A chloroplast DNA marker frequently found in wild peas

 

Bogdanova, V.S. and Kosterin, O.E.                                                                   Inst. of Cytol. and Genet.

                                                                                                 Russian Acad. of Sci., Novosibirsk . Russia

 As part of a study of chloroplast inheritance in pea we searched for a marker that could distinguish the wild pea accession VIR320 (Pisum sativum L. ssp. elatius (Bieb.) Schmahl., Palestine ) from other pea forms. We used a PCR-RFLP approach and tested two pairs of PCR primers in combination with three restriction endonucleases. The combination we report on here involves a primer pair that amplifies a portion of the coding region of the chloroplast rbcL gene (large subunit of ribulose-bisphosphate carboxylase) and the restriction enzyme AspLEI .  The primers were designed to match the X03853 EMBL database accession: 5’-TTATTATACTCCTGACTATCAAACC-3’ and 5’-TACAGAATCATCTCCAAATATCTCG-3’. The following cycling parameters were used: initial denaturation at 95oC for 1 min followed by 35 cycles including denaturation at 94oC 59 sec, annealing at 58oC 59 sec, elongation at 72oC 1 min. A 15 ml sample of the reaction was treated with 20 units of AspLEI endonuclease for 2 hours at 37oC in a volume of 30 ml and electrophoresed in a 1.5% agarose gel in TAE buffer.  The expected size of the amplified product was 1129 bp.

Most cultivated lines generated the expected 1129 bp fragment that lacked a restriction site.  However VIR320 plastid DNA gave two fragments of about 800 and 300 bp after treatment with AspLEI (Fig. 1).  Partial sequencing the rbcL gene in VIR320 showed that the restriction site was produced by a T to C nucleotide substitution in position 393 from the start codon, or position 325 from the beginning of the primer used. We assayed 27 samples of wild and cultivated peas, representing most of the presently recognized taxa. The electrophoretic pattern of DNA fragments formed upon AspLEI restriction of rbcL amplification product is shown in Fig. 1, presence or absence of the restriction site is indicated in Table 1.  In addition, Table 1 includes both the initial taxonomic designation of an accession and its taxonomic status accepted here.  In particular, following Townsend (2) and Davis (1), we consider all wild representatives of Pisum sativum L. (except for P. sativum ssp. abyssinicum (A. Br.) Berger) within the same subspecies P. sativum ssp. elatius (Bieb.) Schmahl.

 

(1A)    (1B)

 

 

Figure 1. Restriction fragments formed upon AspLEI digestion of rbcL amplification product from different pea accessions. Lane numbers correspond to No in Table 1. M – molecular weight marker 100-1000 bp.

We found that the restriction site for AspLEI in the gene rbcL is present in both accessions of Pisum fulvum Sibth et Smith, both accessions of P. sativum ssp. abyssinicum, in 4 of 11 accessions of P. sativum ssp. elatius (Bieb.) Schmahl., in neither of 2 accessions of P. s. ssp. transcaucasicum Govorov and in only one of 10 accessions of P. s. ssp. sativum (see Table 1).  These data lead us to conclude that the restriction site probably was present in the ancestor of the genus Pisum and has been lost in some lineages of P. sativum ssp. elatius.  A majority of the P. sativum ssp. sativum. germplasm appears to have inherited the derived (absence of restriction site) form of the cpDNA genome.

Table1.  Pea accessions tested for the presence of AspLEI recognition site in rbcL gene.

 

No

Accession number

Collection location

Wild or cultivated

Original taxonomic designation

Taxonomic designation used by authors

Presence of AspLEI recognition site

1

L93

Unknown

Wild

Pisum fulvum

Pisum fulvum

YES

2

VIR6070

Palestine, foothills 30 km SW of Jerusalem

Wild

Pisum fulvum

Pisum fulvum

YES

3

L100

Unknown

Wild

Pisum humile

Pisum sativum ssp. elatius

YES

4

JI1794

Golan Heights

Wild

Pisum humile

P. sativum ssp. elatius

no

5

Ps008

Turkey , Siirt (coll. F. Muehlbauer)

Wild

Pisum humile

P. sativum ssp. elatius

no

6

VIR7329

 

Turkey (“received from USA ”)

 

Wild

P. sativum ssp. syriacum

Pisum sativum ssp. elatius

no

7

VIR320

Palestine (received from Sutton , France , by l. N. I. Vavilov in 1922)

Wild

P. sativum ssp. syriacum

P. sativum ssp. elatius

YES

8

VIR320*

- -

Wild (?)

P. sativum ssp. syriacum

P. sativum ssp. elatius

no

9

VIR2521

Palestine , Kinereth, 1923

Wild

P. sativum ssp. syriacum

P. sativum ssp. elatius

YES

10

VIR4014

 

Azerbaijan, Lenkoran, a winter wheat field

Wild

P. sativum ssp. elatius

P. sativum ssp. elatius

no

11

VIR1851

 

Georgia , Tbilisi , Botanical Garden

 

Wild

P. sativum ssp. elatius

P. sativum ssp. elatius

no

12

VIR2524

North Galilea, in maccia Tarschich et Peccia

Wild

P. sativum ssp. elatius

P. sativum ssp. elatius

YES

13

CE1

 

Crimea , Simeiz (coll. Trusov and Kosterin, 1990).

Wild

P. sativum ssp. elatius

P. sativum ssp. elatius

no

14

VIR2759

Unknown

Cultivated

P. sativum ssp. abyssinicum

P. sativum ssp. abyssinicum

YES

15

WL1446

Unknown

Cultivated

P. sativum ssp. abyssinicum

P. sativum ssp. abyssinicum

YES

16

VIR3249

Georgia, Gori

Cultivated

P. sativum ssp. transcaucasicum

P. sativum ssp. sativum

no

17

VIR4871

Georgia

Cultivated

P. sativum ssp. transcaucasicum

P. sativum ssp. sativum

no

18

VIR3913

The Pamirs, Shugnan District, Shakh-Dara, Pavdysh kishlak

Cultivated

P. sativum ssp. asiaticum

P. sativum ssp. sativum

no

19

VIR1975

Afghanistan , Gerat

Cultivated

P. sativum ssp. asiaticum

P. sativum ssp. sativum

YES

20

VIR3954

Tadjikistan, Shugnan District, Emch kishlak

Cultivated

P. sativum ssp. asiaticum

P. sativum ssp. sativum

no

21

Sprint-1

A laboratory line de-rived from cv. Avanti and VIR7036 ( Nepal )

Cultivated

Hybrid P. sativum ssp. asiaticum x

P. sativum ssp. sativum

P. sativum ssp. sativum

no

22

VIR2593

Cyprus , Nikosia

Cultivated

P. sativum ssp. sativum

P. sativum ssp. sativum

no

23

VIR3429

Egypt , Assuan

?

P. sativum ssp. sativum (characters as in “P. jomardi”)

P. sativum ssp. sativum

no

24

VIR7163

Lebanon

Cultivated

P. sativum ssp. sativum

P. sativum ssp. sativum

no

25

VIR7006

Syria , Damask

Cultivated

P. sativum ssp. sativum

P. sativum ssp. sativum

no

26

VIR2516

Palestine

Cultivated

P. sativum ssp. sativum

P. sativum ssp. sativum

no

27

VIR2172

Palestine , Prosch-Pina Agelet Hoschahas

Cultivated

P. sativum ssp. sativum

P. sativum ssp. sativum

no

                        N – lane number of Fig. 1.

 Acknowledgment: This work was supported by the "Biosphere origin and evolution" project of the Russian Academy of Sciences.

 1.  Davis , H.  1970.  Flora of Turkey and the East Aegean Islands . Edinburgh , v. 3.

2.  Townsend, C.  1968.   Kew Bull. Roy . Bot. Gard. 21: 435-358.