Microsatellite and ITS sequence variation in wild species and cultivars of pea


Polans, N.O.                                                                   Northern Illinois University, Dekalb, Illinois, USA

Moreno, R.R.                                                  


Previous investigations by our laboratory (1, 2) have applied the nucleotide sequence variation found in ribosomal internal transcribed spacer (ITS) regions to the systematic analysis of closely related pea taxa, in part due to the high rate of evolutionary change characterizing these DNA regions (3). No attempt was made to infer evolutionary relationships among the 65 taxa, however, given the relatively few informative sites available to the analysis. Despite a paucity of data, several observations could be made respecting the overall level of genetic variation found across the genus and the topological relationships established among the selected groups of taxa. These include: 1) very close genetic affinities throughout Pisum, with P. fulvum exhibiting the greatest degree of genetic divergence based upon the data examined; 2) support for P. fulvum, northern P. humile and southern P. humile as distinct taxonomic categories; 3) the independent evolution of a pea chromosomal translocation; 4) the assignment of JI1794 as a “northern” P. humile; and 5) inconsistent taxonomic assignments for JI241, JI198, JI1398, JI1096 and JI2055. The data were unable to resolve the very close systematic relationships among P. elatius, P. abyssinicum and P. sativum.


More recently, our laboratory has developed and/or characterized a series of pea microsatellite loci (4, 5) to serve as informative neutral molecular markers for a number of project applications. In previous studies involving pea, microsatellite-based molecular markers have been used to determine inter-genera transferability of primers (6), to analyze diversity within the genus Pisum (7) and to estimate the level of microsatellite polymorphism in Pisum sativum L. (8). RAMS (Randomly Amplified MicroSatellites), a novel molecular marker technique (7), applies lower PCR annealing temperatures to microsatellite-specific primers and provides an efficient means to generate greater numbers of  less-specific multi-banded DNA profiles.   


Unfortunately, in the case of a P. sativum ssp. Syriacum accession from the original collection, too few reliable RAMS bands were obtained to include it in the current data set. The goal of the present study, therefore, is to compare and combine RAMS microsatellite variation with nuclear ribosomal DNA variation as a systematic organizing tool for 64 wild and cultivated pea taxa, both to re-examine our previous findings and to explore the results of combining both data sets in a common analysis.


Materials and Methods

Pisum isolates 701-723 are from the Ben Ze’ev and Zohary (1973) collection (courtesy of J.G. Waines), JI accessions are from the John Innes collection (courtesy of M.J. Ambrose), cv. Alaska is from J. Mollema and Son, Inc. (Grand Rapids, MI), cv. (Morse’s) Progress #9 is from Ferry-Morse Seeds (Mountain View, CA)  and accessions 82-14n, A1078-234 and PI 179449 were kindly provided to this project by G. Marx and N. Weeden.


DNAs from 64 pea accessions representing the range of the genus Pisum are amplified with 11 of the 31 primer sets described previously (4, 5) and then separated on polyacrylamide gels to evaluate detectable differences using the RAMS method (see 4). Clearly discernable polymorphic and monomorphic bands between 90-300 bp in size are scored as “present”, “absent” or “missing data” for each accession. Sequence data are analyzed using the PAUP computer package (9).

Results and Discussion

Seventy-eight scoreable DNA bands are produced among the 64 representative pea accessions using primer sets 3, 5, 14, 16, 17, 18, 20, 22, 23, 25 and 31 (see 4, 5 and Table 1), resulting in 4992 total data points. Of the 78 RAMS bands generated, 46 are polymorphic and 44 are informative across all of the accessions. A compilation of the 44 informative bands is delineated for all 64 pea taxa in Table 1. As noted previously (1), the table is organized in accordance with the two commonly recognized species of pea (10, 11, 12), the more divergent P. fulvum (also see 13) and the typically cultivated P. sativum. The former is represented by eight different accessions, while the latter is further differentiated in the table as four subspecies: humile, elatius, abyssinicum and sativum. Subspecies humile is subdivided by northern and southern populations (10). There are six accessions characterized as questionable taxonomic assignments solely based on the RAMS data, as contrasted with five inconsistent assignments (three shared in common) based on the nuclear ribosomal DNA variation reported previously (1).


A Neighbor Joining (NJ) distance analysis of the 44 RAMS data points is presented as a phylogram in Figure 1 to provide a basic illustration of the information presented in Table 1. In the figure, P. fulvum, northern and southern P. humile, P. abyssinicum and a half-dozen P. elatius accessions maintain distinct group associations; although, the P. abyssinicum group includes JI2385, formerly designated as P. sativum (1).  Two P. elatius accessions (JI 1096 and JI 2055) that displayed the largest number of ITS sequence differences with P. fulvum in the ribosomal DNA study now group as part of the six P. elatius accessions. Four other P. elatius, a single putative P. humile and the paired northern P. humile all intersperse with P.  sativum in the figure. A second NJ phylogram (Figure 2) combines the 44 RAMS data points with the 21 informative ITS data points from the earlier study (1). The relationships within and among P. fulvum, southern P. humile, P. abyssinicum and the six P. elatius remain essentially the same as depicted in Figure 1, and the four other P. elatius and one P. humile remain dispersed within an otherwise single block of P. sativum. With the combination of data sets, however, the pair of northern P. humile accessions is associated more closely with the P. elatius group and is not interspersed with P. sativum.



Both the microsatellite and combined data sets presented in Figures 1 and 2, respectively, support the designation of P. fulvum as a distinct taxon; although, the relatively small number of available data points renders any conclusions from this study tentative. Additional support for P. fulvum as a distinct species, however, is presented elsewhere (1, 10, 11, 12, 13). With respect to the remaining taxa, southern P. humile is least closely associated with P. sativum and remains separated from northern P. humile. The P. abyssinicum group is most closely associated with southern P. humile and portions of a dispersed P. elatius group. These relationships are not inconsistent with the proposed placement of P.  abyssinicum between P. elatius and P. sativum (14) given the dispersed nature of the P. elatius subspecies. Northern P. humile has been postulated the closest wild progenitor of the cultivated pea based in part on a shared chromosomal translocation (10) and detailed chloroplast studies (15). The current study lends limited support to this assertion, which was not supported by the ITS data alone (1).


 Acknowledgement: This work was supported by funds from the Department of Biological Sciences, Northern Illinois University.



1.       Polans, N.O. and Saar, D.E. 2002. Pisum Genetics 34:9-14.

2.       Saar, D.E. and Polans, N.O. 2000. Pisum Genetics 32:42-45.

3.       Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S. and Donoghue, M.J. 1995. Annals Missouri Bot. Gard. 82:247-277.

4.       Moreno, R.R. and Polans, N.O. 2006. Pisum Genetics 38:10-14.

5.       Moreno, R.R. and Polans, N.O. 2007. Pisum Genetics 39:10-12.

6.       Pandian, A., Ford, R. and Taylor, P.W.J.  2000. Plant Mol Bio. Rep. 18:395a-395h.

7.       Ford, R., Le Roux, K., Itman, C., Brouwer J.B. and Taylor, P.W.J. 2002. Euphytica 124: 397-405.

8.       Burstin, J., Deniot, G., Potier, J.,Weinachter, C., Aubert, G. and Baranger, A.  2001.  Plant Breeding 120: 311-317.

9.       Swofford, D.L. 1998. PAUP, Version 4.0b4a. Sinauer Associates, Sunderland, Massachusetts.

10.   Ben Ze’ev, N. and Zohary, D. 1973. Israel J. Bot. 22:73-91.

11.   Hoey, B.K., Crowe, K.R., Jones, V.M. and Polans, N.O. 1996. Theor. Appl. Genet. 92:92-100.

12.   Marx, G.A. 1977. In: Physiology of the Garden Pea. Eds. Sutcliffe, J.F. and Pate, J.S., Academic Press, New York, pp. 21-43.

13.   Jing, R., Johnson, R., Seres, A., Kiss, G., Ambrose, M.J., Ellis, T.H.N. and Flavell, A.J. 2007. Genetics 177:2263-2275.

14.   Weeden, N.F. and Wolko, B. 2001. Pisum Genetics 33:21-25.

15.   Palmer, J.D., Jorgensen, R.A. and Thompson, W.F. 1985. Genetics 109:195-213.



Table 1. Variable RAMS sites for wild and cultivated taxa of pea.








Text Box: Pisum fulvum
701                     11001?110100?1101000001100000000011000110110
702                     110?1101000001101000001100000000011000?10101
703                     10011?110000111?10000011000000000110000101??
706                     ????11?1?0011111100011111000000001????01110?
707                     11001111000011101110011100010001110??0011101
708                     1?0011110000111010100011001000000000?0010100
JI224                 11101111?011010?1000001100100001110110011111
JI1006                10101??10001101?1010001100110000001101011100
Pisum sativum
ssp. humile (northern)
716                     000?0?10101110?111010010011100111100111110??
JI1794                0000011011111011111101100011001111011111101?
ssp. humile (southern)
711                     010111101011101?1100001101110000011011011010
712                     01011110101100011100001011100101111?11111010
713                     01011110101000011101000001110101111011011010
714                     01011100111010111000010011110?01111011011010
ssp. elatius
721                     010?1110101110111011010001110001111?11111010
722                     010?1110101110111011011011110001111111111010
JI64                   010011101011101110110000011110010111111110?0
JI261                 0001111010111011101?0100111100011111100110??
JI1096                00011?1011111011001101?0011100111101110?????
JI2055                00111110111110111011010011110010010?11011010
ssp. abyssinicum
JI2                     01001110101110011?001010011111001011110110?0
JI130                 01011110101110111?00100001100100101111011000
JI225                 0101111010111001100010?001100100101111011000
JI2202                  01011110101110111?001010011001011011110110?0
ssp. sativum
82-14n                    010?1110111110?01101001011110011110111?01111
A1078-234              00101110111110011101011001111001110111111011
PI179449                01101110101110?11001001011110011110111111010
Alaska                    0110111010111011111101100111100111?111101011
Progress#9             0?101110111110111?01011001111001111111111011
JI85   Afghanistan 010011101011101111010010111100111111111110?0
JI156  Sudan          0100111010111011110100101111100011?111111011
JI159  Ethiopia      00101010101110?1100101100111000011?111111011
JI181  Keerau Pea   00001110101110111101001011110001110111011011
Text Box: JI185  Wiraig           ????111010111011100100100111000011011?101011
JI196  Georgia          00101110101110110001011001111000111110001010
JI207 choresmicum       01101110101110111101001001111101111111001011
JI209  arvense          011010101011101?1001011001111001110111111011
JI228  Bolivia          00101110101110011001001001110001111111011011
JI245  Russia           011011101011101111?101101111110111?111001001
JI250  (P.jomardii)     01001110101110111101011011110?0111111100100?
JI263  Balkans          011?1110101110111101001011111001111111111011
JI264  Greece           00101110101110101101001011111101111111101011
JI711  Austrian Winter  01101110101110111101001011111001110111111011
JI787  Minerva          011011101011101111010010111110011111111?1011
JI1033 India            001?11101111101111010010111100001101110110?0
JI1035 Turkey           0010111010111011110100100111010?1111110010?0
JI1057 Antioquia I Chilena 01101110101110111101001001111001111111001011
JI1089 Syriacum    00101110101110111101001001110000110111011010
JI1345 Mongolia         011011101011101111011110011110011101111?1011
JI1372 Mummy Pea        01001110111110111101111001111001111111111011
JI1428(P. tibetanicum)  000011101111101?110100100111100?11?111001011
JI1578 China            0000??10111110111101011001110000110111?11010
JI1758 Nepal            00101110111110111101001011111010111111011010
JI1835 Spain            0100111010111011?101001001111011111111111011
JI2116(P.speciosum)     01101010101110111101011001111011111111101011
JI2124 ponderosum       00101110101110111101011011110001111111101011
JI2265 Primitive Albanian 00101110101110111?110010111110111111?1111011
JI2438 Partridge        001011101011101111?1001001111001111111011011
Inconsistent assignments:
JI241   (1)             01?01110101110111011001011110011111111011010
723     (2)             00101110101110111101011001111001111111111010
JI198   (2)             01101110101110111101011001111001111111111010
JI1398  (2)             01?01110111110101101?110011110?1111111011011
JI2201  (2)             00101110101110111101001011110001111111111011
JI2385(P. sp. Yemen) (3)01011110101110111?001010011001011011110110?0
*1=present, 0=absent, ?=missing data.   
(1) JI241 is listed as ssp. humile, but it displays ssp. Sativum characteristics.
(2) 723, JI198, JI1398 and JI2201 are listed as ssp. elatius, but they display ssp. sativum characteristics.
(3) JI2385 is listed as ssp. sativum, but it displays ssp. Abyssinicum characteristics.