Inheritance and genetic mapping of two nuclear genes involved in nuclear–cytoplasmic incompatibility in peas (Pisum sativum L.)

Genetic analysis was performed to finely map and assess the mode of inheritance of two unlinked nuclear genes Scs1 and Scs2 involved in incompatibility of the nuclear genome of the cultivated pea Pisum sativum subsp. sativum with the cytoplasm of the wild pea of the subspecies P. sativum subsp. elatius, accession VIR320. Based on the segregation of genotypes in the progeny of the test-crosses, we concluded that if the cytoplasm was inherited from the wild pea VIR320, the Scs1 allele from the cultivated pea was gametophyte lethal and sporophyte recessive lethal. The Scs2 allele from the cultivated pea reduced male gametophyte viability. In homozygote, Scs2 from cultivated parent brought about nuclear–cytoplasmic conflict manifested as chlorophyll deficiency, reduction of blade organs, and low pollen fertility of about 20%. In heterozygote, Scs1 and Scs2 genes reduced pollen fertility by ca 50 and 30%, respectively. The Scs1 and Scs2 genes involved in nuclear–cytoplasmic incompatibility were genetically mapped. The distance between the markers bordering Scs1 comprised about 2.5 cM on linkage group III. The map distance between the bordering markers in the neighborhood of Scs2 varied substantially from cross to cross in the range of 2.0–15.1 cM on linkage group V.     

Meiotic abnormalities as expression of nuclear-cytoplasmic incompatibility in crosses of <Emphasis Type="Italic">Pisum sativum</Emphasis> subspecies

Meiosis in anthers and mitosis in somatic cells were studied in reciprocal F₁ hybrids of the accession VIR320, which belonged to wild Pisum sativum ssp. elatius (Bieb.) Schmal., and the laboratory line Sprint-1. When VIR320 was used as a maternal form, the hybrids displayed nuclear-cytoplasmic conflict, which caused chlorophyll defects and meiotic abnormalities. One or two chromosomes lagged in the equatorial region during chromosome segregation to the poles, distorting cytokinesis and yielding abnormal microspores. Chlorophyll defects were not observed, and meiotic abnormalities were far less frequent in reciprocal hybrids and in the case of an abnormal paternal inheritance of plastids from Sprint-1. Mitosis lacked overt abnormalities in all of the hybrids.     

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.     

Wild peas vary in their cross-compatibility with cultivated pea (Pisum sativum subsp. sativum L.) depending on alleles of a nuclear–cytoplasmic incompatibility locus

Key message

Divergent wild and endemic peas differ in hybrid sterility in reciprocal crosses with cultivated pea depending on alleles of a nuclear ‘speciation gene’ involved in nuclear–cytoplasmic compatibility.

Background

In hybrids between cultivated and wild peas, nuclear–cytoplasmic conflict frequently occurs. One of the nuclear genes involved, Scs1, was earlier mapped on Linkage Group III.

Results

In reciprocal crosses of seven divergent pea accessions with cultivated P. sativum, some alleles of Scs1 manifested incompatibility with an alien cytoplasm as a decrease in pollen fertility to about 50 % in the heterozygotes and lack of some genotypic classes among F2 segregants. Earlier, we defined monophyletic evolutionary lineages A, B, C and D of pea according to allelic state of three markers, from nuclear, plastid and mitochondrial genomes. All tested representatives of wild peas from the lineages A and C exhibited incompatibility due to Scs1 deleterious effects in crosses with testerlines of P. sativum subsp. sativum (the common cultivated pea) at least in one direction. A wild pea from the lineage B and a cultivated pea from the lineage D were compatible with the testerline in both directions. The tested accession of cultivated P. abyssinicum (lineage A) was partially compatible in both directions. The Scs1 alleles of some pea accessions even originating from the same geographic area were remarkably different in their compatibility with cultivated Pisum sativum cytoplasm.

Conclusion

Variability of a gene involved in reproductive isolation is of important evolutionary role and nominate Scs1 as a speciation gene.     

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.     

Mapping of quantitative trait loci for resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in <Emphasis Type="Italic">Pisum sativum</Emphasis> subsp. <Emphasis Type="Italic">syriacum</Emphasis>

Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings. The wild Pisum sativum subsp. syriacum accession P665 shows good levels of resistance to this pathogen. Knowledge of the genetic factors controlling resistance to M. pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M. pinodes in pea have never included this wild species. The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M. pinodes in P. sativum subsp. syriacum and to compare these with QTLs previously described for the same trait in P. sativum. A population formed by 111 F_6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers. The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes. Six QTLs associated with resistance to M. pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait. While QTLs MpIII.1 and MpIII.2 were detected both for seedlings and field resistance, MpV.1 and MpII.1 were specific for growth chamber conditions and MpIII.3 and MpIV.1 for field resistance. Quantitative trait loci MpIII.1, MpII.1, MpIII.2 and MpIII.3 may coincide with other QTLs associated with resistance to M. pinodes previously described in P. sativum. Four QTLs associated with earliness of flowering were also identified. While dfIII.2 and dfVI.1, may correspond with other genes and QTLs controlling earliness in P. sativum, dfIII.1 and dfII.1 may be specific to P. sativum subsp. syriacum. Flowering date and growth habit were strongly associated with resistance to M. pinodes in the field evaluations. The relation observed between earliness, growth habit and resistance to M. pinodes is discussed.     

Identification and characterisation of antixenosis and antibiosis to pea aphid (Acyrthosiphon pisum) in Pisum spp. germplasm

Acyrthosiphon pisum is a polyphagous aphid of major importance on the pea crop to which few resistant cultivars are available. In this study, we screened a germplasm collection of Pisum spp. under field conditions over two seasons yielding the identification of a number of accessions with intermediate levels of resistance. Selected accessions were further studied under semi‐controlled and controlled conditions in no choice and choice assays to validate the responses, and to further characterise the mechanisms of resistance operative. Results elucidated the valuable resistance of accession P40 (Pisum sativum ssp. sativum) and P665 (P. sativum ssp. syriacum), with the combination of both antixenosis, by reducing aphid preference, and antibiosis, by diminishing aphid proliferation.     

Gibberellin A20 in seed of Pisum sativum L., cv. Alaska

Summary The main gibberellin in immature seed of Pisum sativum L., cv. Alaska, is identified as GA₂₀ by GC-MS. GA₉ may also be present.     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Band composition of electrophoretic spectra of storage proteins in interspecific pea hybrids

Electrophoretic spectra of storage proteins in parental plants and interspecific F₁ and F₂ hybrids Pisum sativum × Pisum fulvum have been studied. Correspondence between the polymorphism levels of protein components among the species and within the species P. sativum was established. Accessions of P. fulvum I609881 and I609885 manifested low polymorphism. Storage proteins of both parents were observed in spectra of F₁ hybrids. F₂ hybrids segregated at a limited set of bands. Accession I609881 of P. fulvum is characterized by unique band 7, which was inherited in F₁.     

Iron Deficiency Induced Changes on Electron Transport Rate in Pisum Sativum Chloroplasts

Iron deficiency induced decrease in the rate of whole electron transport chain in chloroplasts of pea (Pisum sativum L.). Such reduction was mainly due to the loss of photosystem (PS) 2 activity. The same result was obtained when the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m) was evaluated. The loss in PS 2 activity was primarily due to a loss of 33, 23 and 17 kDa polypeptides. In contrast, iron deficiency induced the synthesis of 28 and 29 kDa polypeptides.     

Assignment of linkage groups to pea chromosomes after karyotyping and gene mapping by fluorescent in situ hybridization

Chromosomes of the pea (Pisum sativum L.) were submitted to fluorescent in situ hybridization (FISH) with probes specific for the oligonucleotides (AG)₁₂, (AC)₁₂, (GAA)₁₀, and (GATA)₇ and for the genes encoding 25S rRNA, 5S rRNA and the storage proteins legumin A, K and vicilin. A fourth 5S rRNA gene locus, apparently specific for an accession of the cultivar Grüne Victoria, was newly detected. This allowed all seven chromosome pairs to be distinguished by FISH signals of rRNA genes. The same was possible using a combination of oligonucleotide probes or of oligonucleotides and rRNA gene-specific probes in multicolour FISH. Rehybridization with the 5S rRNA gene-specific probe allowed us to assign vicilin genes to the short arm of chromosome 5, the single legumin A locus to the long arm of chromosome 3 and the legumin B-type genes (exemplified by legumin K) to one locus on the short arm of chromosome 6. Correlation of these data with an updated version of the pea genetic map allowed the assignment of most linkage groups to defined chromosomes. It only remains to be established which of linkage groups IV and VII corresponds to the satellited chromosomes 4 or 7, respectively. Received: 13 February 1998; in revised form: 3 April 1998 / Accepted: 7 April 1998     

Identification of gibberellins A20 and A29 in seed of Pisum sativum cv. Progress No. 9 by combined gas chromatography-mass spectrometry

Summary The gibberellin A₁ (GA₁)-like and GA₅-like fractions from immature seeds of Pisum sativum cv. Progress No. 9 were identified by combined gas chromatography-mass spectrometry as GA₂₉ and GA₂₀ respectively.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Sequential extraction of chlorophyll from chlorophyll-protein complexes in lyophilized pea thylakoids with solvents of different polarity

Lyophilized chloroplasts of Pisum sativum (pea) have been extracted with petroleum ether of different polarity (obtained by adding varying amounts of ethanol to the petroleum ether). Extracted thylakoids have then been solubilized by sodium dodecyl sulphate (SDS) and chlorophyll-protein complexes have been isolated by polyacrylamide gel electrophoresis (PAGE). Absorption- and low temperature fluorescence emission spectro-scopy have been used to characterize thylakoids and purified chlorophyll-protein complexes. Weakly polar solvents extracted mainly chlorophyll a. SDS-PAGE scan profiles of similarly extracted thylakoids contained no photosystem II chlorophyll a reaction center antennae (CP-a_n) and the amount of photosystem I chlorophyll a reaction center antennae (CP-a₁) was reduced as compared with an unextracted control. This was due partly to the extraction of chlorophyll a prior to SDS-PAGE, and partly to the increased solubilization of chlorophyll a by SDS as a result of β-carotene extraction. By increasing the polarity of the solvent CP-a₁ also disappeared in the scan profile, leaving only the light-harvesting chlorophyll a/b-protein complex (CP-a/b) and SDS complexed chlorophyll. From these results we conclude that the chlorophyll molecules in the reaction center antennae are relatively more hydrophobically associated than the molecules in the light-harvesting CP-a/b complex. The chlorophyll a of CP-a_u and the far red absorbing chlorophyll a fraction of CP-a₁ appear to be the most hydrophobically associated chlorophyll molecules.     

QTLs for cell membrane stability and flag leaf area under drought stress in a wheat RIL population

A population of 206 recombinant inbred lines (RILs F₉–F₁₀) derived from wheat cross WL711/C306 was phenotyped for morpho-physiological traits such as flag leaf area (FLA), flag leaf length (FLL), flag leaf width (FLW), and cell membrane stability (CMS) under water deficit stress (WDS) environment. High yielding cultivar, WL711 had higher FLA than the medium yielding cultivar C306 across trials under both environments. Parent cultivar C306 maintained membrane integrity while WL711 showed higher membrane damage under WDS. The RIL population showed considerable variation, normal distribution and transgressive segregation for FLA, FLL, FLW and CMS under WDS. The genetic linkage map of WL711/C306 RIL population was constructed comprising of 346 markers. The total map distance was 4526.8 cM with an averaged interval of 12.9 cM between adjacent markers. Major consistent QTL for FLA, FLL, FLW, and CMS were identified on chromosomes 2DS and 3BS respectively in the WL711/C306 RIL population under WDS. The major QTL for FLA, qFLAWD.2D.1 which expressed in multiple environments and for CMS, qCMSWD.3B.3 and qCMSWD.3B.4, accounted for a large proportion of phenotypic variance (PV) with positive allele being contributed by C306, a drought resistant (DR) parent. QTL qFLAWD.2D.1 for FLA co-located with QTL for grain number (GN) and days to flowering (DTF) while QTL qCMSWD.3B.3 and qCMS.3B.4 co-located with QTL for grain yield and its components, days to flowering, canopy temperature and coleoptiles length as reported in our previous publications on the WL711/C306 population (Shukla et al. in Euphytica 203:449–467, 2015; Singh et al. in J Plant Biochem Biotechnol 24:324–330, 2015). Two candidate genes Ghd7 for grain yield and heading date and OsCDK4 for calcium dependent protein kinases were identified in the 2DS and 3BS QTL regions respectively on comparison with gene content of rice chromosomes 7 and 1 respectively. Hence, QTLs qFLAWD.2D.1 and qCMSWD.3B.3 are potential target regions for fine mapping and marker assisted selection for FLA and CMS respectively in wheat under water deficit environments.

     

Variation in Oidium neolycopersici development on host and non-host plant species and their tissue defence responses

Detailed studies of the infection processes of Oidium neolycopersici (tomato powdery mildew) and plant tissue responses were carried out on 10 Lycopersicon spp. accessions and one of each species of Cucumis sativus, Datura stramonium, Lactuca sativa, Petunia hybrida and Pisum sativum with different levels of resistance. Germination of O. neolycopersici conidia was not inhibited by the host plant. However, in the early stages of O. neolycopersici infection significant differences in conidial germ tube development on resistant and susceptible plant lines were observed. The first substantial differences appeared after 24 h post inoculation (hpi). Mycelia and conidiophores developed on susceptible lines; however, there was no mycelial development on resistant plant lines. The most frequent resistant response in Lycopersicon species was the necrotic (hypersensitive) reaction (HR), occasionally followed by pathogen development. The completely resistant accession L. hirsutum (LA 1347) showed only a limited number of necrotic host cells per infection site (2%). In Oidium resistant tomato lines OR 4061 and OR 960008 the existence of adult resistance was detected. This phenomenon occurred mainly in accession OR 4061. Rapid development and profuse sporulation of O. neolycopersici was observed on juvenile plants (6–8 wk old), however this was in contrast to the slow development and sporadic sporulation observed on 4 month old plants. Evidence of posthaustorial resistance was observed in the interaction of O. neolycopersici with non-host species (Lactuca sativa and Pisum sativum.) This was in contrast to Datura stramonium and Petunia hybrida, where development of powdery mildew was delayed at a later stage in the infection cycle. With the exception of Pisum sativum, the necrotic (hypersensitive) response was observed often.     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

Pigments and Gas Exchange Characteristics in Leaves of Chlorophyll Mutants of Pisum sativum

Quantitative and qualitative characteristics of pigment composition and gas exchange were studied in chlorophyll mutants of pea, Pisum sativum L.: chlorotica 2004 and 2014. The mutant 2004 had light-green color, whereas the mutant 2014 has yellow-green leaves and stems; they contained about 80 and 50% of chlorophyll, respectively, compared to the initial line. cv. Torsdag. Leaves of the mutant 2004 had significantly lower carotene content and accumulated more lutein and violaxanthin. In the mutant 2014, the contents of chlorophyll and all carotenoids were reduced almost proportionally. The quantum efficiency of photosynthesis was by 29–30% lower in the mutants, and it was 1.5–2 times higher in F₁ hybrids, as compared to control plants. Our data allow us to conclude that the impairment of photosynthesis in the mutant 2014 is caused by the changed mesostructure of leaves, whereas in the mutant 2004, it may be caused by an impairment of photosystem reaction centers.