. Fig 3). Variations were also observed

at indel regions #1, #2, #3 and #4 due to deletions (1–2 bp,

1–7 bp, 2–4 bp and 1–2 bp, respectively) among different

Vigna 5S IGS sequences, however, duplication of a 3 bp

sequence motif ‘TTC’ was observed at region #3 in

V. umbellata var. gracilis (NI 1398) (Fig. 4; Supp. Fig 3).

Sequence variation

In addition to the indels, the 5S IGS also showed high

sequence variation (Fig. 4; Supp. Fig 3). Of the total 234

sites in the IGS, 50.8% (119) were polymorphic and 69.7%

of the variable sites were parsimony informative. Both

transversions (50) and transitions (39) were observed and 30

sites showed more than one type of substitution (Table 4).

Fig. 3 Schematic

representation of deletions

observed in the putative 5S

pseudogene units isolated from

some Vigna species and their

comparison with the

corresponding full length 5S

gene units from: a V. radiatavar. setulosa (NI 1135),

b V. mungo var. mungo NI 515

and NI208), c V. umbellata var.

gracilis (NI571) and

d V. glabrescens (NI 532)


123

In spite of high sequence variation, the order of nucleotide

abundance (T [ C [ A [ G) was conserved among all the

Vigna species analyzed (Table 3, Supp. Table 1B). IGS

region of V. radiata was found to be ‘T-rich’ compared to

rest of the Vigna species indicating that the deletions might

be localized to ‘G-rich’ regions (Table 3).

Fig. 4 Schematic representation of the variations in the 5S gene unit

(5S gene ? IGS) sequences from Vigna accessions including the

‘intragenomic 5S Types’ of V. nakashimae and V. riukiuensis. Coding

and intergenic spacer regions are indicated. Vertical arrows on the top

of the figure indicate the polymorphic sites, while dashed regions

indicate deletions. The numbers #1 to #5 are the major indel regions

(longer than 2 bp) whereas #5a–#5d are small indels within the indel

region #5

Table 4 Intra-species variation in the 5S intergenic spacer (IGS) region of the Vigna species analyzed in this study

Species No. of

AccessionsaVariation in the Intergenic Spacer (IGS) region

Transitionsb Transversionsb Length variation

(in bp)AG CT AC AT GC GT

V. radiatac 6 6 (0) 3(0) 0 (0) 0 (0) 0 (0) 2 (0) 0–1

V. mungod 5 1 (1) 6 (6) 3 (3) 1 (1) 5 (5) 0 (0) 0–1

V. umbellata 4 5 (2) 10 (5) 4 (4) 3 (1) 5 (4) 4 (1) 0–14

V. trilobata 3 1 (1) 6 (4) 2 (2) 4 (2) 5 (4) 3 (2) 0

V. angularis 2 1 (1) 4 (1) 0 (0) 0 (0) 0 (0) 2 (1) 8

V. minima 2 2 (0) 2 (1) 1 (1) 1 (0) 3 (2) 1 (0) 3

V. nakashimae 1 2 (2) 3 (2) 0 (0) 1 (0) 6 (3) 4 (0) 1–10

V. riukiuensis 1 4 (2) 4 (3) 2 (2) 4 (0) 4 (3) 1 (0) 0–10

All speciese 26 30 (14) 41 (25) 27 (18) 26 (09) 30 (21) 30 (13) 0–128

a Total number of accessions of a species used for analysisb Numbers outside the parenthesis indicate number of substitutions in the total IGS region, whereas the numbers inside the parenthesis indicates

the substitutions present only in the indel region ‘#5’c V. radiata sequences also include the one reported by Hemleben and Werts (1988)d In V. mungo all the variable sites were located only in the in the mid spacer region of IGS. Intra-genomic variants were observed only in

V. nakashimae and V. riukiuensis (data shown in bold)e Total number of substitutions (transitions and transversions) observed among all the accessions belonging to 10 different Vigna species


123

Substantial, intra- and inter-species sequence divergence

was observed in IGS from Vigna species (Supp. Table 2).

Maximum intra-species sequence divergence was observed

among V. umbellata accessions (0.5–11.5%). The cultivated

types (var. umbellata) showed low sequence divergence

(0.5%), the wild (var. gracilis) types showed relatively a

high sequence divergence (5.9%), whereas, the divergence

between the wild and cultivated types was 11.5%. Among

V. radiata accessions, the cultivated and wild types exhib-

ited different divergence and among them var. radiata was

closer to var. sublobata (5.2–6.3%) than to var. setulosa

(8.3–9.4%). Intra-species sequence divergence among the

V. trilobata accessions varied from 0–9.9%, with two

accessions from India and Indonesia showing high simi-

larity. The cultivated types (var. mungo) of V. mungo

showed lesser divergence (0–0.5%) than the wild types (var.

silvestris, 6.1%) and between the wild and cultivated types

of V. mungo a sequence divergence of 6.6% was noted.

V. minima and V. angularis showed a IGS sequence diver-

gence of 4.6 and 3.3%, respectively. The sequence divergence

among intra-genomic 5S types from V. nakashimae (6.8%)

and V. riukiuensis (7.4%) was higher than the intra-species

divergence detected among some species

Phylogenetic analysis

5S IGS sequences were used for inferring phylogenetic

relationships among the ten Vigna species by neighbor-

joining (Fig. 5a) and maximum-parsimony methods

(Fig. 5b) as detailed in materials and methods. Since the

‘complete deletion’ option (which exclude indels) in

MEGA was used for inferring NJ tree, region #5 was also

excluded in which case *50% of variable and parsimony

informative sites, among species other than V. radiata

(Table 4), would not be available for calculations. Hence,

the relationship among the remaining 20 accessions

(excluding the V. radiata accessions) belonging to nine

other Vigna species was also analyzed to infer species

relationships (Fig. 6). The values of consistency index (CI:

0.806 and 0.831), retention index (RI: 0.894 and 0.860) and

rescaled consistency index (RCI: 0.721 and 0.715) for

maximum-parsimony analysis of both the data sets

(including and excluding V. radiata) indicated low homo-

plasy and hence high phylogenetic utility of the 5S IGS.

The species based on both the type of analysis, were

divided into two major clusters: I and II (Figs. 5, 6). Cluster

I included most of the species belonging to sections

Ceratotropis and Aconitifoliae, viz. V. radiata, V. mungo,

V. aconitifolia, V. trilobata and surprisingly V. glabrescens.

Cluster I was further divided into three distinct sub-clusters.

The V. radiata accessions, including the wild and cultivated

types grouped together in one sub-cluster, however, they

(var. radiata, var. sublobata and var. setulosa) showed

distinct lines of divergence from the common progenitor.

The second sub-cluster included the wild (var. silvestris)

and cultivated (var. mungo) types of V. mungo. An inter-

esting observation was the presence of V. glabrescens, the

tetraploid species, in this sub-cluster since earlier studies

had placed it in section Angulares (see ‘‘Discussion’’).

Although V. glabrescens was closer to V. mungo, it showed

a different path of divergence. The third sub-cluster repre-

sented V. aconitifolia and three accessions of V. triolobata

(Figs. 5b, 6). V. aconitifolia showed a different path of

divergence whereas among V. trilobata, accessions from

Indonesia (NI 1439) and India (NI251) were closer to each

other than with the accession from Sri Lanka.

Relationships among the Vigna species in cluster I did

not change substantially, after V. radiata accessions were

excluded, except in case of V. mungo (compare Figs. 5b,

6). This could be attributed to the presence of variable sites

only in the mid-spacer region of IGS (that included region

#5) among V. mungo, whereas in the remaining species

variations were distributed throughout the IGS (Table 4).

The indels along with indel region #5 were automatically

excluded (as complete deletion option was used) when

V. radiata accessions were present, leading to the differences

observed in V. mungo.

Cluster II included V. minima, V. umbellata, V. angu-

laris, V. nakashimae and V. riukiuensis, the species

belonging to section Angulares except V. glabrescens. The

accessions belonging to this group were divided into five

small sub-clusters (Fig. 6). The first sub-cluster was spe-

cific to V. minima. The cultivated types (var. umbellata) of

V. umbellata were present in a second sub-cluster. The wild

types (var. gracilis), however, were not present in this

cluster. The third sub-cluster included the wild (var. nip-

ponensis) and cultivated (var. angularis) type of V. angu-

laris. The remaining two sub-clusters included more than

one species. The fourth sub-cluster included the wild type

of V. umbellata (var. gracilis, NI 1398), intra-genomic 5S

types ‘C’ and ‘D’ of V. riukiuensis and type ‘C’ from V.

nakashimae. The last sub-cluster included the second wild

type of V. umbellata (var. gracilis, NI 571) along with

most of the intra-genomic 5S types (‘A’, ‘B’ and ‘D’) of

V. nakashimae and type ‘A’ from V. riukiuensis. Intra-

genomic 5S type ‘B’ from V. riukiuensis was present

between these two sub-clusters. The wild and cultivated

types belonging to most of the Vigna species were clustered

closely except in case of V. umbellata.

Discussion

The organization of 5S rDNA unit has not been character-

ized among Vigna species (subgenus Ceratotropis), except

for V. radiata (Hemleben and Werts, 1988). Hence, the


123

Fig. 5 a Neighbor-joining

dendrogram generated using 5S

IGS sequences of species

belonging to subgenus

Ceratotropis analyzed in this

study with Glycine max as an

outgroup. The species are

divided into two major clusters

(I and II). The numbers at the

nodes represents bootstrap

values (in %) for a 1000

replicate analysis. b Consensus

parsimony tree (CI: 0.806; RI:

0.894; RCI: 0.721) of 552 most

parsimonious trees generated

using IGS sequences of species

belonging to subgenus

Ceratotropis analyzed in this

study with Glycine max as an

outgroup. The numbers at the

nodes represents bootstrap

values (in %) for a 500 replicate

analysis


123

phylogenetic potential of this region has remained unuti-

lized. The 5S gene unit was found to be highly variable in

both length and sequence among the Vigna species. The

coding region was well conserved, whereas IGS was highly

variable and was sufficient for inferring phylogenetic rela-

tionships among the closely related Ceratotropis species.

5S rDNA is a useful tool for understanding species

relationships. The coding region is conserved across

diverse taxa and is useful in studying higher order rela-

tionships (Hori and Osawa 1988). The IGS, however has

been useful for inferring relationships among closely

related species (Appels et al. 1989, 1992; Reddy and

Appels 1989; Baum and Appels 1992; Moran et al. 1992;

Playford et al. 1992; McIntyre et al. 1992; Capesius 1993;

Udovicic et al. 1995; Yang et al. 1998; Roser et al. 2001),

for discrimination of breeding lines (Volkov et al. 2001)

and to confirm the presence of parental genome in the

somatic hybrids (Zanke et al. (1995).

5S gene

5S gene has been characterized and analyzed at both struc-

tural and functional levels in several organisms including

plants (Barciszewska et al. 1994a, b; Barciszewska et al.

2001). Due to structural and functional constraints, the

coding region is generally highly conserved across diverse

taxa. Though the 5S gene showed high sequence similarity

among the ten Vigna species, a few differences were

observed (Fig. 4, Supp. Fig. 3). The beginning of the coding

region generally shows some family specificity (Volkov

et al. 2001) and the analysis shows that 5S gene from

Ceratotropis species started with ‘AGG’ and this matched

with that reported for species belonging to Fabaceae

(Hemleben and Werts 1988; Barciszewska et al. 1994b). The

only exception was V. aconitifolia where the gene started

with ‘GGG’ and the reason for this is not known. In all the

species, the 5S gene ended with ‘CCT’ without any excep-

tion. The recognition site for RNA polymerase III (internal

control region, ICR) is also present within the 5S gene and

has three regions that are essential for formation of the ini-

tiation complex required for transcription (Pieler et al. 1987;

Wolffe 1994; Nolte et al. 1998). Some substitutions were

also localized in the ICR region (Supp. Fig. 3). However,

none of the sequences, which showed variations at ICR,

harbored more than one substitution.

Intergenic spacer (IGS)

The highly variable IGS is broadly divided into three

regions, 30 downstream, 50 upstream and mid-spacer region

(Sastri et al. 1992). The 30 ‘T-rich’ region immediately

downstream to the 5S gene is known to be essential for

transcription termination (Roser et al. 2001; Korn 1982;

Hemleben and Werts 1988). Among the Vigna species, this

region started with CTTTTT, except in V. trilobata (NI

451) it was TTTTTT. In addition to the substitutions, this

region also exhibited length variation (35–44 bp) due to the

presence of three indels (#1, #2, #3). The region actually

comprised two direct repeats arranged in head-to-tail

fashion (DR I and DR II) (Supp. Fig. 3). The similarity

between the two direct repeats within different accessions

ranged from 66.6% (in V. trilobata) to as high as 88.8% (in

V. angularis var. angularis).

The 50 region upstream to the 5S gene was 49 bp in

length and showed absolutely no length variation. However,

*50% of the sites were found to be polymorphic (Supp.

Fig. 3). The region started with ‘TTAT/TTGT’ in most of

the species except V. umbellata var. umbellata (CTAT) and

ended with GAC in most of the species, CAC in V. radiata

(except NI 634, CGC) and GGC in V. minima, V. umbellata

var. umbellata. This region in intra-genomic 5S types (A, B

and D) of V. nakashimae ended with ‘GAC’ (and GGC in

type ‘C’), whereas, in V. riukiuensis, it ended with ‘GGC’

(except GAC in type ‘A’). A highly conserved ‘AT-rich’

motif (consensus: ATATAT) was present in the second half

of the 50 upstream region (position 324–329, Fig. 4). This

Fig. 6 Consensus parsimony tree (CI: 0.831; RI: 0.860; RCI: 0.715)

of 30 most parsimonious trees generated using IGS sequences of

Vigna species analyzed in this study, except V. radiata accessions

with Glycine max as an outgroup. The numbers at the nodes

represents bootstrap values (in %) for a 500 replicate analysis


123

motif is also observed in the 5S IGS of several plants and is

known as the ‘TATA box’ (Tyler 1986; Hemleben and

Werts 1988; Venkateswarlu et al. 1991; Roser et al. 2001).

The mid-spacer region among the Vigna species was

found to be highly variable both in length and sequence

(Fig. 4, Supp. Fig. 3). It was smallest (11 bp) among the

V. radiata accessions due to deletion of region #5. Besides,

mid-spacer region from rest of the Vigna species carried

small indels (#5a to #5d) that resulted in the length varia-

tion among them. In addition, indel #5 of the mid-spacer

region from the Vigna species contained *50% of the total

polymorphic sites of IGS making it the most variable

spacer region. Mid-spacer region from several plant species

have been reported to be highly variable and contains many

indels (Venkateswarlu et al. 1991; Scoles et al. 1988;

Moran et al. 1992; Volkov et al. 2001; Roser et al. 2001).

Complete deletion of region #5 in V. radiata and presence

of smaller indels in some other Vigna species suggest that

the mid-spacer might not be important for the expression of

the 5S genes in plants, unlike 30 downstream and 50

upstream regions (Scoles et al. 1988). Presence of promoter

elements (ICR) within the 5S gene (Pieler et al. 1987) and

extremely small spacers observed in some other species

(Cox et al. 1992) also suggest that most of the IGS region

may be non-functional (Allaby and Brown 2001).

Intra-genomic, divergent 5S rDNA units

in V. nakashimae and V. riukiuensis

The 5S rDNA is generally homogenized like other multi-

gene families as a result of molecular mechanisms like

unequal crossing over or gene conversion (Smith 1976;

Birky and Skarvil 1976; Ohta 1984) that are collectively

referred to as ‘molecular drive’ (Dover 1986). However,

variations in the 5S rDNA, as a result of incomplete

homogenization, have been reported in several plant spe-

cies (Appels and Clarke 1992; McIntyre et al. 1992; Roser

et al. 2001; Scoles et al. 1988; Kellogg and Appels 1995;

Cronn et al. 1996 and present study). Present study

detected divergent, intra-genomic 5S rDNA units only in

V. nakashimae and V. riukiuensis and this suggested that

the region is being homogenized at different rates among

the ten Vigna species. Since these intragenomic 5S types

did not contain deletion in coding and essential spacer

regions, they were not considered as ‘pseudogenes’.

Analysis of pattern of sequence variations (substitution

and indels) among the intragenomic 5S units revealed

several interesting findings: (1) Among the four intrage-

nomic 5S types from V. riukiuensis, type ‘A’ shared

high similarity with two 5S types (‘A’ and ‘B’) of

V. nakashimae and one of the wild type V. umbellata (var.

gracilis, NI 571) (Table 5). (2) Among the 5S types from

V. nakashimae, type ‘C’ shared high similarity with two

5S types (‘C’ and ‘D’) from V. riukiuensis and the other

wild type V. umbellata (var. gracilis, NI 1398) (Table 5).

These results show that interspecific hybridization is

responsible for the observed intragenomic heterogeneity in

the 5S rDNA of V. nakashimae and V. riukiuensis. Inter-

specific hybridization is reported from section Angulares

(subgenus Ceratotropis) (Sawa 1983; Siriwardhane et al.

1991), and many of them also grow sympatrically (Vaughan

et al. 2000).

Table 5 Comparison of specific variations (substitution and indels) in the 5S IGS region among the ‘intragenomic 5S Types’ from

V. nakashimae, V. riukiuensis and IGS sequences of wild types (var. gracilis) of V. umbellata

Species Polymorphic sites

128 140 148 162 181 #5a

(181–186)

#5b

(189–195)

216 225 #5c

(226–230)

235 240 #5d

(257–259)

260 289 348 351

V. u. gracilis (NI 571) C G T T – – – C C ? C G – A T A A

V. nakashimae (A) T G T T – – – C C ? C G – G T A A

V. nakashimae (B) C T T T – – – C C ? C G – G T A A

V. riukiuensis (A) C – T T – – – C C ? C G – A T A A

V. nakashimae (C) G T G C G ? ? G T – G C ? A C T G

V. riukiuensis (C) G T G C A ? ? G T – G C ? A C T G

V. riukiuensis (D) G T G C G ? ? G T – G C ? A C T G

V. u. gracilis (NI 1398) C T T C G ? ? G T – G C ? A C T A

V. nakashimae (D) G – G C G ? ? G C ? C G – G T A A

V. riukiuensis (B) G T G C G ? ? C C ? C G – A C T G

Similar sequences are placed next to each other for comparison

‘–’ or ‘?’ indicates deletion or insertion at the indel regions


123

The sequence analysis showed that among the intra-

genomic variants of 5S rDNA, type ‘D’ from V. nakashi-

mae and type ‘B’ from V. riukiuensis are recombinant

sequences. The recombinants harbored variations (indels

and substitutions) specific to both the ‘parental 5S types’

likely to be involved in the crossover event (Table 5;

Fig. 7). Recombination in nuclear rDNA is known to

generate chimeric molecules with a sequence intermediate

between two ancestral sequences (Muir et al. 2001). The

type ‘D’ from V. nakashimae is likely to be a product of a

single cross over event between 5S type ‘A’/‘B’ and type

‘C’ (which is closer to 5S types from V. riukiuensis) and

the probable region of the crossover lies between position

216 and 225 (Table 5; Fig. 7), whereas, 5S type ‘B’ from

V. riukiuensis is likely to be the product of a double

recombination event between type ‘D’ and type ‘A’ (which

is closer to 5S types from V. nakashimae). Analysis indi-

cates that the position of first crossover lies between indel

region #5b and 216 whereas the position of second one

lies between indel region #5d and 260 (Table 5; Fig. 7).

The recombinant sequences may arise as a result of intro-

gression or migration, which introduces new variants faster

than they can be homogenized within the genome

(Sanderson and Doyle 1992). Recombinant sequences in

nuclear ribosomal DNA regions are also reported in fungi

and several angiosperm groups (Hughes and Paterson

2001; Buckler et al. 1997; Muir et al. 2001).

The incomplete homogenization leading to the presence

of recombinant 5S sequences could be result of, (a) rate of

introgression of variants is faster than the rate of homog-

enization, (b) introgression or inter-specific hybridization

has occurred very recently, (c) the absence of genetic

exchanges between the chromosome specific arrays as

noticed in Triticeae (Scoles et al. 1988; Kellogg and Ap-

pels 1995) and Gossypium (Cronn et al. 1996), (d) presence

of large number of loci present in the genome and (e) the

process of homogenization is lower due to any of the above

reasons. It is likely that the presence of intra-genomic 5S

variants in V. nakashimae, V. riukiuensis could be due

to either (a) or (b) as these phenomena have been shown

to be responsible to intra-genomic rDNA heterogeneity

(Sanderson and Doyle 1992; Jobst et al. 1998).

Comparison of phylogenetic utility of 5S IGS

with atpB-rbcL of cpDNA and ITS

The 5S IGS was evaluated for the phylogenetic information

and compared to ITS and atpB-rbcL spacer of cpDNA.

Both these regions were used for understanding species’

relationships among Vigna species (Doi et al. 2002; Goel

et al. 2002). The data from previous studies on Vigna

species show that the atpB-rbcL spacer, though compara-

ble to ITS in size exhibits lesser polymorphism (Doi et al.

2002). The present study demonstrates that 5S IGS is

substantially more informative than atpB-rbcL in number

of variable and parsimony informative sites. The IGS is

comparable to ITS1 and ITS2 in size and number of vari-

able sites though it had substantially more parsimony

informative sites (Table 6). As it is known that different

sequences have different rates of evolution and that 5S IGS

is highly informative, the analysis could give additional

insights into evolution of a group of species as evident

from the insights obtained on the evolution of the tetraploid

species, V. glabrescens.

Phylogenetic analysis and implications

Phylogenetic analysis based on 5S IGS divided the Vigna

species into two major clusters. Previous studies such as

analysis of morphological characters (Maekawa 1955;

Dana 1980) and cytogenetical analysis (Egawa et al. 1988)

had suggested that the Vigna species belonging to subgenus

Ceratotropis could be divided into two major groups that

are known to be isolated by reproductive barriers (Chen

Fig. 7 Schematic

representation of generation of

‘recombinant 5S units’

(indicated as Rec.) in the two

Vigna species, which also

harbored multiple 5S types: (a)

in V. nakashimae and (b) in

V. riukiuensis. Numbers on topindicates the position of the

variable sites and - or ?

indicates absence or presence of

indels. The likely parental type

5S units involved and the sites

of crossover are also indicated


123

et al. 1983). Later analysis by RFLP (Fatokun et al. 1993)

and RAPD (Kaga et al. 1996) confirmed the earlier clas-

sification of subgenus Ceratotropis. However, in these

earlier molecular studies, several species such as V. trilo-

bata, V. minima and the tetraploid species V. glabrescens

were not included and hence their relative taxonomic

positions were not confirmed. A recent analysis of the

Phaseolus–Vigna complex using ITS that included these

species also suggested two major groups in the subgenus,

where V. aconitifolia and V. trilobata were close to

V. radiata and V. mungo, however, V. glabrescens and

V. minima were included in the section Angulares (Goel et al.

2002). Recently, a third group (section Aconitifoliae) has

been proposed in the subgenus Ceratotropis that included

species such as V. aconitifolia, V. trilobata, V. aridicola and

V. stipulacea (Tomooka et al. 2002; Doi et al. 2002).

Though the phylogenetic analysis using 5S IGS divided

the Vigna species into two major clusters (known to

be reproductively isolated), the three different sections,

Ceratotropis, Aconitifoliae and Angulares were clearly dif-

ferentiated (Figs. 5b, 6). However, some important excep-

tions are observed: (1) V. glabrescens, a tetraploid, known to

belonging to section Angulares (Egawa et al. 1988; Doi et al.

2002; Goel et al. 2002) was placed close to V. mungo in

section Ceratotropis, (2) wild and cultivated types of

V. umbellata showed a high divergence and they clustered

with different species and (3) intra-genomic heterogeneity

in the 5S rDNA was identified in V. nakashimae and

V. riukiuensis.

Section Ceratotropis

Vigna mungo and V. radiata are closely related species as

shown by analysis of F1 hybrids (Egawa et al. 1988), RFLP

(Fatokun et al. 1993), RAPD (Kaga et al. 1996) and ITS

analysis (Doi et al. 2002; Goel et al. 2002; Saini et al.

2008). Both the species were placed into two distinct

sub-clusters (Fig. 5a, b) and this was attributed to the

considerable divergence in the IGS (both length and

sequence) (Fig. 4, Supp. Fig. 3). The wild and cultivated

types of V. radiata showed substantial divergence. Vigna

radiata var. sublobata and var. setulosa are two distinct

wild types of V. radiata (mungbean) existing in India

(Arora 1985). The relationship of var. setulosa with the two

types (var. radiata and var. sublobata) was based on the

earlier morphological studies (Hara 1955; Ohwi and

Ohashi 1969). This is the first report on the studies on var.

setulosa by modern molecular techniques. The results

suggested that var. setulosa, var. sublobata and var. radiata

have evolved from a common ancestor and among the wild

types var. setulosa seems to have diverged from the com-

mon ancestor earlier than var. sublobata and var. radiata is

recently evolved. In a few recent studies, var. setulosa has

been designated as a different species, V. subramaniana

(Doi et al. 2002; Tomooka et al. 2003). Present analysis,

however, contradicts the placement of V. radiata var.

setulosa as a separate species. The results obtained raise

doubts about the new taxonomic designation of var.

setulosa used in some recent studies (Doi et al. 2002;

Tomooka et al. 2003) and suggest a more detailed analysis

to assess the proper taxonomic status of this wild type of

V. radiata. Analysis of these Vigna species by 18S-5.8S-26S

rDNA ITS (Saini et al. 2008) and AP-PCR (unpublished data)

have also shown similar observations as obtained in 5S IGS

analysis.

Unlike V. radiata, there was less divergence between

cultivated and wild type of V. mungo though the accessions

originated in diverse geographical locations. There was no

clear-cut separation between wild (var. silvestris) and

cultivated (var. mungo) types (Fig. 6). The most striking

observation in V. mungo subcluster in section Ceratotropis

was the presence of V. glabrescens (Fig. 6). Initially,

V. glabrescens was reported as an allotetraploid species of

uncertain origin (Marechal et al. 1978). The analysis of the

Table 6 Comparison of phylogenetic informativeness of ITS1, ITS2, atpB-rbcL spacer (of cpDNA) and intergenic spacer of 5S rRNA gene unit

Genomic region

analyzed

Length variation

(in bp)

No. of

polymorphic sites

Average genetic

distance valueaNo. of parsimony

informative sites

rDNA ITSb 611–652 280 0.076 111

ITS-1b 185–211 126 0.119 47

ITS-2b 206–211 124 0.112 59

atpB-rbcL spacera 687–700 104 0.019 22

5S Intergenic spacerc 96–224 119 0.198d 83d

a Genetic distances were estimated using Kimura two-parameter model (Kimura 1980)b Analysis of ITS, ITS1, ITS2 and atpB-rbcL spacer in subgenus Ceratotropis by Doi et al. (2002)c Analysis of the 5S IGS region in subgenus Ceratotropis (present study)d Average genetic distance and number of parsimony informative sites were highest in 5S IGS region compared to the ITS1, ITS2 (which are of

comparable size) and atpB-rbcL spacer (which is more than twice in length)


123

F1 hybrid suggested V. angularis and V. umbellata as likely

diploid genome donors (Egawa et al. 1988; Chen et al.

1983). However, Tateishi (1985) and Egawa et al. (1996)

suggested V. hirtella and V. trinervia as the ancestral dip-

loid progenitors of V. reflexo-pilosa, the tetraploid wild

progenitor of V. glabrescens. Closeness between V. reflexo-

pilosa and V. trinervia was also observed in proteinase

inhibitor studies (Konarev et al. 2002). In the present

analysis, 5S IGS sequence of V. glabrescens was found to

be divergent from the species belonging to section Angu-

lares (Supp. Table 2). Since Angulares is known to be the

most recently evolved section in the subgenus (Doi et al.

2002), it is highly unlikely that the IGS of V. glabrescens

has accumulated substantial variations and has significantly

diverged from the suggested progenitors (based on previ-

ous studies), most of which belong to section Angulares.

However, it is possible that one of the diploid progeni-

tors of the tetraploid may belong to section Ceratotropis.

This progenitor may not be a recently evolved species such

as V. radiata as suggested by low frequency of bivalents

(Egawa et al. 1988), but some extinct species or still an

unknown species. Our efforts to detect the second 5S gene

sequence (from the second diploid progenitor) or recom-

binants from V. glabrescens by sequencing a larger number

of clones, failed. However, several truncated 5S fragments

(pseudogenes) were identified. The 5S rDNA from the

second progenitor of V. glabrescens might have been

homogenized or lost, as both these phenomena are well

known in many other polyploid plants (Wendel 2000).

Complete loss of 18S-5.8S-26S and/or 5S rDNA arrays

from a progenitor upon polyploidization have been repor-

ted in Triticeae (Dubcovsky and Dvorak 1995), Nicotiana

(Volkov et al. 1999), Festuca (Thomas et al. 1997),

Brassica (Snowdown et al. 1997), Glycine (Danna et al.

1996; Shi et al. 1996) and Scilla (Vaughan et al. 1993). The

loss of rDNA array occurs either as a result of a single

deletion event or a slow decay process leading to pseudo-

gene formation (Wendel 2000). Detection of several

truncated 5S fragments (pseudogenes, Figs. 2, 3) in

V. glabrescens suggested the likelihood of a slow decay

process of 5S array from one of the progenitors subsequent

to polyploidization event during the course of evolution.

The other possibility is inter-locus homogenization of

the rDNA arrays as a result of ‘molecular drive’ (Dover

1986). The homogenization of rDNA repeats subsequent to

polyploidization has been shown among allopolyploids in

Gossypium (Hanson et al. 1996; Wendel et al. 1995) and

among polyploid plants such as Microseris (Roelofs et al.

1997; Van Houten et al. 1993), Paeonia (Sang et al. 1995;

Zhang and Sang 1998), and Saxifraga (Brockmann et al.

1996). The similarity in ITS of V. glabrescens to species

belonging to section Angulares (Goel et al. 2002; Saini

et al. 2008) and that of 5S IGS to species in section

Ceratotropis (present study) suggested that V. glabrescens,

subsequent to the polyploidization, has retained 18S-5.8S-

26S rRNA rDNA region specific to one of the diploid

parents and the 5S rDNA specific to the other. This may be

possible since both these rDNA regions are usually present

at different chromosomal locations and the homogenization

can occur in the direction of any of the parental type after

the allopolyploid formation (Wendel 2000).

The results presented in this manuscript and in the

previous reports (Goel et al. 2002; Doi et al. 2002) show

that one of the diploid genome donors to V. glabrescens is

V. umbellata/V. angularis (section Angulares) and the other

is from the section Ceratotropis. The analysis of cp DNA

(atpB-rbcL intergenic spacer) by Doi et al. (2002) had also

suggested the closeness of V. glabrescens to V. trinervia, a

species intermediate to section Ceratotropis and Angul-

ares. More detailed analysis is still needed, including

analysis of 5S IGS from species such as V. trinervia,

V. hirtella, V. reflexo-pilosa, etc. and analysis of some

more loci for inferring the progenitors of V. glabrescens

with certainity.

Section Aconitifoliae

Vigna aconitifolia and V. trilobata though present in cluster

I, formed a distinct subcluster representing section Aconi-

tifoliae (Fig. 6). Vigna trilobata accessions from Indonesia

(NI 1439) and India (NI 251) showed high similarity,

whereas the accession from Sri Lanka (NI 451) was clearly

divergent from the other two. Some accessions of V. trilobata

have been recently identified as V. stipulacea (Tomooka et al.

2003), including accession NI 251 used in this study. It is

likely that the accession NI 1439 is also V. stipulacea since it

shows high similarity with NI 251 in the 5S IGS region

(Table 4). This explains the high divergence between these

two accessions and accession NI 451. Present analysis

suggested that more than one accession of each species

should be used for inferring relationships as the misclassified

accessions can be rapidly identified and hence could prevent

phylogenetic problems.

Section Angulares

Most of the species that are known to belong to section

Angulares were grouped as a second major cluster, except

V. glabrescens that was clustered in section Ceratotropis

(Fig. 6). Earlier studies have shown that V. minima,

V. nakashimae and V. riukiuensis are closely related spe-

cies referred to as ‘V. minima complex’ (Yoon et al. 2000).

Vigna minima was shown to be related to the wild type

(var. gracilis) of V. umbellata (Marechal et al. 1978)

and even considered the wild relative of V. umbellata

(Gopinathan and Babu 1986). Relationships inferred by 5S


123

IGS suggested a close relationship among V. nakashimae,

V. riukiuensis and V. umbellata var. gracilis, however, both

the V. minima accessions were clearly divergent and were

present as a separate sub-cluster.

The two V. minima accessions showed some sequence

divergence in the 5S IGS (Supp. Table 2). Considerable

intraspecific heterogeneity among V. minima accessions

have been reported earlier (Yoon et al. 2000) but it should

be carefully verified, since cases of wrongly identified

V. minima have been reported (Egawa et al. 1996; Doi et al.

2002). Like, V. trilobata accessions mentioned above,

some V. minima accessions have also been renamed, such

as NI 970 and NI 1377 (also used in this study) has been

identified as V. nepalensis and V. hirtella, respectively

(Tomooka et al. 2003). Our analysis of ITS also indicates

the closeness of NI 970 to V. nepalensis. However, NI 1377

was close to NI 1376 (Goel et al. 2002), but both the

accessions were not close to V. hirtella (Saini et al. 2008).

Hence, the above-mentioned results were actually due to

wrongly named V. minima accessions. This also explains

why these two accessions were not clustered along with the

species belonging to ‘V. minima complex’ in the present 5S

IGS analysis. The wild (var. nipponensis) and cultivated

(var. angularis) types of V. angularis were closely related

(Fig. 6) and the results were in agreement with earlier

studies (Yamaguchi 1992; Kaga et al. 1996; Doi et al.

2002).

Wild and cultivated types of most of the species were

clustered together in the present analysis, except

V. umbellata (Fig. 6). The cultivated types (var. umbellata)

were clustered together, whereas the wild ancestral types

(var. gracilis) were present in different sub-clusters. This

could be attributed to different geographical origin of

the two wild type accessions NI 571 (Lao) and NI 1398

(Thailand). Cultivated V. umbellata from different loca-

tions has shown high divergence in the proteinase inhi-

bitor studies among the Vigna species (Konarev et al.

2002). Interestingly, the wild V. umbellata accessions used

in the present study clustered with the diverse intra-geno-

mic 5S rDNA types identified in V. nakashimae and

V. riukiuensis.

Multiple, diverse intra-genomic 5S rDNA units were

detected in V. nakashimae and V. riukiuensis. Surprisingly,

not all the 5S types identified in a species were clustered

together (Fig. 6). One sub-cluster included 5S types ‘A’

and ‘B’ from V. nakashimae, type ‘A’ from V. riukiuensis

and V. umbellata var. gracilis (NI 571), while the second

one included 5S types ‘C’ and ‘D’ from V. riukiuensis, type

‘C’ from V. nakashimae and the second wild V. umbellata

accession (NI 1398). The relative position of 5S type ‘B’

from V. nakashimae and type ‘D’ from V. riukiuensis as

compared to other 5S sequences in the dendrogram was

attributed to the fact that these were recombinant

sequences. Analysis showed horizontal gene transfer as a

result of inter-species hybridization between V. nakashimae

and V. umbellata var. gracilis (NI 571) and also between

V. riukiuensis and of V. umbellata var. gracilis (NI 1398).

This inference is also supported by the fact that several

species belonging to section Angualres are known to be

cross-compatible and also grow sympatrically (Sawa 1983;

Siriwardhane et al. 1991; Vaughan et al. 2000), horizontal

gene-transfer may occur between diploid taxa belonging to

the subgenus Ceratotropis (as explained above). A case of

inter-species gene-transfer has also been reported between

two species, Vicia sativa and Vicia segetalis (Potokina et al.

2000). In addition, earlier reports also suggest that species

of ‘V. minima complex’ (V. nakashimae, V. riukiuensis,

V. minima) could act as bridging species for the gene

transfer between V. angularis and V. umbellata (Tomooka

et al. 2000).

Conclusions

1. The 5S IGS is highly informative (comparable to ITS

and much better than atpB-rbcL spacer of cpDNA) and

could effectively be used for inferring species rela-

tionship among Vigna species in the subgenus Cera-

totropis. The 5S IGS is small but sufficiently

polymorphic to be used for evaluating other Vigna

species.

2. An extraordinary long deletion ([100 bp) in IGS

specific to V. radiata was an interesting observation.

The ITS of V. radiata was the largest among the

species belonging to the subgenus Ceratotropis, due to

deletions in ITS1 and ITS2 of other species (Doi et al.

2002; Goel et al. 2002; Saini et al. 2008), whereas, the

5S IGS of V. radiata was the smallest in the subgenus.

Some other Vigna species also showed small indels at

specific regions within the region (#5). This raises a

question whether IGS in remaining Vigna species is

also heading towards small size by deletions in mid-

spacer region, which does not have a known function.

A similar case of small 5S repeat units in Solanum

species had also suggested that reduction of 5S repeat

length is a general direction of molecular evolution of

5S rDNA in the genus Solanum (Volkov et al. 2001).

3. The 5S rDNA showed different rate of evolution

among different species as indicated by low intra-

species divergence in most of the species such as,

V. trilobata, V. mungo, etc. and incomplete homoge-

nization in V. nakashimae and V. riukiuensis.

4. The 5S rDNA analysis provided important clue on

the diploid progenitors of the tetraploid species

V. glabrescens and suggested detailed analysis of

more number of loci.


123

Acknowledgments Grateful thanks are due to Dr. T. Vanderborght,

National Botanic Garden of Belgium from whom we obtained the

seed material used in this study.

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