New microsatellite markers for pigeonpea (cajanus cajan (L.) millsp.)
BMC Research Notes
BioMed Central
Short Report
Open Access
New microsatellite markers for pigeonpea (cajanus cajan (L.)
millsp.)
DA Odeny*1,3, Jayashree B2, C Gebhardt3 and J Crouch4
Address: 1University of Bonn, Centre for Development Research (ZEFc), Walter-Flex Str.3 53113 Bonn, Germany, 2International Crops Research
Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Hyderabad, India, 3Max-Planck Institute for Plant Breeding Research (MPIZ),
50829 Köln, Germany and 4International Maize and Wheat Improvement Centre (CIMMYT), CP 56130, El Batán, Mexico
Email: DA Odeny* - ; Jayashree B - ; C Gebhardt - ;
J Crouch -
* Corresponding author
Published: 6 March 2009
BMC Research Notes 2009, 2:35
doi:10.1186/1756-0500-2-35
Received: 29 December 2008
Accepted: 6 March 2009
This article is available from: http://www.biomedcentral.com/1756-0500/2/35
© 2009 Odeny et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Pigeonpea is a nutritious tropical legume with several desirable characteristics but
has been relatively neglected in terms of research. More efficient improvement can be achieved in
this crop through molecular breeding but adequate molecular markers are lacking and no linkage
map has been developed so far. Microsatellites remain the markers of choice due to their high
polymorphism and their transferability from closely related genera. The overall objective of this
study was to develop microsatellite markers from an enriched library of pigeonpea as well as testing
the transferability of soybean microsatellites in pigeonpea.
Results: Primers were designed for 113 pigeonpea genomic SSRs, 73 of which amplified
interpretable bands. Thirty-five of the primers revealed polymorphism among 24 pigeonpea
breeding lines. The number of alleles detected ranged from 2 to 6 with a total of 110 alleles and an
average of 3.1 alleles per locus. GT/CA and GAA class of repeats were the most abundant dinucleotide and tri-nucleotide repeats respectively. Additionally, 220 soybean primers were tested
in pigeonpea, 39 of which amplified interpretable bands.
Conclusion: Despite the observed morphological diversity, there is little genetic diversity within
cultivated pigeonpea as revealed by the developed microsatellites. Although some of the tested
soybean microsatellites may be transferable to pigeonpea, lack of useful polymorphism may hinder
their full use. A robust set of markers will still have to be developed for pigeonpea genome if
molecular breeding is to be achieved.
Background
The increasing concern of the effect of global climate
change and its likely impact on agriculture has stimulated
scientists to search for crops that can withstand extreme
environmental conditions. Among legumes, pigeonpea
{Cajanus cajan (L.) Millspaugh} (2n = 22) has attracted
attention as being both drought-tolerant [1] and highly
nutritious [2]. Extensive morphological variation within
the genus Cajanus as a whole and in cultivated species in
particular has always led to the assumption that there
exists abundant genetic diversity within the cultivated species. To the contrary, molecular studies have reported
extremely low levels of polymorphism within the cultivated species compared to its wild relatives [3,4]. Such
findings suggest that efforts towards the development of a
linkage map of pigeonpea should focus on the use of an
Page 1 of 5
(page number not for citation purposes)
BMC Research Notes 2009, 2:35
http://www.biomedcentral.com/1756-0500/2/35
interspecific cross, and the development of a substantially
high number of markers. We report the development of
new 36 polymorphic simple sequence repeat (SSR) markers that will be an asset in characterising and understanding the nature of diversity within Cajanus species.
Results
A total of 641 non-redundant contigs were generated from
2,131 sequenced clones reflecting an overall redundancy
level of 70%. Of the 641 contigs, 117 sequences (20%) contained a microsatellite. The average size of each contig was
500 bp. This library thus covered an estimated 320,500 bp
of pigeonpea genome. On the whole, di-nucleotide repeats
were the longest (average 27 bp long) and also the most
abundant followed closely by tri-nucleotide repeats (average 25 bp long). The longest motif was a 258 bp perfect
hexa-nucleotide (AAACCC) repeat while a GT had the longest uninterrupted repeat of 74. A list of all designed pigeon-
pea primer sequences, SSR motifs and PCR programmes
used for amplification is provided [see Additional file 1].
Table 1 gives a detailed characterisation of 35 pigeonpea
primers that were polymorphic among the 24 diverse genotypes. Di-nucleotide repeats formed the highest proportion of polymorphic markers followed by tri-nucleotide
repeats. The number of alleles detected ranged from 2 – 6
at each of the 35 polymorphic loci with a total of 110 alleles and an average of 3.1 alleles per locus. Gene diversity
values ranged from 0.07–0.76 with an average of 0.41.
While TG class of repeats formed the highest proportion
(40%) of all the polymorphic loci, the highest number of
alleles (6) was observed from a perfect tri-nucleotide
repeat (CCttc019). The most informative marker with polymorphism information content (PIC) of 0.76 was a trinucleotide compound repeat (CCttc005), which was also
the longest motif.
Table 1: PCR conditions, allele sizes and core motifs of new microsatellite loci that amplified in 24 different genotypes of pigeonpea
SSR Name
Core Motif
Allele size range
No. of alleles
PIC1
CCttc002
CCttc004
CCac003
CCttc005
CCac004
CCttc007
CCtc003
CCtc005
CCtta006
CCcttc001
CCgaaa001
Cccta001
CCac006
CCgtt002
CCttc012
CCac0071
CCgtt003
CCtc007
Ccac010
Ccac011
CCttc017
Ccat006
Cccta003
CCac015
CCtc009
CCac0171
CCac018
CCac019
CCac026
CCac030
CCttc018
CCac027
CCttc019
CCttc020
CCac029
(gaa)5g(gaa)5
(gaa)6
(ca)8
(gaa)11gag(gaa)5gaggaagag(gaa)17
(ta)5(tg)7ta(tg)4
(aga)5
(tc)8
(ag)20
(att)21
(cttc)4
(cttt)4
(gat)5(tct)(gat)4
(ca)10cg(ca)6
(tgt)4
(ttc)7
(tg)(tc)2(tg)7
(ttg)5(ttc)7
(tc)6
(ca)7
(gt)7
(aga)11(ggag)(gaa)4ga(gga)3a(gaa)16
(ta)7(ca)6
(gat)4
(ac)4aa(ac)38c(ca)7
(tc)6
caccac(a)5(ca)6c(a)4
(ac)6a
(tg)6
(ac)7
(cata)3ta(tg)6
(aga)5
(tg)7
(aag)13
(ctt)8
(caa)(ca)6caa
184–215
240–250
175–198
305–320
243–245
265–275
175–190
165–185
290–310
260–270
220–225
275–302
295–350
210–235
170–190
275–280
165–180
310–330
191–198
227–270
145–150
220–277
420–450
135–145
200–220
215–227
200–210
130–135
278–295
236–244
275–288
295–300
220–245
236–242
160–180
5
2
5
5
2
4
2
4
5
4
2
3
5
3
3
2
2
4
3
2
3
3
3
2
4
2
3
2
4
2
2
2
6
2
3
0.63
0.08
0.72
0.76
0.08
0.54
0.07
0.59
0.71
0.28
0.37
0.29
0.49
0.40
0.42
0.08
0.37
0.68
0.50
0.36
0.37
0.54
0.39
0.14
0.45
0.24
0.54 (...truncated)
