Vera S. Bogdanova

Inheritance of organelle DNA markers in a pea cross associated with nuclear-cytoplasmic incompatibility

An unusual biparental mode of plastid inheritance was found in pea, in a cross associated with nuclear-cytoplasmic incompatibility manifested as deficiency of chlorophyll pigmentation. Plastid DNA marker trnK and mitochondrial DNA marker cox1 were analyzed in F1 progeny that received cytoplasm from an accession of a wild subspecies Pisum sativum ssp. elatius. Plants with sectors of green tissue on leaves and seed cotyledons with green patches on an otherwise chlorotic background were found to carry paternally inherited plastid DNA, suggesting that photosynthetic function was affected by nuclear-cytoplasmic conflict and required proliferation of paternally inherited plastids for normal performance. The paternally inherited plastid DNA marker was also observed in the roots. The presence of the paternal marker in cotyledons, roots and leaves was independent of each other. Inheritance of the mitochondrial DNA marker cox1 appeared to be of the maternal type.

Relationship of wild and cultivated forms of Pisum L. as inferred from an analysis of three markers, of the plastid, mitochondrial and nuclear genomes

Eighty-nine accessions of wild and cultivated peas (12 Pisum fulvum Sibth. et Smith., 7 P. abyssinicum A. Br., 31 wild and 42 cultivated forms of P. sativum L.) were analysed for presence of the variants of three functionally unrelated polymorphic markers referring to different cellular genomes. The plastid gene rbcL either contains or not the recognition site for restriction endonuclease AspLEI (rbcL+ vs. rbcL−); the mitochondrial gene cox1 either contains or not the recognition site for restriction endonuclease PsiI (cox1+ vs. cox1−); the nuclear encoded seed albumin SCA is represented by slow (SCAS) or fast (SCAF) variant. Most of the accessions possessed either of two marker combinations: 24 had SCAFcox1+ rbcL+ (combination A) and 49 accessions had SCAScox1− rbcL− (combination B), 16 accessions represented 5 of the rest 6 possible combinations. All accessions of P. fulvum and P. abyssinicum had combination A, the overwhelming majority of cultivated forms of P. sativum had combination B while wild representatives of P. sativum had both combinations A and B, as well as rare combinations. This pattern indicates that combination A is the ancestral state in the genus Pisum L., inherited by P. fulvum and P. abyssinicum, while combination B seems to have arisen in some lineage of wild P. sativum which rapidly fixed mutational transitions of the three markers studied, most probably via a bottleneck effect during the Pleistocene. Then this ‘lineage B’ spread over Mediterranean and also gave rise to cultivated forms of P. sativum. Rare combinations may have resulted from occasional crosses between ‘lineage A’ and ‘lineage B’ in nature or during cultivation, or represent intermediate evolutionary lineages. The latter explanation seems relevant for an Egyptian cultivated form ‘Pisum jomardii Schrank’ (SCAFcox1− rbcL−) which is here given a subspecies rank. Wild representatives of P. sativum could be subdivided in two subspecies corresponding to ‘lineage A’ and ‘lineage B’ but all available subspecies names seem to belong to lineage B only. Presently all wild forms would better be considered within a fuzzy paraphyletic subspecies P. sativum subsp. elatius (Bieb.) Schmalh. s. l.

Genetic analysis of nuclear-cytoplasmic incompatibility in pea associated with cytoplasm of an accession of wild subspecies Pisum sativum subsp. elatius (Bieb.) Schmahl.

The genetic basis of nuclear-cytoplasmic incompatibility was examined using the wild pea (Pisum sativum subsp. elatius) accession VIR320. When this accession is used as the female parent in crosses with domesticated peas (Pisum sativum subsp. sativum) the F1 is highly sterile and displays chlorophyll deficiency, chlorophyll variegation, reduction of leaflets and stipulae while the reciprocal cross produces hybrids that appear normal. A mapping recombinant inbred line (RIL) population was established based on a cross in a compatible direction of a tester line WL1238 with VIR320. The ability to cause nuclear-cytoplasmic conflict was analysed by crossing individual RIL plants as pollen parents with VIR320 as donor of cytoplasm and scoring each F1 for major signs of the conflict. It is concluded that two unlinked nuclear genes are involved in the genetic control of the observed incompatibility. One of the genes, denoted as Scs1, is closely linked to the PhlC gene on linkage group III and the other, denoted as Scs2, is closely linked to the gp gene on linkage group V. Alleles of both genes in WL1238 are dominant and appear to be lethal in the homozygous condition in the VIR320 cytoplasm background.

Histone H1 of the garden pea (Pisum sativum L.) ; composition, developmental changes, allelic polymorphism and inheritance

Abstract Histone H1 of the garden pea ( Pisum sativum L. ), as a rule, is represented by seven subtypes, as revealed by the high resolution acetic acid/urea polyacrylamide gel electrophoresis. Each subtype has molecular variants differing in the electrophoretic mobility and is encoded by a single gene. Gene His1 , coding for the slowest subtype 1, was mapped on chromosome 5. A complex locus His(2–6) , containing closely linked genes encoding subtypes 2 to 6, and the gene His7 of the subtype 7 were mapped on chromosome 1, with the distance of approximately 30 cM between them. Histone H1 spectrum changes during the maturation of tissues: subtype 7 is relatively abundant in all young tissues and gradually disappears in the course of their maturation, while the relative amount of subtype 6 increases. Several other legume species tested have H1 spectra homologous to that of the garden pea.

New data on three molecular markers from different cellular genomes in Mediterranean accessions reveal new insights into phylogeography of Pisum sativum L. subsp. elatius (Bieb.) Schmalh.

Twelve accessions classified as Pisum sativum subsp. elatius, mostly from West and Central Mediterranean, were analysed for three markers from different cellular genomes: rbcL (plastid genome), coxI (mitochondrial genome) and SCA (nuclear genome). Based on geographical distribution of their allele combinations analysed in this and the earlier study, we suggest a putative history of wild representatives of P. sativum. The ancestor of this species belonged to lineage A (coxI+, rbcL+, SCAf); it appeared in East Mediterranean, then spread westward most probably during one of the Pleistocene coolings when the sea was smaller, so that representatives of lineage A remained in the Eastern Mediterranean and on the islands of Sicily and Menorca. Mutation leading to the loss of the restriction site for PsiI in coxI−, gave rise to lineage C (coxI−, rbcL+, SCAf) which spread widely in the Mediterranean and is now found in France, Greece and Ethiopia. Mutation leading to rbcL− gave rise to lineage D (coxI−, rbcL−, SCAf), now found in Egypt (P. sativum subsp. jomardii) and Spain. Mutational transition of SCAf to SCAs most probably took place in North-Eastern Mediterranean since the resulting lineage B (coxI−, rbcL−, SCAs) now occupies the Tauro-Caucasian area. In Asia Minor and North Israel, line B met the ancestral line A so that both lines coexist there presently. The lineage B gave rise to the cultivated P. sativum subsp. sativum.

Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene.

A phylogenetic analysis of the genus Pisum (peas), embracing diverse wild and cultivated forms, which evoke problems with species delimitation, was carried out based on a gene coding for histone H1, a protein that has a long and variable functional C-terminal domain. Phylogenetic trees were reconstructed on the basis of the coding sequence of the gene His5 of H1 subtype 5 in 65 pea accessions. Early separation of a clear-cut wild species Pisum fulvum is well supported, while cultivated species Pisum abyssinicum appears as a small branch within Pisum sativum. Another robust branch within P. sativum includes some wild and almost all cultivated representatives of P. sativum. Other wild representatives form diverse but rather subtle branches. In a subset of accessions, PsbA-trnH chloroplast intergenic spacer was also analysed and found less informative than His5. A number of accessions of cultivated peas from remote regions have a His5 allele of identical sequence, encoding an electrophoretically slow protein product, which earlier attracted attention as likely positively selected in harsh climate conditions. In PsbA-trnH, a 8bp deletion was found, which marks cultivated representatives of P. sativum.

Large changes in the structure of the major histone H1 subtype result in small effects on quantitative traits in legumes

Electrophoretic analysis of the most abundant subtype of histone H1 (H1-1) of 301 accessions of grasspea (Lathyrus sativus) and 575 accessions of lentil (Lens culinaris) revealed allelic variants which most probably arose due to recent mutations. In each species, a single heterozygote for a mutation was taken for construction of isogenic lines carrying different H1-1 variants. Sequencing of alleles encoding H1-1 in lentil, grasspea, pea and Lathyrus aphaca showed the presence of an extended region in C-terminal tail which we termed ‘regular zone’ (RZ). It consists of 14 6-amino-acid units of which 12 (pea and Lathyrus species) or 13 (lentil) are represented by an AKPAAK sequence. The structure of the hypervariable unit 8 is species-specific. At the DNA level most AKPAAK units differ in the third codon positions, implying the action of natural selection preserving the RZ organization. In lentil, the fast variant lost two units (including unit 8), while one AKPAAK repeat of the slow variant is transformed into an anomalous SMPAAK. The mutant variant of the grasspea H1-1 differs from the standard one by duplication of an 11-amino-acid segment in N-terminal tail. The isogenic lines of lentil and grasspea were compared for a number of quantitative traits, some of them showing small (1–8%) significant differences.

Phenotypic effect of substitution of allelic variants for a histone H1 subtype specific for growing tissues in the garden pea (Pisum sativum L.)

In pea, subtype H1-7 of histone H1 is specific for young actively growing tissues and disappears from chromatin of mature tissues. We sequenced the alleles coding for three main variants, numbered according to the increase of the electrophoretic mobility. Allele 1 differs from the most common allele 2 by eight nucleotide substitutions, two of them associated with amino acid replacements, His->Tyr in the globular domain and Ala->Val in the C-terminal domain. Allele 3 differs from alleles 1 and 2 by a 24-bp deletion in the part coding for the C-terminal domain. In three greenhouse experiments, we compared quantitative traits in nearly isogenic lines differing by these H1-7 variants. In experiment 1, three lines bearing either of the three allelic variants were compared, the other experiments involved pairs of lines bearing variants 1 and 3. In all experiments, statistically significant differences between the lines were registered, mostly related to the plant size. The most prominent effect was associated with plant growth dynamics. Plants of line 3, carrying the 8-amino acid deletion in histone H1-7, on average grew slower. In two experiments, the differences of the mean stem length persisted throughout plant growth while in experiment 2 differences disappeared upon maturity. The H1-7 subtype is supposed to be related to maintenance of chromatin state characteristic for cell growth and division.

Nuclear-cytoplasmic conflict in pea (Pisum sativum L.) is associated with nuclear and plastidic candidate genes encoding acetyl-CoA carboxylase subunits.

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

Reciprocal compatibility within the genus Pisum L. as studied in F1 hybrids: 1. Crosses involving P. sativum L. subsp. sativum

Seven diverse accessions of peas including Pisum fulvum Boiss. et Noë (WL2140), Pisum abyssinicum A. Br. (VIR2759) and Pisum sativum L. subsp. elatius (Bieb.) Schmahl. were crossed reciprocally with a testerline WL1238 of the cultivated pea, P. sativum L. subsp. sativum. Efficiency of crosses (the average number of hybrid seeds per cross) in reciprocal directions and general characteristics of reciprocal F1 hybrids, such as F1 seed mass, pollen and seed fertility, height, yield and biomass, were compared. P. fulvum and P. abyssinicum showed strong reproductive barriers with P. sativum subsp. sativum, both prezygotic, as estimated by crossing efficiency, and postzygotic, manifested as hybrid sterility and weakness. Among crosses of five accessions of wild P. sativum subsp. elatius with the testerline, only hybrids with CE1 (Crimea) showed no difference in reciprocal combinations. Reciprocal hybrids with CE1 and JI1794 (Holan Heights) showed fully fertile pollen. In other cases reciprocal differences were registered as to pollen fertility and general vigour. Two special cases with wild peas as seed parents were observed: abnormal hybrids with VIR320 (Palestine) and arrest of embryo development in crosses with L100 (Be’er Sheva). In all cases of reciprocal differences except for those involving accession 721 (Mt. Carmel), vigour and fertility of hybrids were higher when WL1238 (P. sativum subsp. sativum) was used as seed parent. Paternal plastid DNA was registered in hybrids with WL2140 and VIR320 as seed parents. Reproductive barriers within P. sativum L. showed a complicated pattern scarcely corresponding to any intraspecies taxonomy.