Garden pea (Pisum sativum L.) represents one of the most important models for studying plant developmental genetics. This species serves as a model object for most investigations on the genetic control of formation of compound inflorescence, compound leaf and of symbiotic interaction with nitrogen-fixing bacteria (nodulation). The latter phenomenon is typical for most legumes and is of significant theoretical and practical interest. By now, multiple genes involved in the genetic control of nodulation have been identified (for review see (2)).

One of the key processes in plant development is the regulation of stem apical meristem (SAM) activity. Studies on the model plant Arabidopsis thaliana (L.) Heynh. (Brassicaceae) demonstrated that equilibrium between SAM proliferation and keeping its volume stable is reached via the CLAVATA-WUSCHEL regulatory feedback loop. Expression of the gene WUSCHEL (WUS) maintains the meristematic condition of cells and activates expression of the CLAVATA (CLV1, CLV2 and CLV3) gene family. CLV proteins negatively regulate expression of WUS thus limiting its expression pool (14). Mutations in CLV genes lead to abnormal enlargement of the WUS-expressing zone resulting in flower and stem fasciation. Genes of the FASCIATA (FAS1 and FAS2) family also serve as negative regulators of

WUS (5).

Although no functionally confirmed homologs of CLV, FAS or WUS genes were identified in pea previously, some data exist suggesting that the same genes are involved in both nodulation processes and control of SAM activity in legumes (10). In pea, there are two gene mutations which are known to cause stem fasciation and hypernodulation: Nod4 (11) and Sym28 (9). Nod4 was localized earlier in linkage group (LG) V (11). There is also some evidence that Sym28 also belongs to LGV (J. Weller, pers. comm.). Further dissection of the activity, interactions and expression of these genes may provide greater understanding of the link between SAM activity and symbiotic nodule formation. Major macrosynteny observed between the chromosomes of pea and alfalfa (Medicago sativa L.) (4) together with the availability of a nearly complete genome sequence of Medicago truncatula Gaertn. is expected to be helpful in identifying the candidate genes and their map-based cloning in pea.

Our current study was aimed at the genetic mapping of Sym28 along with the putative pea homologs from A. thaliana for CLV2 and FAS1, which can be considered as candidate loci responsible for the fasciation trait. A brief characteristic of these fragments is presented in Table 1. The allelism test between nod4 and sym28 mutants was also performed in order to clarify the relationships between these genes.

Table 1. Putative pea homologs of CLV2 and FAS1 used as markers.


Fragment	Length, b.p.	Nucleotide sequence homology with *Arabidopsis*	Translated sequence homology*
*PsCLV2*	835	*92% (2 10^-140)**	87%
*PsFASI*	933	*67% (3 10^-9)**	71%

*, synonymic replacements left out of account. E- value for homology to Arabidopsis nucleotide sequences is presented in parentheses.

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Research Papers

Materials and Methods

Plant material and growth conditions

The following lines were used as material for this study: K301 (nod4) from the Institute of Cytology and Genetics (Novosibirsk, Russia) collection; P64 (sym28) from the All-Russia Research Institute of Agricultural Microbiology (Pushkin, Russia) collection and marker line WL1238 from the Genetics Dept. of Moscow State University (MSU) collection. Lines and filial progeny were planted in an experimental field on a territory of S.N. Skadovskii Biological Station of MSU (Moscow District, Russia) in 2007-2008 and also in greenhouse conditions. In total, 158 F₂ plants from the cross P64/WL1238 were examined for morphological traits and 87 were sampled for further DNA analysis. This sample included all fasciated recombinants together with a group of non-fasciated plants.

DNA extraction, PCR conditions and genetic mapping

DNA was extracted from freshly collected leaflets of parental lines and individual F₂ progeny from the cross P64/WL1238 using a modified CTAB procedure (12). PCR was carried out in a MC2+ thermal cycler (DNA Technology, Russia) according to the protocol described earlier (6). Both morphological (gp, te, r, tl) and molecular (see Table 2) markers of LGV were used to localize gene Sym28 more precisely. Two of the CAPS markers used in our study, PsCLV2 and PsFAS1, represent putative pea homologs of the genes CLV2 and FAS1 of A. thaliana respectively (unpublished data) thus being candidate genes for the fasciated phenotype in P64. The CAPS markers have been tested for polymorphism between parental lines using 12 restriction endonucleases: Tru9I, TaqI, RsaI, HaeIII, AluI, BstFNI, HpaII, HinfI, PspN4I, BstDEI, Bst4CI, and AspLEI (SibEnzyme, Russia). PsCLV2 turned out to be monomorphic in the cross P64/WL1238 (data not shown). Distances between markers were calculated using Mapmaker/EXP 3.0 software package using the Kosambi mapping function and LOD score threshold set at 3.0 (7).

Table 2. Characteristics of molecular markers used for mapping.


Marker / Endonuclease*	Genbank entry	Primer sequences	Marker type	Source
*PsCLV2 / -*	DQ478949	F: 5'- CCTGAGAGTTTRCTKTATTTGAAGTC- 3' R: 5'- GAGAAAGATACYTGAGGTTKGWCC- 3'	CAPS	This study
*PsFAS1 / TaqI*	DQ480717	F: 5'- CGTTGTCKAAGCTTGTWGATG- 3' R: 5'- AGCTTCWCWTCTATTTYTMTCC- 3'	CAPS	This study
*Fbpp / Hpal* 1	L34806	F: 5'- CCTTACTCTCCTTCACGTCT- 3' R: 5'- CTTTTCAACCTTCTCCACCT- 3'	CAPS	(3)
*AD79*	-	F: 5'- ACAAGACTTCCAGAAATTTTGCAT- 3' R: 5'- AGGACTGATGACGGAGACAAAG- 3'	SSR	(8)
*AD175*	-	F: 5'- CTTGTGCAGAAGCATTTGATTA- 3' R: 5'- AGAGACAATGGATGCTCATAGT- 3'	SSR	(8)

*, for CAPS markers only. Note: in degenerate primer sequences R stands for A or G, K for G or T,

W for A or T, Y for C or T.

Results and Discussion

Allelism tests

Sym28 has been shown to have significant linkage with markers of LGV (see below; J. Weller, pers. comm.); therefore, it was proposed that sym28 may be allelic to another mutation causing both stem fasciation and hypernodulation, nod4, since nod4 is known to be on LGV (11). To test this, the cross was made between lines P64 and K301. These lines possess somewhat different manifestation of fasciation. Phyllotaxis abnormalities are observed beginning from node 16.65 ± 4.61 in P64 and from 3.82 ± 0.88 in K301 (mean ± standard deviation). Axillary flower-bearing axes terminate with an abnormal flower in K301 (resembling ectopic flowers described in (1), while no such phenomena were observed in P64. This evidence supports the idea that fasciation is caused by different mutations in these lines and this hypothesis was confirmed by allelism tests: all F₁ progeny were non-fasciated thus evidencing for different genetic mechanisms underlying the described abnormalities.

Pisum Genetics

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Research Papers

Mapping of Sym28

Significant linkage was found between Sym28 and LGV markers. Based on the linkage data, a map fragment surrounding Sym28 was constructed (Fig. 1b). The arrangement of sequence-tagged markers (CAPS) in the resulting map was compared with the positions of putative homologous loci (according to BLAST search results) on the physical map of Medicago truncatula Gaertn. chromosome 7 (Fig. 1c) which was reported to be syntenic with LGV of pea (4). The obtained map was also compared with the one containing Nod4 and morphological markers, as reported in (11). Map position of genes Sym28 and Nod4 was similar. Obviously, more linkage analyses are needed to reveal a more certain position of Nod4 in relation with Sym28 and possible correspondence between the genes studied in this report and known genes controlling meristem identity in Arabidopsis.

Figure 1. Correspondence between regions of pea LGV containing genes nod4 (a, after (11)), sym28 (b) and a physical map of chromosome 7 of Medicago truncatula (c) generated by Medicago CviT- BLAST tool (http://www.medicago.org/genome/cvit_blast.php). Chromosome orientation was established according to (13). Numbers between markers designate distances, cM; numbers in parentheses stand for LOD score.

Acknowledgements: The authors are indebted to Dr. Viktor E. Tsyganov (All-Russia Research Institute of Agricultural Microbiology) and to Dr. Klavdia K. Sidorova (Institute for Cytology and Genetics òî÷íåå) for generously provided seeds of studied lines P64 and K301, respectively. The authors also thank Olga A. Ash and Galina A. Khartina for assistance in field work. The work was supported by Russian Foundation for Basic Research (grant no. 07-04-00652).

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Research Papers

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